CROSS REFERENCE TO RELATED APPLICATIONS
This application is the U.S. national phase application under 35 U.S.C. § 371 of PCT International Application No. PCT/US2020/018195, filed February 13, 2020, claiming the benefit of U.S. Provisional Application No. 62/805,238, filed February 13, 2019; 62/805,271, filed February 13, 2019; 62/852,224, filed May 23, 2019; 62/852,228, filed May 23, 2019; 62/931,722, filed November 6, 2019; 62/941,569, filed November 27, 2019; and 62/966,526 filed January 27, 2020, the contents of which are incorporated herein by reference in their entirety.
SEQUENCE LIST
The present application contains a sequence listing filed electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy created on March 27, 2020 is called 180802-041902PCTSequenceListing.txt and is 818,399 bytes in size.
RECORDING BY REFERENCE
All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application were expressly and individually indicated as being incorporated by reference. Unless otherwise noted, publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety.
BACKGROUND OF THE DISCLOSURE
In healthy individuals, alpha-1-antitrypsin (A1AT) is produced by hepatocytes in the liver and secreted into the systemic circulation where it acts as a protease inhibitor. It is a particularly good inhibitor of neutrophil elastase, protecting tissues and organs such as the lungs from elastin breakdown. In patients with alpha-1 antitrypsin deficiency (A1AD), mutations in the gene that codes for A1AT lead to decreased protein production. Consequently, elastin in the lungs is more easily degraded by neutrophil elastase, and over time the loss of lung elasticity develops into chronic obstructive pulmonary disease (COPD).
The most common pathogenic A1AT variant is a guanine to adenine mutation resulting in a glutamate to lysine substitution at amino acid 342. This substitution leads to misfolding and polymerization of the protein in the hepatocytes, and ultimately the toxic aggregates can lead to liver damage and cirrhosis. While liver toxicity could be addressed by gene knockout (CRISPR/ZFN/TALEN) or gene knockdown (siRNA), neither approach addresses lung pathology. Although pulmonary pathology can be addressed with protein replacement therapy, this therapy also does not address liver toxicity. Gene therapy would also be inappropriate to address the A1AT genetic defect. Since the liver of patients with A1AD is already heavily burdened with endogenous A1AT, gene therapy that increases A1AT in the liver would be counterproductive.
Therefore, there is a need for a method of treating patients with A1AD that addresses both pulmonary pathology and liver toxicity.
SUMMARY
As described below, the present invention provides compositions and methods for editing deleterious mutations associated with alpha-1-antitrypsin deficiency (A1AD). In certain embodiments, the invention provides methods of treating A1AD using a modified adenosine deaminase designated "ABE8" that has unprecedented levels (e.g., >60-70%) of efficiency and specificity to target A1AD-associated mutations correct.
In one aspect, the invention provides a method of editing an alpha-1-antitrypsin polynucleotide containing a single nucleotide polymorphism (SNP) associated with alpha-1-antitrypsin deficiency, the method comprising contacting the polynucleotide with one or more guide RNAs and a base editor contains a polynucleotide-programmable DNA binding domain and at least one base editor domain which is an adenosine deaminase variant containing an alteration at amino acid position 82 or 166
wherein the guide RNA targets the base editor to cause a change in the SNP associated with alpha-1 antitrypsin deficiency.
In another aspect, the invention provides a method of editing an alpha-1-antitrypsin polynucleotide containing a single nucleotide polymorphism (SNP) associated with alpha-1-antitrypsin deficiency, the method comprising contacting an alpha -1-antitrypsin polynucleotide with one or more guide RNAs and a fusion protein containing a polynucleotide-programmable DNA-binding domain containing the following sequence:
wherein the bold sequence denotes a Cas9-derived sequence, the italic sequence denotes a linker sequence, and the underlined sequence denotes a bipartite nuclear localization sequence, and at least one base editor domain containing an adenosine deaminase variant containing a change at amino acid position 82 or 166 of
In another aspect, the invention provides a base editing system comprising the fusion protein of any preceding aspect and a guide RNA comprising a nucleic acid sequence selected from
In another aspect, a cell made by introducing into the cell or a precursor thereof is:
a base editor, a polynucleotide encoding the base editor, to the cell, the base editor including a polynucleotide-programmable DNA binding domain and an adenosine deaminase domain described in any one of the preceding aspects; and one or more leader polynucleotides that target the base editor to effect an A⋅T to G⋅C change in the SNP associated with alpha-1-antitrypsin deficiency. In one embodiment, the cell produced is a hepatocyte or a progenitor thereof. In another embodiment, the cell is from an alpha-1-antitrypsin deficient patient. In another embodiment, the cell is a mammalian cell or human cell.
In various embodiments of the above aspects, the gRNA further includes a nucleic acid sequence
In a still further aspect, the invention provides a method of treating alpha-1-antitrypsin deficiency in a patient, comprising administering to the patient a cell of any preceding aspect. In one embodiment, the cell is autologous or allogeneic to the subject.
In a still further aspect, the invention provides an isolated cell or cell population propagated or expanded from the cell of the aspects and embodiments described above.
In yet another aspect, the invention provides a method for preparing a hepatocyte, comprising: (a) introducing a base editor or a polynucleotide encoding the base editor into a hepatocyte containing an alpha-1-antitrypsin deficiency-associated SNP, wherein the Base Editor comprises a polynucleotide-programmable nucleotide binding domain and an adenosine deaminase variant domain described in any of the aspects and embodiments described above; and one or more leader polynucleotides, wherein the one or more leader polynucleotides target the base editor to effect an A⋅T to G⋅C change in the SNP associated with alpha-1 antitrypsin deficiency.
In various embodiments, the hepatocyte is a mammalian cell or a human cell.
In other embodiments of the above aspects, the adenosine deaminase variant comprises changes at amino acid positions 82 and 166. In other embodiments of the above aspects, the adenosine deaminase variant comprises a V82S change. In other embodiments of the above aspects, the adenosine deaminase variant comprises a T166R alteration. In other embodiments of the above aspects, the adenosine deaminase variant includes V82S and T166R alterations. In other embodiments of the above aspects, the adenosine deaminase variant further comprises one or more of the following alterations: Y147T, Y147R, Q154S, Y123H and Q154R. In other embodiments of the above aspects, the adenosine deaminase variant comprises the following changes: Y147T + Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. In one embodiment of the aspects described above, the adenosine deaminase variant comprises Y147R + Q154R + Y123H. In one embodiment of the aspects described above, the adenosine deaminase variant comprises Y147R + Q154R + I76Y. In one embodiment of the aspects described above, the adenosine deaminase variant comprises Y147R + Q154R + T166R. In one embodiment of the aspects described above, the adenosine deaminase variant comprises Y147T + Q154R. In one embodiment of the aspects described above, the adenosine deaminase variant comprises Y147T + Q154S. In one embodiment of the aspects described above, the adenosine deaminase variant comprises Y147R + Q154S. In one embodiment of the aspects described above, the adenosine deaminase variant comprises V82S + Q154S. In one embodiment of the aspects described above, the adenosine deaminase variant comprises V82S + Y147R. In one embodiment of the aspects described above, the adenosine deaminase variant comprises V82S + Q154R. In one embodiment of the aspects described above, the adenosine deaminase variant comprises V82S + Y123H. In one embodiment of the aspects described above, the adenosine deaminase variant comprises I76Y + V82S. In one embodiment of the aspects described above, the adenosine deaminase variant comprises V82S + Y123H + Y147T. In one embodiment of the aspects described above, the adenosine deaminase variant comprises V82S + Y123H + Y147R. In one embodiment of the aspects described above, the adenosine deaminase variant comprises V82S + Y123H + Q154R. In one embodiment of the aspects described above, the adenosine deaminase variant comprises Y123H + Y147R + Q154R + I76Y. In one embodiment of the aspects described above, the adenosine deaminase variant comprises V82S + Y123H + Y147R + Q154R. In one embodiment of the aspects described above, the adenosine deaminase variant comprises I76Y + V82S + Y123H + Y147R + Q154RI. In other embodiments of the above aspects, the adenosine deaminase variant comprises a deletion of the C-terminus beginning with a residue selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156 and 157. In other embodiments of the above aspects, the base editor domain comprises a single adenosine deaminase variant containing V82S and T166R. In other embodiments of the above aspects, the base editor domain comprises a wild-type adenosine deaminase domain and a variant adenosine deaminase. In other embodiments of the above aspects, the adenosine deaminase variant further comprises an alteration selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R, Q154R. In other embodiments of the above aspects, the basic editor domain comprises a TadA7.10 domain and an adenosine deaminase variant. In other embodiments of the above aspects, the adenosine deaminase variant further comprises an alteration selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R, Q154R. In other embodiments of the above aspects, the base editor comprises a TadA7.10 domain and an adenosine deaminase variant containing changes selected from the group consisting of Y147T + Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. In other embodiments of the above aspects, the base editor is an ABE8 comprising or consisting essentially of the following sequence or a fragment thereof having adenosine deaminase activity:
In other embodiments of the above aspects, the adenosine deaminase variant comprises a truncated ABE8 missing 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,6 , 17, 18, 19, or 20 N-terminal amino acid residues relative to full-length ABE8. In other embodiments of the above aspects, the adenosine deaminase variant is a truncated ABE8 lacking 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,6 , 17, 18, 19, or 20 C-terminal amino acid residues relative to full-length ABE8.
In other embodiments of the above aspects, the change from A.T to G.C at the SNP associated with alpha-1-antitrypsin deficiency changes a glutamic acid to a lysine in the alpha-1-antitrypsin polypeptide. In other embodiments of the above aspects, the SNP associated with alpha-1-antitrypsin deficiency results in the expression of an alpha-1-antitrypsin polypeptide having a lysine at amino acid position 342. In other embodiments of the above aspects, the SNP associated with alpha-1-antitrypsin Deficiency-associated SNP 1 antitrypsin deficiency replaces a glutamic acid with a lysine. In other embodiments of the above aspects, the cell is selected for the change in SNP from A*T to G*C associated with alpha-1-antitrypsin deficiency. In other embodiments of the above aspects, the polynucleotide-programmable DNA binding domain is modifiedStaphylococcus aureusCas9 (SaCas9),Streptococcus thermophilus1 Cas9 (St1Cas9), a modifiedStreptococcus pyogenesCas9 (SpCas9) or variants thereof. In other embodiments of the above aspects, the polynucleotide-programmable DNA binding domain comprises a variant of SpCas9 having an altered protospacer adjacent motif (PAM) specificity or specificity for a non-G-PAM. In other embodiments of the above aspects, the altered PAM has specificity for the nucleic acid sequence 5'-NGC-3'. In other embodiments of the above aspects, the modified SpCas9 comprises the amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E and T1337R or corresponding amino acid substitutions thereof. In other embodiments of the above aspects, the polynucleotide-programmable DNA binding domain is a nuclease-inactive or nickase variant. In other embodiments of the above aspects, the nickase variant comprises an amino acid substitution D10A or a corresponding amino acid substitution thereof. In other embodiments of the above aspects, the base editor further comprises a zinc finger domain. In other embodiments of the above aspects, the adenosine deaminase domain is capable of deaminating adenine in deoxyribonucleic acid (DNA). In other embodiments of the above aspects, the one or more guide RNAs comprise a CRISPR RNA (crRNA) and a transcoded small RNA (tracrRNA), wherein the crRNA comprises a nucleic acid sequence complementary to an alpha-1-antitrypsin nucleic acid sequence the SNP associated with alpha-1 antitrypsin deficiency. In other embodiments of the above aspects, the base editor and the one or more lead polynucleotides form a complex in the cell. In other embodiments of the above aspects, the base editor is in complex with a single guide RNA (sgRNA) containing a nucleic acid sequence complementary to an alpha-1-antitrypsin nucleic acid sequence encoding the alpha-1-antitrypsin deficiency-associated SNP contains.
In another aspect, there is provided a method of treating alpha-1-antitrypsin deficiency (A1AD) in a subject, the method comprising administering a fusion protein comprising an adenosine deaminase variant inserted into a Cas9 or a Cas12 Polypeptide comprising the subject, or a polynucleotide encoding the fusion protein thereof; and one or more leader polynucleotides to target the fusion protein to effect an A⋅T to G⋅C change of a single nucleotide polymorphism (SNP) associated with A1AD, thereby treating A1AD in the patient.
In another aspect, there is provided a method of treating alpha-1-antitrypsin deficiency (A1AD) in a subject, the method comprising administering an adenosine base editor, ABE8, or a polynucleotide encoding the base editor, to the subject, wherein the ABE8 comprises an adenosine deaminase variant inserted into a Cas9 or Cas12 polypeptide; and one or more leader polynucleotides that target ABE8 to effect an A⋅T to G⋅C change of a SNP associated with A1AD, thereby treating A1AD in the subject.
In one embodiment of the methods described above, the ABE8 is selected from ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, ABE8.5-m, ABE8.6-m, ABE8. 7-m, ABE8.8-m, ABE8.9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.13-m, ABE8.14-m, ABE8 .15- m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8.23-m, ABE8.24-m, ABE8.1-d, ABE8.2-d, ABE8.3-d, ABE8.4-d, ABE8.5-d, ABE8.6-d, ABE8.7-d , ABE8. 8-d, ABE8.9-d, ABE8.10-d, ABE8.11-d, ABE8.12-d, ABE8.13-d, ABE8.14-d, ABE8.15-d, ABE8 .16- d, ABE8.17-d, ABE8.18-d, ABE8.19-d, ABE8.20-d, ABE8.21-d, ABE8.22-d, ABE8.23-d or ABE8. 24-d. In one embodiment of the methods described above, the adenosine deaminase variant comprises the amino acid sequence of:
and wherein the amino acid sequence comprises at least one change. In one embodiment, the adenosine deaminase variant includes changes at amino acid position 82 and/or 166. In one embodiment, the at least one change includes: V82S, T166R, Y147T, Y147R, Q154S, Y123H, and/or Q154R.
In one embodiment of the methods described above, the adenosine deaminase variant comprises one of the following combinations of changes: Y147T + Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. In one embodiment of the methods described above, the adenosine deaminase variant is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA * 8.22, TadA*8.23 or TadA*8.24. In one embodiment, the adenosine deaminase variant comprises a deletion of the C-terminus beginning at a residue selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156 and 157. In one embodiment, the adenosine deaminase variant is an adenosine deaminase monomer comprising a TadA*8 adenosine deaminase variant domain. In one embodiment, the adenosine deaminase variant is an adenosine deaminase heterodimer comprising a wild-type adenosine deaminase domain and a TadA*8 adenosine deaminase variant domain. In one embodiment, the adenosine deaminase variant is an adenosine deaminase heterodimer comprising a TadA domain and a TadA*8 adenosine deaminase variant domain. In one embodiment of the methods described above, changing from A*T to G*C at the A1AD-associated SNP changes a glutamic acid at amino acid position 342 to lysine. In one embodiment of the methods described above, the SNP associated with A1AD results in expression of an alpha-1-antitrypsin polypeptide having a lysine at amino acid position 342. In one embodiment of the methods described above, the SNP associated with the SNP at alpha-1- In antitrypsin deficiency, a glutamic acid is replaced by a lysine.
In one embodiment of the methods described above, the variant adenosine deaminase is inserted into a flexible loop, an alpha-helix region, an unstructured portion, or a solvent-accessible portion of the Cas9 or Cas12 polypeptide. In one embodiment of the methods described above, the adenosine deaminase variant is flanked by an N-terminal fragment and a C-terminal fragment of Cas9 or Cas12 polypeptide.
In one embodiment of the methods described above, the fusion protein or ABE8 comprises the structure NH2-[N-terminal fragment of Cas9 or Cas12 polypeptide]-[variant adenosine deaminase]-[C-terminal fragment of Cas9 or Cas12 polypeptide]-COOH, wherein each instance of 14'' is an optional linker. In one embodiment, the C-terminus of the N-terminal fragment or the N-terminus of the C-terminal fragment comprises part of a flexible loop of the Cas9 or Cas12 polypeptide. In one embodiment, the flexible loop includes an amino acid near the target nucleobase when the adenosine deaminase variant deaminates the target nucleobase.
In one embodiment of the methods described above, the methods further comprise administering to the subject a guide nucleic acid sequence to cause deamination of the target SNP nucleobase associated with A1AD. In one embodiment of the methods described above, deamination of the SNP target nucleobase replaces the target nucleobase with a wild-type nucleobase or with a non-wild-type nucleobase, and wherein deamination of the target nucleobase improves symptoms of A1AD. In one embodiment of the methods described above, deamination of the A1AD-associated SNP replaces a glutamic acid with lysine.
In one embodiment of the methods described above, the target nucleobase is 1-20 nucleobases away from a PAM sequence in the target polynucleotide sequence. In one embodiment, the target nucleobase is 2-12 nucleobases upstream of the PAM sequence. In one embodiment of the methods described above, the N-terminal fragment or the C-terminal fragment of the Cas9 or Cas12 polypeptide binds to the target polynucleotide sequence. In certain embodiments, the N-terminal fragment or the C-terminal fragment comprises a RuvC domain; the N-terminal fragment or the C-terminal fragment comprises an HNH domain; neither the N-terminal fragment nor the C-terminal fragment comprises an HNH domain; or neither the N-terminal fragment nor the C-terminal fragment comprises a RuvC domain. In one embodiment, the Cas9 or Cas12 polypeptide comprises a partial or complete deletion in one or more structural domains and wherein the deaminase is inserted at the partial or complete deletion position of the Cas9 or Cas12 polypeptide. In certain embodiments, the deletion occurs within a RuvC domain; the deletion is within an HNH domain; or the deletion bridges a RuvC domain and a C-terminal domain.
In one embodiment of the methods described above, the fusion protein or ABE8 comprises a Cas9 polypeptide. In one embodiment, the Cas9 polypeptide is aStreptococcus pyogenesCas9 (SpCas9),Staphylococcus aureusCas9 (SaCas9),Streptococcus thermophilus1 Cas9 (St1Cas9) or variants thereof. In one embodiment, the Cas9 polypeptide comprises the following amino acid sequence (Cas9 reference sequence):
(single underline: HNH domain; double underline: RuvC domain; (Cas9 reference sequence) or a corresponding region thereof. In certain embodiments, the Cas9 polypeptide comprises a deletion of amino acids 1017-1069, as in the Cas9 polypeptide reference sequence numbered or corresponding amino acids thereof; the Cas9 polypeptide comprises a deletion of amino acids 792-872 as numbered in the Cas9 polypeptide reference sequence, or corresponding amino acids thereof; or the Cas9 polypeptide comprises a deletion of amino acids 792-906 as in Cas9 polypeptide numbers reference sequence or corresponding amino acids thereof.
In one embodiment of the methods described above, the adenosine deaminase variant is inserted into a flexible loop of the Cas9 polypeptide. In one embodiment, the flexible loop comprises a region selected from the group consisting of amino acid residues at positions 530-537, 569-579, 686-691, 768-793, 943-947, 1002-1040, 1052-1077, 1232-1248 and 1298-1300 as numbered in the Cas9 reference sequence, or corresponding amino acid positions thereof.
In one embodiment of the methods described above, the deaminase variant is inserted between amino acid positions 768-769, 791-792, 792-793, 1015-1016, 1022-1023, 1026-1027, 1029-1030, 1040-1041, 1052-1053, 1054-1055, 1067-1068, 1068-1069, 1247-1248 or 1248-1249 as numbered in the Cas9 reference sequence, or corresponding amino acid positions thereof. In one embodiment of the methods described above, the deaminase variant is inserted between amino acid positions 768-769, 792-793, 1022-1023, 1026-1027, 1040-1041, 1068-1069 or 1247-1248 as numbered in the Cas9 reference sequence or corresponding amino acid positions thereof. In one embodiment of the methods described above, the deaminase variant is inserted between amino acid positions 1016-1017, 1023-1024, 1029-1030, 1040-1041, 1069-1070 or 1247-1248 as numbered in the Cas9 reference sequence or corresponding amino acid positions thereof. In one embodiment of the methods described above, the adenosine deaminase variant is inserted within the Cas9 polypeptide at the loci identified in Table 13A. In one embodiment, the N-terminal fragment comprises amino acid residues 1-529, 538-568, 580-685, 692-942, 948-1001, 1026-1051, 1078-1231 and/or 1248-1297 of Cas9 reference sequence or corresponding residues of that. In one embodiment, the C-terminal fragment comprises amino acid residues 1301-1368, 1248-1297, 1078-1231, 1026-1051, 948-1001, 692-942, 580-685 and/or 538-568 of Cas9 reference sequence or residues corresponding of that.
In one embodiment of the methods described above, the Cas9 polypeptide is a modified Cas9 and has specificity for an altered PAM or a non-G-PAM. In one embodiment of the methods described above, the Cas9 polypeptide is a nickase or wherein the Cas9 polypeptide is nuclease-inactive. In one embodiment of the methods described above, the Cas9 polypeptide is a modified SpCas9 polypeptide. In one embodiment, the modified SpCas9 polypeptide comprising the amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E and T1337R (SpCas9-MQKFRAER) and having specificity for the altered PAM 5'-NGC-3'.
In another embodiment of the methods described above, the fusion protein or ABE8 comprises a Cas12 polypeptide. In one embodiment, the adenosine deaminase variant is inserted into the Cas12 polypeptide. In one embodiment, the Cas12 polypeptide is Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, or Cas12i. In one embodiment, the adenosine deaminase variant is inserted between amino acid positions: a) 153-154, 255-256, 306-307, 980-981, 1019-1020, 534-535, 604-605 or 344-345 of BhCas12b or a corresponding amino acid residue of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h or Cas12i; b) 147 and 148, 248 and 249, 299 and 300, 991 and 992 or 1031 and 1032 of BvCas12b or a corresponding amino acid residue of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h or Cas12i; or c) 157 and 158, 258 and 259, 310 and 311, 1008 and 1009 or 1044 and 1045 of AaCas12b or a corresponding amino acid residue of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h or Cas12i. In one embodiment, the adenosine deaminase variant is inserted within the Cas12 polypeptide at the loci identified in Table 13B. In one embodiment, the Cas12 polypeptide is Cas12b. In one embodiment, the Cas12 polypeptide comprises a BhCas12b domain, a BvCas12b domain, or an AACas12b domain.
In one embodiment of the methods described above, the guide RNA comprises a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). In one embodiment of the methods described above, the subject is a mammal or a human.
In another aspect there is provided a pharmaceutical composition comprising a base editing system according to any of the methods, aspects and embodiments described above and a pharmaceutically acceptable carrier, vehicle or excipient.
In one aspect there is provided a pharmaceutical composition comprising the cell of the aspects and embodiments described above and a pharmaceutically acceptable carrier, vehicle or excipient.
In another aspect, a kit is provided that includes the basic editing system according to any of the methods, aspects, and embodiments described above.
In another aspect, there is provided a kit comprising the cell of any of the aspects and embodiments described above. In one embodiment of the kits, the kit further comprises a package insert with instructions for use.
In one aspect, there is provided herein a base editor comprising a polynucleotide-programmable DNA binding domain and at least one base editor domain comprising an adenosine deaminase variant comprising an alteration at amino acid position 82 or 166
In one aspect, a base editor system comprises the base editor described above and a target RNA, wherein the target RNA targets the base editor to cause an alteration in the SNP associated with alpha-1 antitrypsin deficiency. In some embodiments, the adenosine deaminase variant comprises a V82S mutation and/or a T166R mutation. In some embodiments, the adenosine deaminase variant further comprises one or more of the following changes: Y147T, Y147R, Q154S, Y123H, and Q154R. In some embodiments, the base editor domain comprises an adenosine deaminase heterodimer comprising a wild-type adenosine deaminase domain and a variant adenosine deaminase. In some embodiments, the adenosine deaminase variant is a truncated TadA8, dem 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18 lacks 19 or 20 N-terminal amino acid residues relative to full-length TadA8. In some embodiments, the adenosine deaminase variant is a truncated TadA8, dem 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18 lacks 19 or 20 C-terminal amino acid residues relative to full-length TadA8. In some embodiments, the polynucleotide-programmable DNA binding domain is modifiedStaphylococcus aureusCas9 (SaCas9),Streptococcus thermophilus1 Cas9 (St1Cas9), a modifiedStreptococcus pyogenesCas9 (SpCas9) or variants thereof. In some embodiments, the polynucleotide-programmable DNA-binding domain is a variant of SpCas9 with an altered protospacer adjacent motif (PAM) specificity or specificity for a non-G-PAM. In some embodiments, the polynucleotide-programmable DNA binding domain is a nuclease-inactive Cas9. In some embodiments, the polynucleotide-programmable DNA binding domain is a Cas9 nickase.
In one aspect, there is provided herein a base editor system comprising one or more guide RNAs and a fusion protein comprising a polynucleotide-programmable DNA binding domain comprising the following sequence:
wherein the bold sequence denotes a Cas9 derived sequence, the italic sequence denotes a linker sequence and the underlined sequence denotes a bipartite nuclear localization sequence and at least one base editor domain comprising an adenosine deaminase variant comprising a change at amino acid position 82 and/or 166 of
In one aspect there is provided a cell comprising any of the basic editor systems described above. In some embodiments, the cell is a human cell or a mammalian cell. In some embodiments, the cell is ex vivo, in vivo, or in vitro.
The invention provides compositions and methods for editing mutations associated with alpha-1-antitrypsin deficiency (A1AD). Compositions and articles defined by the invention have been isolated or otherwise prepared in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description and from the claims.
The definition
The following definitions are supplementary to those in the prior art and refer to the current application and are not to be attributed to a related or unrelated instance, e.g. B. a patent or application that is in common ownership. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one skilled in the art to which this invention pertains. The following references provide those skilled in the art with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd Edition 1994); The Cambridge Dictionary of Science and Technology (ed. Walker, 1988); The Glossary of Genetics, 5th ed., R. Rieger et al. (ed.), Springer-Verlag (1991); and Hale & Marham, The HarperCollins Dictionary of Biology (1991).
In this application, use of the singular includes the plural unless expressly stated otherwise. It is to be noted that as used in the specification, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. In this application, unless otherwise specified, the use of "or" means "and/or" and is intended to be inclusive. Additionally, the use of the term "including" as well as other forms such as "include," "includes," and "contain" is not limiting.
As used in this specification and claim(s), the words "comprising" (and any form of including, such as "include" and "include") mean "having" (and any form of having, such as "having" and "has"), "including" (and any form of including, such as "contains" and "contains"), or "contains" (and any form of contain, such as "contains" and " contains") are inclusive or open-ended and do not exclude additional, uncited elements or method steps. It is contemplated that any embodiment discussed in this specification may be implemented with respect to any method or composition of the present disclosure, and vice versa. Additionally, compositions of the present disclosure can be used to achieve methods of the present disclosure.
The term "about" or "approximately" means within an acceptable range of error for the particular value, as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i. H. the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation according to prior art practice. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a particular value. Alternatively, particularly in relation to biological systems or processes, the term may mean within an order of magnitude, such as within 5 times or within 2 times a value. When specific values are described in the application and claims, unless otherwise noted, the term "about" should be taken to mean within an acceptable range of error for the specific value.
The ranges provided here are shorthand for all values within the range, including the first and last values. For example, a range from 1 to 50 is understood to mean any number, number combination or partial range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50.
Reference in the specification to "some embodiments," "an embodiment," "an embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is present in at least some Embodiments are included, but not necessarily all, embodiments of the present disclosures.
By "adenosine deaminase" is meant a polypeptide or fragment thereof capable of catalyzing the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine or of deoxyadenosine to deoxyinosine. In some embodiments, adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g. engineered adenosine deaminases, evolved adenosine deaminases) may be derived from any organism such as a bacterium.
In some embodiments, the adenosine deaminase comprises an alteration in the following sequence:
(also called TadA*7.10).
In some embodiments, TadA*7.10 includes at least one change. In some embodiments, TadA*7.10 includes an alteration at amino acid 82 and/or 166. In certain embodiments, a variant of the above sequence includes one or more of the following changes: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R. The change Y123H is also referred to herein as H123H (the change H123Y in TadA*7.10 reverted to Y123H (wt)). In other embodiments, a variant of the TadA*7.10 sequence comprises a combination of changes selected from the group consisting of: Y147T + Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.
In other embodiments, the invention provides adenosine deaminase variants comprising deletions, e.g. B. TadA*8, comprising a deletion of the C-terminus beginning at residue 149, 150, 151, 152, 153, 154, 155, 156 or 157. In other embodiments, the adenosine deaminase variant is a TadA (e.g. B. TadA*8) monomer comprising one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R and/or Q154R. In other embodiments, the adenosine deaminase variant is a monomer comprising a combination of changes selected from the group consisting of: Y147T + Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.
In still other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (eg, TadA*8) each containing one or more of the following alterations Y147T, Y147R, Q154S, Y123H, V82S, T166R and/or Q154R. In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8) each having a combination of changes selected from the group consisting of: Y147T + Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.
In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type TadA adenosine deaminase domain and a variant adenosine deaminase domain (eg, TadA*8) comprising one or more of the following alterations Y147T, Y147R, Q154S, Y123H, V82S , T166R and/or Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type TadA adenosine deaminase domain and a variant adenosine deaminase domain (e.g., TadA*8) comprising a combination of alterations selected from the group of: Y147T + Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.
In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (eg, TadA*8) comprising one or more of the following alterations Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (eg, TadA*8) comprising a combination of the following changes: Y147T + Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; or I76Y+V82S+Y123H+Y147R+Q154R.
In one embodiment, the adenosine deaminase is a TadA*8 comprising or consisting essentially of the following sequence or a fragment thereof having adenosine deaminase activity:
In some embodiments, TadA*8 is truncated. In some embodiments, the truncated TadA*8 is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N- terminal amino acid residues relative to full-length TadA*8. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C- terminal amino acid residues relative to full-length TadA*8. In some embodiments, the adenosine deaminase variant is a full-length TadA*8.
In certain embodiments, an adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from one of the following:
"Adenosine deaminase base editor 8 (ABE8) polypeptide" is a base editor (BE) as defined and/or described herein, comprising an adenosine deaminase variant comprising a change at amino acid position 82 and/or 166 the following reference sequence:
In some embodiments, ABE8 includes other changes relative to the reference sequence.
By "adenosine deaminase base editor 8 (ABE8) polynucleotide" is meant a polynucleotide (polynucleotide sequence) encoding an ABE8 polypeptide.
"Administering" is referred to herein as providing one or more compositions described herein to a patient or subject. By way of example and not limitation, composition administration, e.g. injection, by intravenous (IV) injection, subcutaneous (SC) injection, intradermal (ID) injection, intraperitoneal (IP) injection or intramuscular (IM) injection. Injection. One or more such routes can be used. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively or simultaneously, administration may be by the oral route.
By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
By "alpha-1-antitrypsin (A1AT) protein" is meant a polypeptide or fragment thereof that shares at least about 95% amino acid sequence identity with UniProt accession number P01009. In certain embodiments, an A1AT protein includes one or more changes relative to the following reference sequence. In a particular embodiment, an A1AT protein associated with A1AD comprises an E342K mutation. An exemplary A1AT amino acid sequence is provided below.
In the A1AT protein sequence above, the first 24 amino acids form the signal peptide (underlined). Position 342 of the sequence mutated in A1AD (i.e. E342K) is determined by placing the amino acid residue "E" after the signal sequence as amino acid "1".
By "change" is meant a change (e.g., increase or decrease) in the structure, expression levels, or activity of a gene or polypeptide, as detected by methods known in the art, such as those described herein. As used herein, a change includes a change in a polynucleotide or polypeptide sequence or a change in expression levels, such as a 25% change, a 40% change, a 50% change or more.
By "better" is meant to reduce, suppress, mitigate, reduce, arrest, or stabilize the development or progression of a disease.
By "analogue" is meant a molecule that is not identical but has analogous functional or structural features. For example, a polynucleotide or polypeptide analog retains the biological activity of a corresponding naturally occurring polynucleotide or polypeptide while having certain modifications that enhance the analog's function relative to a naturally occurring polynucleotide or polypeptide. Such modifications could increase the analog's affinity for DNA, efficiency, specificity, protease or nuclease resistance, membrane permeability, and/or half-life without altering, for example, ligand binding. An analog can include an unnatural nucleotide or an amino acid.
By "base editor (BE)" or "nucleobase editor (NBE)" is meant an agent that binds a polynucleotide and has nucleobase modifying activity. In various embodiments, the base editor comprises a nucleobase-modifying polypeptide (e.g., a deaminase) and a nucleic acid-programmable nucleotide binding domain in conjunction with a guide polynucleotide (e.g., guide RNA). In various embodiments, the agent is a biomolecular complex comprising a protein domain with base-editing activity, i. H. a domain capable of substituting a base (eg, A, T, C, G, or U) within a nucleic acid molecule (eg, DNA). In some embodiments, the polynucleotide-programmable DNA binding domain is fused or linked to a deaminase domain. In one embodiment, the agent is a fusion protein comprising a domain with base-editing activity. In another embodiment, the protein domain with base-editing activity is linked to the guide RNA (e.g., via an RNA-binding motif on the guide RNA and an RNA-binding domain fused to the deaminase). In some embodiments, the domain with base-editing activity is capable of deamination of a base within a nucleic acid molecule. In some embodiments, the base editor is capable of deaminating one or more bases within a DNA molecule. In some embodiments, the base editor is capable of deaminating an adenosine (A) within DNA. In some embodiments, the base editor is an adenosine base editor (ABE).
In some embodiments, base editors (e.g. ABE8) are generated by cloning a variant adenosine deaminase (e.g. TadA*8) into a scaffold containing a circular permutant Cas9 (e.g. spCAS9 or saCAS9) and a two-part nuclear localization sequence. Circular permutant Cas9s are known in the art and are described, for example, in Oakes et al., Cell 176, 254-267, 2019. Exemplary circular permutants follow, with the bold sequence indicating a Cas9-derived sequence, the italic sequence denoting a linker sequence, and the underlined sequence denoting a bipartite nuclear localization sequence.
CP5 (mit MSP „NGC=Pam Variant with mutations Regular Cas9 likes NGG“ PID=Protein Interacting Domain und „D10A“ Nickase):
In some embodiments, the ABE8 is selected from a base editor from Tables 6-9, 13 or 14 below. In some embodiments, ABE8 contains an adenosine deaminase variant developed from TadA. In some embodiments, the adenosine deaminase variant of ABE8 is a TadA*8 variant, as described in Table 7, 9, 13 or 14 below. In some embodiments, the adenosine deaminase variant is a TadA*7.10 variant (e.g., TadA*8) comprising one or more of an alteration selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R and/or Q154R. In various embodiments, ABE8 comprises a TadA*7.10 variant (e.g. TadA*8) with a combination of changes selected from the group of Y147T + Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. In some embodiments, ABE8 is a monomeric construct. In some embodiments, ABE8 is a heterodimeric construct. In some embodiments, the ABE8 comprises the sequence:
In some embodiments, the polynucleotide-programmable DNA binding domain is a CRISPR-associated (e.g., Cas or Cpf1) enzyme. In some embodiments, the base editor is a catalytically dead Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. Details of basic editors are described in PCT International Application Nos. PCT/2017/045381 (WO 2018/027078) and PCT/US2016/058344 (WO 2017/070632), each of which is incorporated herein by reference in its entirety. See also Komor, A.C., et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, N.M., et al., "Programmable base editing of A⋅T to G⋅C in genomic DNA without DNA cleavage" Nature 551, 464-471 (2017); Komor, A.C., et al., "Improved inhibition of base excision repair and bacteriophage Mu-Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity" Science Advances 3:eaao4774 (2017) and Rees, HA, et al., "Basic editing: precision chemistry on the genome and transcriptome of living cells." Nat. Rev. Genet. 2018 December; 19(12):770-788. doi: 10.1038/s41576-018-0059-1, the entire contents of which are hereby incorporated by reference.
For example, the adenine base editor (ABE) as used in the base editing compositions, systems, and methods described herein has the nucleic acid sequence (8877 base pairs), (Addgene, Watertown, Mass.; Gaudelli N.M., et al., Nature 2017 November 23, 551(7681):464-471, doi:10.1038/nature24644, Koblan LW et al., Nat Biotechnol 2018 October, 36(9):843-846, doi:10.1038 /nbt .4172.) as given below. Polynucleotide sequences with at least 95% or more identity to the ABE nucleic acid sequence are also included.
By "base editing activity" is meant that a base within a polynucleotide is chemically altered. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is adenosine or adenine deaminase activity, e.g. B. Conversion of A⋅T to G⋅C. The base editing activity may also involve adenosine or adenine deaminase activity, e.g. B. Conversion of A⋅T to G⋅C, and cytidine deaminase activity, e.g. B. Conversion of target C⋅G to T⋅A. In some embodiments, the base editing activity is evaluated by the efficiency of the editing. Baseline editing efficiency can be measured by any suitable means, such as Sanger sequencing or next-generation sequencing. In some embodiments, the base editing efficiency is measured by the percentage of total sequencing reads with nucleobase conversion effected by the base editor, for example the percentage of total sequencing reads with target AT base pair converted into a G.C. base pair . In some embodiments, base editing efficiency is measured as a percentage of all cells with nucleobase conversion caused by the base editor when base editing is performed on a population of cells.
The term "base editor system" refers to a system for editing a nucleobase of a target nucleotide sequence. In various embodiments, the base editor system comprises (1) a polynucleotide-programmable nucleotide binding domain (e.g., Cas9); (2) a deaminase domain (e.g. an adenosine deaminase) for deamination of the nucleobase; and (3) one or more guide polynucleotides (eg, guide RNA). In some embodiments, the polynucleotide-programmable nucleotide binding domain is a polynucleotide-programmable DNA binding domain. In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor system is ABE8.
In some embodiments, a base editor system may include more than one base editing component. For example, a base editor system may contain more than one deaminase. In some embodiments, a base editor system may contain one or more adenosine deaminases. In some embodiments, a single target polynucleotide can be used to target different deaminases to a target nucleic acid sequence. In some embodiments, a single pair of guide polynucleotides can be used to direct different deaminases to a target nucleic acid sequence.
The deaminase domain and the polynucleotide-programmable nucleotide binding component of a base editor system may be associated with each other covalently or non-covalently, or with any combination of associations and interactions thereof. For example, in some embodiments, a deaminase domain can be targeted to a target nucleotide sequence by a polynucleotide-programmable nucleotide binding domain. In some embodiments, a polynucleotide-programmable nucleotide binding domain can be fused or linked to a deaminase domain. In some embodiments, a polynucleotide-programmable nucleotide binding domain can target a deaminase domain to a target nucleotide sequence through non-covalent interaction with or association with the deaminase domain. For example, in some embodiments, the deaminase domain may comprise an additional heterologous portion or domain that can interact, associate, or form a complex with an additional heterologous portion or domain that is part of a polynucleotide-programmable nucleotide bond domain . In some embodiments, the additional heterologous portion may be capable of binding, interacting, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a leader polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous part can be a protein domain. In some embodiments, the additional heterologous portion may be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu-Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif, and a Ku -protein be B. a telomerase Sm7 binding motif and Sm7 protein or an RNA recognition motif.
A base editor system may further comprise a lead polynucleotide component. It is understood that components of the base editor system may be linked to each other via covalent bonds, non-covalent interactions, or any combination of associations and interactions thereof. In some embodiments, a deaminase domain can be targeted to a target nucleotide sequence by a target polynucleotide. For example, in some embodiments, the deaminase domain may include an additional heterologous portion or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) that is capable of interacting with, associating with, or having a To form a complex therewith, a portion or segment (e.g., a polynucleotide motif) of a lead polynucleotide. In some embodiments, the additional heterologous portion or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or linked to the deaminase domain. In some embodiments, the additional heterologous portion may be capable of binding, interacting, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a leader polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous part can be a protein domain. In some embodiments, the additional heterologous portion may be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu-Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif, and a Ku -protein be B. a telomerase Sm7 binding motif and Sm7 protein or an RNA recognition motif.
In some embodiments, a base editor system may further comprise a base excision repair (BER) component inhibitor. It is understood that components of the base editor system may be linked to each other via covalent bonds, non-covalent interactions, or any combination of associations and interactions thereof. The BER component inhibitor may comprise a BER inhibitor. In some embodiments, the inhibitor of BER can be a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of BER can be an inosine BER inhibitor. In some embodiments, the inhibitor of BER can be targeted to the target nucleotide sequence through the polynucleotide-programmable nucleotide binding domain. In some embodiments, a polynucleotide-programmable nucleotide binding domain can be fused or linked to an inhibitor of BER. In some embodiments, a polynucleotide-programmable nucleotide binding domain can be fused or linked to a deaminase domain and an inhibitor of BER. In some embodiments, a polynucleotide-programmable nucleotide binding domain can target an inhibitor of BER to a target nucleotide sequence by non-covalently interacting with or associating with the inhibitor of BER. For example, in some embodiments, the inhibitor of the BER component may comprise an additional heterologous portion or domain capable of interacting with an additional heterologous portion or portion or domain that is part of a programmable polynucleotide to associate with or form a complex with a nucleotide binding domain.
In some embodiments, the inhibitor of BER can be targeted to the target nucleotide sequence by the leader polynucleotide. For example, in some embodiments, the inhibitor of BER may include an additional heterologous portion or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) capable of interacting with, binding to associate or form a complex with a portion or segment (e.g., a polynucleotide motif) of a leader polynucleotide. In some embodiments, the additional heterologous portion or domain of the leader polynucleotide (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or linked to the inhibitor of BER. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a leader polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous part can be a protein domain. In some embodiments, the additional heterologous portion may be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu-Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif, and a Ku -protein be B. a telomerase Sm7 binding motif and Sm7 protein or an RNA recognition motif.
The term "Cas9" or "Cas9 domain" refers to an RNA-directed nuclease that comprises a Cas9 protein or fragment thereof (e.g., a protein that contains an active, inactive, or partially active DNA cleavage domain of Cas9 and/or the gRNA comprises binding domain of Cas9). A Cas9 nuclease is also sometimes referred to as a Casn1 nuclease or a CRISPR (clustered regular interspaced short palindromic repeat) associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to preceding mobile elements, and targeted invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In Type II CRISPR systems, correct processing of pre-crRNA requires a transcoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-assisted processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand that is not complementary to the crRNA is first cut endonucleolytically and then trimmed exonucleolytically from 3'-5'. In nature, DNA binding and cleavage typically requires protein and both RNAs. However, single-guide RNAs (“sgRNA” or simply “gNRA”) can be engineered to integrate aspects of both crRNA and tracrRNA into a single RNA species. See e.g. B. Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821 (2012), the entire contents of which are hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeats (the PAM or protospacer-adjacent motif) to help distinguish self from non-self. Cas9 nuclease sequences and structures are well known to those skilled in the art (see e.g. "Complete genome sequence of an M1stamm ofStreptococcus pyogenes." Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G, Lyon K, Primeaux C, Sezate S, Suvorov AN, Kenton S, Lai HS, Lin SP, Qian Y, Jia HG., Najar FZ, Ren Q, Zhu H, Song L, White J, Yuan X, Clifton SW, Roe BA, McLaughlin RE, Proc. national Academic Science. DEER. 98:4658-4663 (2001); "CRISPR RNA maturation by transcoded small RNA and host factor RNase III." Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E, Nature 471:602-607 (2011); and "A programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E.Science337:816-821 (2012), the entire contents of which are incorporated herein by reference). Cas9 orthologues have been described in a variety of ways including, but not limited to,S. pyogenesAndS. thermophilus. Other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci described in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immune systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.
An example is Cas9Streptococcus pyogenesCas9 (spCas9) whose amino acid sequence is given below:
A nuclease-inactivated Cas9 protein may be interchangeably referred to as "dCas9" protein (for nuclease-"dead" Cas9) or catalytically inactive Cas9. Methods for generating a Cas9 protein (or a fragment thereof) with an inactive DNA cleavage domain are known (see, e.g., Jinek et al.,Science.337:816–821 (2012); Qi et al., „Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression“ (2013)Cell.28; 152(5):1173-83, the entire contents of which are incorporated herein by reference). For example, the Cas9 DNA cleavage domain is known to comprise two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the gRNA complementary strand, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence Cas9 nuclease activity. For example, mutations D10A and H840A completely inactivate nuclease activityS. pyogenesCas9 (Jinek et al., Science. 337:816-821 (2012); Qi et al., Cell. 28; 152(5):1173-83 (2013)). In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase referred to as an "nCas9" protein (for "nickase" Cas9). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as "Cas9 variants". A Cas9 variant has homology to Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical % identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9.
In some embodiments, the Cas9 variant comprises a fragment of Cas9 (eg, a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical % identical is % identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild-type Cas9.
In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 , 1200, 1250 or at least 1300 amino acids in length.
In some embodiments, wild-type Cas9 corresponds to Cas9 ofStreptococcus pyogenes(NCBI reference sequence: NC 017053.1, nucleotide and amino acid sequences as follows).
In some embodiments, wild-type Cas9 corresponds to or includes the following nucleotide and/or amino acid sequences:
In some embodiments, wild-type Cas9 corresponds to Cas9 ofStreptococcus pyogenes(NCBI reference sequence: NC_002737.2 (nucleotide sequence as follows); and Uniprot reference sequence: Q99ZW2 (amino acid sequence as follows).
In some embodiments, Cas9 refers to Cas9 of:Corynebacterium ulcerans(NCBI references: NC_015683.1, NC_017317.1);Corynebacterium Diphtherie(NCBI references: NC_016782.1, NC_016786.1);Spiroplasma syrphidicola(NCBI reference: NC_021284.1);Prevotella intermedia(NCBI reference: NC_017861.1);Spiroplasma taiwanense(NCBI reference: NC_021846.1);Streptococcus iniae(NCBI reference: NC_021314.1);Baltic Warbler(NCBI reference: NC_018010.1);Psychroflexus twists(NCBI reference: NC_018721.1);Streptococcus thermophilus(NCBI reference: YP 820832.1),harmless listeria(NCBI reference: NP 472073.1),Campylobacter jejuni(NCBI Ref: YP_002344900.1) odermeningococci(NCBI Ref: YP_002342100.1) or to a Cas9 from any other organism.
In some embodiments, Cas9 is ameningococciCas9 (NmeCas9) or a variant thereof. In some embodiments, NmeCas9 has specificity for a NNNNGYTT PAM, where Y is C or T and W is A or T. In some embodiments, NmeCas9 has specificity for a NNNNGYTT PAM, where Y is C or T and W is A or T. The NmeCas9 has specificity for a NNNNGTCT PAM. In some embodiments, the NmeCas9 is a Nme1-Cas9. In some embodiments, NmeCas9 has specificity for a NNNNGATT PAM, a NNNNCCTA PAM, a NNNNCCTC PAM, a NNNNCCTT PAM, a NNNNCCTG PAM, a NNNNCCGT PAM, a NNNNCCGGPAM, a NNNNCCCA PAM, a NNNNCCCT PAM, a NNNNCCCC PAM, a NNNNCCAT on PAM, a NNNNCCAG PAM, a NNNNCCAT PAM, or a NNNGATT PAM. In some embodiments, Nme1Cas9 has specificity for a NNNNGATT PAM, a NNNNCCTA PAM, a NNNNCCTC PAM, a NNNNCCTT PAM, or a NNNNCCTG PAM. In some embodiments, NmeCas9 has specificity for a CAA-PAM, a CAAA-PAM, or a CCA-PAM. In some embodiments, the NmeCas9 is a Nme2-Cas9. In some embodiments, NmeCas9 has specificity for an NNNNCC (N4CC) PAM, where N is one of A, G, C, or T, an NNNNCCCT PAM, an NNNNCCCC PAM, an NNNNCCAT PAM, an NNNNCCAG PAM, an NNNNCCAT PAM , or an NNNGATT PAM. In some embodiments, NmeCas9 is Nme3Cas9. In some embodiments, NmeCas9 has specificity for a NNNNCAAA PAM, a NNNNCC PAM, or a NNNNCNNN PAM. In some embodiments, the PAM-interacting domains for Nme1, Nme2, or Nme3 are N4GAT, N4CC, and N4CAAA, respectively. Additional NmeCas9 features and PAM sequences are described in Edraki et al., A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing, 1999.Mol. Cell.(2019) 73(4):714-726, which is incorporated herein by reference in its entirety.
An exemplarymeningococciCas9 protein, Nme1Cas9, (NCBI reference: WP_002235162.1; Type II CRISPR RNA-driven endonuclease Cas9) has the following amino acid sequence:
Another exemplary onemeningococciCas9 protein, Nme2Cas9, (NCBI reference: WP_002230835; Type II CRISPR RNA guided endonuclease Cas9) has the following amino acid sequence:
In some embodiments, dCas9 corresponds to, includes part or all of a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, a dCas9 domain comprises D10A and an H840A mutation or corresponding mutations in another Cas9. In some embodiments, the dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A):
In some embodiments, the Cas9 domain comprises a D10A mutation while the residue at position 840 remains a histidine in the amino acid sequence provided above or at corresponding positions in any of the amino acid sequences provided herein.
In other embodiments, dCas9 variants are provided with mutations other than D10A and H840A, e.g. B. lead to nuclease-inactivated Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions at D10 and H840 or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain). In some embodiments, variants or homologues of dCas9 are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, about 99 % identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants of dCas9 are provided that have amino acid sequences that are shorter or longer by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.
In some embodiments, the Cas9 fusion proteins provided herein comprise the complete amino acid sequence of a Cas9 protein, e.g. B. one of the Cas9 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas9 sequence, but only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable Cas9 domain and fragment sequences will be apparent to those skilled in the art.
It is understood that additional Cas9 proteins (eg, a nuclease-dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease-active Cas9), including variants and homologues thereof, are within the scope of this disclosure lay. Exemplary Cas9 proteins include, without limitation, those provided below. In some embodiments, the Cas9 protein is a nuclease-dead Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease-active Cas9.
The amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows:
The amino acid sequence of an exemplary Cas9 catalytic nickase (nCas9) is as follows:
The amino acid sequence of an exemplary catalytically active Cas9 is as follows:
In some embodiments, Cas9 refers to a Cas9 of archaea (e.g., nanoarchaea), which is a domain and kingdom of unicellular prokaryotic microbes. In some embodiments, Cas9 refers to CasX or CasY, such as those described in Burstein et al., New CRISPR-Cas systems from uncultivated microbes. cell res. 2017 Feb. 21. doi:10.1038/cr.2017.21, the entire contents of which are hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems have been identified, including the first reported Cas9 in the archaeal domain of life. This aberrant Cas9 protein has been found to be part of an active CRISPR-Cas system in understudied nanoarchaea. Two previously unknown systems have been discovered in bacteria, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems discovered to date. In some embodiments, Cas9 refers to CasX or a variant of CasX. In some embodiments, Cas9 refers to a CasY or a variant of CasY. It is understood that RNA-directed DNA-binding proteins other than nucleic acid-programmable DNA-binding protein (napDNAbp) can be used and are within the scope of this disclosure.
In some embodiments, Cas9 is a Cas9 variant with specificity for an altered PAM sequence. In some embodiments, the additional Cas9 variants and PAM sequences are described in Miller et al., Continuous evolution of SpCas9 variants compatible with non-G PAMs.Nat. Biotechnol(2020). doi.org/10.1038/s41587-020-0412-8, the entirety of which is incorporated herein by reference. In some embodiments, a Cas9 variant has no specific PAM requirements. In some embodiments, a Cas9 variant, e.g. a SpCas9 variant, specificity for an NRNH-PAM where R is A or G and H is A, C or T. In some embodiments, the SpCas9 variant has specificity for a PAM sequence AAA, TAA, CAA, GAA, TAT, GAT, or CAC. In some embodiments, the SpCas9 variant includes an amino acid substitution at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1218, 1219, 1221, 1249, 1256, 1264, 1290, 1318, 13207, 1321, 1323, 1332, 1333, 1335, 1337 or 1339 as numbered relative to the reference sequence below, or a corresponding position thereof.
In some embodiments, the SpCas9 variant includes an amino acid substitution at position 1114, 1135, 1218, 1219, 1221, 1249, 1320, 1321, 1323, 1332, 1333, 1335, or 1337 as numbered relative to the reference sequence above, or a corresponding position of that. In some embodiments, the SpCas9 variant includes an amino acid substitution at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1219, 1221, 1256, 1264, 1290, 1318, 1317, 1320, 1333, as numbered relative to the reference sequence above, or a corresponding position thereof. In some embodiments, the SpCas9 variant includes an amino acid substitution at position 1114, 1131, 1135, 1150, 1156, 1180, 1191, 1218, 1219, 1221, 1227, 1249, 1253, 1286, 1293, 1320, 1321, 13352, 1339 as numbered relative to the reference sequence above, or a corresponding position thereof. In some embodiments, the SpCas9 variant includes an amino acid substitution at position 1114, 1127, 1135, 1180, 1207, 1219, 1234, 1286, 1301, 1332, 1335, 1337, 1338, 1349 as numbered relative to the reference sequence above. Exemplary amino acid substitutions and PAM specificity of SpCas9 variants are shown in Tables AD
In certain embodiments, napDNAbps useful in the methods of the invention include circular permutants that are known in the art and are described, for example, by Oakes et al., Cell 176, 254-267, 2019. The sequence in bold denotes one of Cas9 derived sequence, the italicized sequence denotes a linker sequence and the underlined sequence denotes a bipartite nuclear localization sequence, CP5 (with MSP "NGC=Pam Variant with mutations Regular Cas9 likes NGG" PID=Protein Interacting Domain and "D10A" nickase).
Non-limiting examples of a polynucleotide-programmable nucleotide binding domain that can be incorporated into a base editor include a CRISPR protein-derived domain, a restriction nuclease, a meganuclease, TAL nuclease (TALEN), and a zinc finger nuclease (ZFN).
In some embodiments, the nucleic acid-programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% or at least 99.5% identical to a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp is a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% or at least 99.5% identical to any CasX or CasY protein described herein. It is understood that Cas12b/C2c1, CasX and CasY from other bacterial species can also be used in accordance with the present disclosure.
The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid having a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G.E. and Schirmer, R.H., Principles of Protein Structure, Springer-Verlag, New York (1979) ). According to such analyses, groups of amino acids can be defined in which amino acids within a group are preferentially exchanged with one another and are therefore most similar in their influence on the overall protein structure (Schulz, G.E. and Schirmer, R.H., supra). Non-limiting examples of conservative mutations include amino acid substitutions of amino acids, e.g., lysine for arginine and vice versa, so that a positive charge can be maintained; glutamic acid for aspartic acid and vice versa, so that a negative charge can be maintained; serine for threonine so that a free -OH can be maintained; and glutamine for asparagine, leaving a free -NH2can be maintained.
The term "coding sequence" or "protein coding sequence" as used interchangeably herein refers to a segment of a polynucleotide that encodes a protein. The region or sequence is bounded by a start codon nearer the 5' end and by a stop codon nearer the 3' end. Coding sequences can also be referred to as open reading frames.
The term "deaminase" or "deaminase domain" as used herein refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, adenosine deaminase catalyzes the hydrolytic deamination of adenosine into deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g. engineered adenosine deaminases, evolved adenosine deaminases) may be derived from any organism such as a bacterium. In some embodiments, the adenosine deaminase is from a bacterium, such asEscherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, orCaulobacter crescentus.
In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is a TadA variant. In some embodiments, the TadA variant is a TadA*8. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, bovine, canine, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to a naturally occurring deaminase. For example, deaminase domains are described in PCT International Application Nos. PCT/2017/045381 (WO 2018/027078) and PCT/US2016/058344 (WO 2017/070632), each of which is incorporated herein by reference in its entirety. See also Komor, A.C., et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, N.M., et al., "Programmable base editing of A⋅T to G⋅C in genomic DNA without DNA cleavage" Nature 551, 464-471 (2017); Komor, A.C., et al., "Improved inhibition of base excision repair and bacteriophage Mu-Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity" Science Advances 3:eaao4774 (2017)), and Rees, H.A., et al., "Basic editing: precision chemistry on the genome and transcriptome of living cells." Nat. Rev. Genet. 2018 December; 19(12):770-788. doi: 10.1038/s41576-018-0059-1, the entire contents of which are hereby incorporated by reference.
"Detecting" refers to identifying the presence, absence, or amount of the analyte to be detected. In one embodiment, a sequence change in a polynucleotide or polypeptide is detected. In another embodiment, the presence of indels is detected.
By "detectable label" is meant a composition which, when bound to a molecule of interest, renders the latter detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful labels include, for example, radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron dense reagents, enzymes (such as those commonly used in an ELISA), biotin, digoxigenin, or haptens.
By "disease" is meant any condition or disorder that damages or disrupts the normal functioning of a cell, tissue, or organ. In one embodiment, the disease is A1AD.
The term "effective amount" as used herein refers to an amount of a biologically active agent sufficient to elicit a desired biological response. In certain embodiments, an effective amount is an amount of a base editor system (e.g., a fusion protein comprising a programmable DNA binding protein, a nucleobase editor, and gRNA) sufficient to induce an A1AT mutation in a cell to change in order to achieve a therapeutic effect. Such a therapeutic effect may not be sufficient to alter an A1AD in all cells of a tissue or organ, but only in about 1%, 5%, 10%, 25%, 50%, 75% or more of the cells present in a subject , tissue or organ. In one embodiment, an effective amount is sufficient to ameliorate one or more symptoms of A1AD. The effective amount of one or more active ingredients used to practice the present invention for the therapeutic treatment of a disease will vary depending on the mode of administration, age, body weight, and general health of the subject. Ultimately, the treating physician or veterinarian will decide the appropriate amount and dosing regimen. This amount is referred to as the "effective" amount. In one embodiment, an effective amount is that amount of a base editor of the invention (e.g., a fusion protein comprising a programmable DNA-binding protein, a nucleobase editor, and gRNA) sufficient to introduce an alteration in a gene of interest into a cell to introduce (e.g. a cell in vitro or in vivo). In one embodiment, an effective amount is the amount of a base donor required to produce a therapeutic effect (e.g., to reduce or control a disease or symptom or condition thereof).
By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference nucleic acid molecule or polypeptide. A fragment can contain 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides or amino acids.
By "guide RNA" or "gRNA" is meant a polynucleotide that may be specific for a target sequence and capable of forming a complex with a polynucleotide-programmable nucleotide-binding domain protein (e.g., Cas9 or Cpf1). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs or as a single RNA molecule. gRNAs that exist as a single RNA molecule can be referred to as single guide RNAs (sgRNAs), although "gRNA" is used interchangeably to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules exist. Typically, gRNAs, which exist as single RNA species, comprise two domains: (1) a domain that shares homology with a target nucleic acid (e.g., and directs the binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNAs (e.g., those containing domain 2) can be found in US provisional patent application, US serial no. No. 61/874,682, filed September 6, 2013, entitled "Switchable Cas9 Nucleases and Uses of There," and US Provisional Patent Application, US Ser. Serial No. 61/874,746, filed September 6, 2013, entitled "Delivery System For Functional Nucleases," the entire contents of each are hereby incorporated by reference in their entirety. In some embodiments, a gRNA comprises two or more of domains (1) and (2) and may be referred to as an "extended gRNA". An extended gRNA binds two or more Cas9 proteins and binds a target nucleic acid at two or more different regions, as described herein. The gRNA includes a nucleotide sequence that complements a target site that mediates binding of the nuclease/RNA complex to the target site, thereby providing sequence specificity of the nuclease:RNA complex. As will be appreciated by those skilled in the art, RNA polynucleotide sequences, e.g. B. gRNA sequences, the nucleobase uracil (U), a pyrimidine derivative, rather than the nucleobase thymine (T), which is contained in DNA polynucleotide sequences. In RNA, the uracil base pairs with adenine and replaces thymine during DNA transcription.
"Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
The term "base repair inhibitor" or "IBR" refers to a protein capable of inhibiting the activity of a nucleic acid repair enzyme, such as a base excision repair (BER) enzyme. In some embodiments, the IBR is an inhibitor of inosine base excision repair. Exemplary base repair inhibitors include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGG1, hNEIL1, T7 Endo1, T4PDG, UDG, hSMUG1 and hAAG. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is a catalytically inactive EndoV or a catalytically inactive hAAG. In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, the base repair inhibitor is a catalytically inactive EndoV or a catalytically inactive hAAG.
In some embodiments, the base repair inhibitor is uracil glycosylase inhibitor (UGI). UGI refers to a protein capable of inhibiting a uracil DNA glycosylase base excision repair enzyme. In some embodiments, a UGI domain comprises a wild-type UGI or a fragment of a wild-type UGI. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to a UGI or a UGI fragment. In some embodiments, the base repair inhibitor is an inosine base excision repair inhibitor. In some embodiments, the base repair inhibitor is a "catalytically inactive inosine-specific nuclease" or "dead inosine-specific nuclease". Without wishing to be bound by any particular theory, catalytically inactive inosine glycosylases (e.g., alkyladenine glycosylase (AAG)) can bind inosine but not create an abasic site or remove the inosine, thereby sterically blocking the newly formed inosine unit from DNA damage. repair mechanisms. In some embodiments, the catalytically inactive inosine-specific nuclease may be able to bind an inosine in a nucleic acid but does not cleave the nucleic acid. Non-limiting example catalytically inactive inosine-specific nucleases include catalytically inactive alkyladenosine glycosylase (AAG nuclease), for example from a human, and catalytically inactive endonuclease V (EndoV nuclease), for example fromE coli. In some embodiments, the catalytically inactive AAG nuclease comprises an E125Q mutation or a corresponding mutation in another AAG nuclease.
By "increase" is meant a positive change of at least 10%, 25%, 50%, 75% or 100%.
An "intein" is a fragment of a protein that is capable of excising itself and joining the remaining fragments (the exteins) with a peptide bond in a process known as protein splicing. Inteins are also known as "protein introns". The process by which an intein excises itself and joins the remaining parts of the protein is referred to herein as "protein splicing" or "intein-mediated protein splicing". In some embodiments, a precursor protein intein (an intein-containing protein prior to intein-mediated protein splicing) is derived from two genes. Such an intein is referred to herein as a split intein (eg, split intein-N and split intein-C). For example, in cyanobacteria, DnaE, the catalytic subunit a of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c. The intein encoded by the dnaE-n gene may be referred to herein as "Intein-N". The intein encoded by the dnaE-c gene may be referred to herein as "intein-C".
Other Intel systems can also be used. For example, a synthetic intein based on the dnaE intein, the intein pair Cfa-N (e.g. split intein-N) and Cfa-C (e.g. split intein-C), has been described (e.g. in Stevens et al., J Am Chem Soc 2016 Feb 24, 138(7):2162-5, incorporated herein by reference). Non-limiting examples of pairs of inteins that may be used according to the present disclosure include: Cfa-DnaE intein, Ssp-GyrB intein, Ssp-DnaX intein, Ter-DnaE3 intein, Ter-ThyX intein, Rma -DnaB intein and Cne-Prp8 intein (e.g. as described in US Patent No. 8,394,604, incorporated herein by reference.
Exemplary nucleotide and amino acid sequences of inteins are provided.
Intein-N and intein-C can be fused to the N-terminal part of cleaved Cas9 and the C-terminal part of cleaved Cas9, respectively, to join the N-terminal part of cleaved Cas9 and the C-. End section of split Cas9. For example, in some embodiments an intein-N is fused to the C-terminus of the N-terminal part of split Cas9, i. H. to a structure of the N-[N-terminal part of split Cas9]-[intein-N]-C. In some embodiments, an intein-C is fused to the N-terminus of the C-terminal portion of the cleaved Cas9, i. H. to form a structure of N-[Intein-C]-[C-terminal part of cleaved Cas9]-C. The mechanism of intein-mediated protein splicing to join the proteins to which the inteins are fused (e.g. cleaved Cas9) is known in the art, e.g. B. as in Shah et al., Chem Sci. 2014; 5(1): 446-461, incorporated herein by reference. Methods for designing and using inteins are known in the art and are described, for example, in WO2014004336, WO2017132580, US20150344549 and US20180127780, each of which is incorporated herein by reference in its entirety.
The terms "isolated," "purified," or "biologically pure" refer to material that is, to varying degrees, free of constituents that normally accompany it in its original state. "Isolate" denotes a degree of separation from the original source or environment. "Purification" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free from other materials that any contaminants do not materially affect the protein's biological properties or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified when it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized . Purity and homogeneity are typically determined using analytical chemical techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that a nucleic acid or protein forms a substantial band in an electrophoretic gel. For a protein that can undergo modifications, such as phosphorylation or glycosylation, different modifications can lead to different isolated proteins that can be purified separately.
By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of genes that flank the gene in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. The term thus includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (e.g., a cDNA or genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene that encodes an additional polypeptide sequence.
By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60% by weight free of proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation consists of at least 75%, more preferably at least 90% and most preferably at least 99% by weight of a polypeptide according to the invention. An isolated polypeptide of the invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any suitable method, for example column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
The term "linker" as used herein can refer to a covalent linker (e.g. covalent bond), a non-covalent linker, a chemical group or a molecule connecting two molecules or entities, e.g. B. two components of a protein complex or a ribonucleocomplex, or two domains of a fusion protein, such as a polynucleotide-programmable DNA-binding domain (e.g. dCas9) and a deaminase domain (e.g. an adenosine deaminase or an adenosine -deaminase and a cytidine deaminase, B. as described in PCT/US19/44935). A linker can connect different components or different parts of components of a base editor system. For example, in some embodiments a linker can carry a leader polynucleotide Binding domains of a connect a polynucleotide programmable nucleotide binding domain and a catalytic domain of a deaminase. In some embodiments, a linker can connect a CRISPR polypeptide and a deaminase. In some embodiments, a linker can connect a Cas9 and a deaminase deaminase. In some embodiments, a linker can connect an nCas9 and a deaminase. In some embodiments, a linker can connect a leader polynucleotide and a deaminase. In some embodiments, a linker can connect a deaminating component and a polynucleotide-programmable nucleotide binding component of a base editor system. In some embodiments, a linker can connect an RNA-binding portion of a deaminating component and a polynucleotide-programmable nucleotide-binding component of a base editor system. In some embodiments, a linker can connect an RNA-binding portion of a deaminating component and an RNA-binding portion of a polynucleotide-programmable nucleotide-binding component of a base editor system. A linker can be positioned between or flanked by two groups, molecules or other entities and linked together via a covalent bond or non-covalent interaction, thereby joining the two. In some embodiments, the linker can be an organic molecule, group, polymer, or chemical entity. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, a linker can include an aptamer capable of binding to a ligand. In some embodiments, the ligand can be carbohydrate, a peptide, a protein, or a nucleic acid. In some embodiments, the linker may include an aptamer, which may be derived from a riboswitch. The riboswitch from which the aptamer is derived can be selected from a theophylline riboswitch, a thiamine pyrophosphate (TPP) riboswitch, an adenosine cobalamin (AdoCb1) riboswitch, an S-adenosylmethionine (SAM) riboswitch, an SAH riboswitch, a flavin mononucleotide (FMN) riboswitch, a tetrahydrofolate riboswitch, a lysine riboswitch, a glycine riboswitch, a purine riboswitch, a GlmS riboswitch, or a pre-queosine1 (PreQ1) riboswitch. In some embodiments, a linker can include an aptamer attached to a polypeptide or protein domain, such as a polypeptide ligand. In some embodiments, the polypeptide ligand may be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu-Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif, and a Ku protein be. a telomerase Sm7 binding motif and a Sm7 protein or an RNA recognition motif. In some embodiments, the polypeptide ligand can be part of a base editor system component. For example, a nucleobase-editing component can include a deaminase domain and an RNA recognition motif.
In some embodiments, the linker can be one or more amino acids (e.g., a peptide or protein). In some embodiments, the linker can be about 5-100 amino acids long, for example about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20 -30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or 90-100 amino acids in length. In some embodiments, the linker can be about 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 amino acids in length. Longer or shorter linkers can also be considered.
In some embodiments, a linker connects a gRNA-binding domain of an RNA-programmable nuclease, including a Cas9 nuclease domain, and the catalytic domain of a nucleic acid-editing protein (e.g., adenosine deaminase). In some embodiments, a linker connects a dCas9 and a nucleic acid editing protein. For example, the linker is positioned between or flanked by two groups, molecules or other entities and is connected to each by a covalent bond, thereby joining the two. In some embodiments, the linker is one or more amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical entity. In some embodiments, the linker is 5-200 amino acids long, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90, 95, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 175, 180, 190 or 200 amino acids in length. Longer or shorter linkers are also contemplated.
In some embodiments, the domains of the nucleobase editor are fused via a linker comprising the amino acid sequence of SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 48), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 49), or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGGTPGSEGPATSGGTPGSEGPATSGGTPGSEGPATSGGTGPGSEGATSGG). . In some embodiments, domains of the nucleobase editor are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 51), which may also be referred to as the XTEN linker. In some embodiments, a linker comprises the amino acid sequence SGGS (SEQ ID NO: 52). In some embodiments, a linker (SGGS) comprisesN(SEQ ID NO: 53), (GGGS)N(SEQ ID NO: 54), (GGGGS)N(SEQ ID NO: 55), (G)N(SEQ ID NO: 56), (EAAAK)N(SEQ ID NO: 57), (GGS)N(SEQ ID NO: 58), SGSETPGTSESATPES (SEQ ID NO: 51) oder (XP)NMotif (SEQ ID NO: 59), or a combination of any thereof, where n is independently an integer between 1 and 30 and where X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 60). In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 61). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS (SEQ ID NO: 62). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker includes the amino acid sequence
By "marker" is meant any protein or polynucleotide that exhibits a change in expression level or activity that is associated with a disease or disorder.
The term "mutation" as used herein refers to a substitution of a residue within a sequence, e.g. B. a nucleic acid or amino acid sequence, by another residue or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue, followed by the residue's position within the sequence and the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art and are provided, for example, by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2012)). In some embodiments, the base editors disclosed herein can efficiently create a "intended mutation," such as a point mutation, in a nucleic acid (e.g., a nucleic acid within a subject's genome) without creating a significant number of unintended mutations. like accidental point mutations. In some embodiments, a targeted mutation is a mutation generated by a specific base editor (e.g., adenosine base editor) linked to a leader polynucleotide (e.g., gRNA) specifically designed to generate the targeted mutation .
In general, mutations made or identified in a sequence (eg, an amino acid sequence described herein) are numbered relative to a reference (or wild-type) sequence, i. H. a sequence that does not contain the mutations. One skilled in the art would readily understand how to determine the position of mutations in amino acid and nucleic acid sequences relative to a reference sequence.
The term "non-conservative mutations" includes amino acid substitutions between different groups, for example lysine for tryptophan or phenylalanine for serine etc. In this case it is preferred that the non-conservative amino acid substitution does not interfere with, or the biological activity of, the inhibit functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the wild-type protein.
The term "nuclear localization sequence", "nuclear localization signal" or "NLS" refers to an amino acid sequence that promotes the import of a protein into the cell nucleus. Nuclear localization sequences are known in the art and are described, for example, in Plank et al., PCT International Application, PCT/EP2000/011690, filed November 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein is incorporated by reference for its disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS, described for example by Koblan et al., Nature Biotech. 2018 doi:10.1038/nbt.4172. In some embodiments, an NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO: 64), KRPAATKKAGQAKKKK (SEQ ID NO: 65), KKTELQTTNAENKTKKL (SEQ ID NO: 66), KRGINDRNFWRGENGRKTR (SEQ ID NO: 67), RKSGKIAAIVVKRPRK (SEQ ID NO: 68), PKKKRKV (SEQ ID NO: 69) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 70).
The terms "nucleic acid" and "nucleic acid molecule" as used herein refer to a compound comprising a nucleobase and an acidic moiety, e.g. B. a nucleoside, a nucleotide or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g. B. Nucleic acid molecules comprising three or more nucleotides, linear molecules in which adjacent nucleotides are linked by a phosphodiester bond. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more single nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably to refer to a polymer of nucleotides (e.g., a chain of at least three nucleotides). In some embodiments, "nucleic acid" includes both RNA and single and/or double stranded DNA. Nucleic acids can be naturally occurring, for example, associated with a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule can be a non-naturally occurring molecule, e.g. a recombinant DNA or RNA, an artificial chromosome, an engineered genome or a fragment thereof, or a synthetic DNA, RNA, a DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. In addition, the terms "nucleic acid", "DNA", "RNA" and/or similar terms include nucleic acid analogs, e.g. B. Analogs that have something other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. If necessary, e.g. in the case of chemically synthesized molecules, nucleic acids may include nucleoside analogs, such as analogs with chemically modified bases or sugars and backbone modifications. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise specified. In some embodiments, a nucleic acid is or includes natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); Nucleoside analogues (eg, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromomuridine, C5-fluorouridine, C5-ioduridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g. methylated bases); intercalated bases; modified sugars (2'-e.g. fluororibose, ribose, 2'-deoxyribose, arabinose and hexose); and/or modified phosphate groups (e.g. phosphorothioates and 5'-N-phosphoramidite linkages).
The term "nucleic acid-programmable DNA-binding protein" or "napDNAbp" can be used interchangeably with "polynucleotide-programmable nucleotide-binding domain" to refer to a protein that binds to a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid, acid, or guide polynucleotide (e.g., gRNA) that directs the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide-programmable nucleotide binding domain is a polynucleotide-programmable DNA binding domain. In some embodiments, the polynucleotide-programmable nucleotide binding domain is a polynucleotide-programmable RNA binding domain. In some embodiments, the polynucleotide-programmable nucleotide binding domain is a Cas9 protein. A Cas9 protein can associate with a guide RNA, which directs the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Non-limiting examples of nucleic acid-programmable DNA binding proteins include Cas9 (eg, dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, and Cas12i. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cash, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Type II Cas Effector Proteins, Type V Cas Effector Proteins, Type VI Cas effector proteins, CARF, DinG, homologues thereof, or modified or engineered versions thereof. Other nucleic acid-programmable DNA-binding proteins are also within the scope of this disclosure, although they may not be specifically listed in this disclosure. See e.g. B. Makarova et al. "Classification and nomenclature of CRISPR-Cas systems: Where from?"Crisp J.2018 October; 1:325-336. doi: 10.1089/crispr.2018.0033; Yan et al., "Functionally diverse type V CRISPR-Cas systems" Science. January 4, 2019; 363(6422):88-91. doi: 10.1126/science.aav7271, the entire contents are hereby incorporated by reference.
The term "nucleobase", "nitrogenous base" or "base", used interchangeably herein, refers to a nitrogenous biological compound that forms a nucleoside, which in turn is a component of a nucleotide. The ability of nucleobases to base pair and stack on top of each other leads directly to long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Five nucleobases - adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) - are referred to as primary or canonical. Adenine and guanine are derived from purine, and cytosine, uracil, and thymine are derived from pyrimidine. DNA and RNA can also contain other (non-primary) bases that are modified. Non-limiting exemplary modified nucleobases may include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydromethylcytosine. Hypoxanthine and xanthine can be generated by mutagen, both by deamination (replacing the amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can result from deamination of cytosine. A “nucleoside” consists of a nucleobase and a five-carbon sugar (either ribose or deoxyribose). Examples of a nucleoside include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. Examples of a nucleoside having a modified nucleobase include inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (T). A “nucleotide” consists of a nucleobase, a five-carbon sugar (either ribose or deoxyribose), and at least one phosphate group.
The terms "nucleobase-editing domain" or "nucleobase-editing protein" as used herein refer to a protein or enzyme that can catalyze nucleobase modification in RNA or DNA, such as e.g. B. deamination of adenine (or adenosine) to hypoxanthine (or inosine), as well as non-template nucleotide additions and insertions. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., an adenine deaminase or an adenosine deaminase). In some embodiments, the nucleobase editing domain is more than one deaminase domain (e.g. an adenine deaminase or an adenosine deaminase and a cytidine or a cytosine deaminase, e.g. as described in PCT/US19/44935 ). In some embodiments, the nucleobase editing domain can be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase editing domain may be an engineered or evolved nucleobase editing domain from the naturally occurring nucleobase editing domain. The nucleobase editing domain can be from any organism, such as a bacterium, human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse.
As used herein, as in "obtaining an agent," "obtaining" includes synthesizing, purchasing, creating, preparing, or otherwise acquiring the agent.
A "patient" or "subject" as used herein refers to a mammalian subject or individual who has been diagnosed, is at risk of having or developing, or is suspected of having, a disease or disorder has or is developing a disease or disorder. In some embodiments, the term "patient" refers to a mammalian subject with a higher-than-average likelihood of developing a disease or disorder. Exemplary subjects can be humans, non-human primates, cats, dogs, pigs, cattle, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), and other mammals that can benefit from the therapies disclosed herein. Exemplary human subjects can be male and/or female.
"Patient in need" or "subject in need" is referred to herein as a patient or individual who has been diagnosed with, is at risk of, is susceptible to, is predestined for, or suspected of having a disease or disorder.
The terms pathogenic mutation, pathogenic variant, disease-causing mutation, disease-causing variant, deleterious mutation, or predisposing mutation refer to a genetic alteration or mutation that determined an individual's susceptibility or predisposition to a illness or disorder. In some embodiments, the pathogenic mutation comprises at least one wild-type amino acid replaced by at least one pathogenic amino acid in a gene-encoded protein.
The terms "protein", "peptide", "polypeptide" and their grammatical equivalents are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide or polypeptide is at least three amino acids in length. A protein, peptide or polypeptide can refer to a single protein or a collection of proteins. One or more of the amino acids in a protein, peptide or polypeptide can be modified, for example by adding a chemical moiety such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group such as a fatty acid group, a linker for conjugation, functionalization or other modifications, etc. A protein, peptide or polypeptide can also be a single molecule or a multimolecular complex. A protein, peptide or polypeptide can only be a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide can be naturally occurring, recombinant, or synthetic, or any combination thereof. The term "fusion protein" as used herein refers to a hybrid polypeptide comprising protein domains from at least two different proteins. A protein may be localized to the amino-terminal (N-terminal) portion of the fusion protein or to the carboxy-terminal (C-terminal) protein, thereby forming an amino-terminal fusion protein and a carboxy-terminal fusion protein, respectively. A protein can comprise different domains, for example a nucleic acid binding domain (e.g. the gRNA binding domain of Cas9, which directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a nucleic acid editing protein. In some embodiments, a protein comprises a proteinaceous portion, e.g. B. an amino acid sequence forming a nucleic acid binding domain and an organic compound, e.g. B. a compound that can act as a nucleic acid cleaving agent. In some embodiments, a protein is in a complex with a nucleic acid, e.g. B. RNA or DNA, or is associated with. Any of the proteins provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced via recombinant protein expression and purification, which is particularly appropriate for fusion proteins comprising a peptide linker. Methods for expressing and purifying recombinant proteins are well known and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). , the entire content of which is incorporated herein by reference.
Polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) may comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-Benzyl-N'-methyl-lysine, N',N'-Dibenzyl-lysine, 6-Hydroxylysine, Ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid B. α-aminocycloheptane carboxylic acid, α-(2- Amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine. The polypeptides and proteins may be associated with post-translational modifications of one or more amino acids of the polypeptide constructs. Non-limiting examples of post-translational modifications include phosphorylation, acylation including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation including methylation and ethylation, ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bonds, sulfation, myristoylation , palmitoylation, isoprenylation, farnesylation, geranylation, glyphylation, lipoylation and iodination.
The term "recombinant" as used herein in relation to proteins or nucleic acids refers to proteins or nucleic acids that do not occur in nature but are the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any naturally occurring sequence.
By "reduced" is meant a negative change of at least 10%, 25%, 50%, 75% or 100%.
By "reference" is meant a standard or control condition. In one embodiment, the reference is a wild-type or healthy cell. In other embodiments, and without limitation, a reference is an untreated cell that is not subjected to any test condition or is exposed to placebo or normal saline, medium, buffer, and/or a control vector that does not harbor a polynucleotide of interest.
A "reference sequence" is a defined sequence used as the basis for a sequence comparison. A reference sequence can be a subset or all of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the entire cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence is generally at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence is generally at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides, or any integer thereof or in between. In some embodiments, a reference sequence is a wild-type sequence of a protein of interest. In other embodiments, a reference sequence is a polynucleotide sequence encoding a wild-type protein.
The terms "RNA-programmable nuclease" and "RNA-directed nuclease" are used with (e.g., bind or associate) one or more RNA(s) that is not a target for cleavage. In some embodiments, when an RNA-programmable nuclease is in a complex with an RNA, it may be referred to as a nuclease:RNA complex. Typically, the bound RNA(s) is referred to as guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs or as a single RNA molecule. gRNAs that exist as a single RNA molecule can be referred to as single guide RNAs (sgRNAs), although "gRNA" is used interchangeably to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules exist. Typically, gRNAs, which exist as single RNA species, comprise two domains: (1) a domain that shares homology with a target nucleic acid (e.g., and directs the binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNAs (e.g., those containing domain 2) can be found in US provisional patent application, US serial no. No. 61/874,682, filed September 6, 2013, entitled "Switchable Cas9 Nucleases and Uses of There," and US Provisional Patent Application, US Ser. Serial No. 61/874,746, filed September 6, 2013, entitled "Delivery System For Functional Nucleases," the entire contents of each are hereby incorporated by reference in their entirety. In some embodiments, a gRNA comprises two or more of domains (1) and (2) and may be referred to as an "extended gRNA". For example, an extended gRNA z. B. two or more Cas9 proteins and binds a target nucleic acid at two or more different regions, as described herein. The gRNA includes a nucleotide sequence that complements a target site that mediates binding of the nuclease/RNA complex to the target site, thereby providing sequence specificity of the nuclease:RNA complex.
In some embodiments, the RNA-programmable nuclease is the (CRISPR-associated system) Cas9 endonuclease, for example Cas9 (Casn1).Streptococcus pyogenes(See e.g. “Complete genome sequence of an M1 strain ofStreptococcus pyogenes." Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G, Lyon K, Primeaux C, Sezate S, Suvorov AN, Kenton S, Lai HS, Lin SP, Qian Y, Jia HG, Najar FZ, Ren Q, Zhu H, Song L, White J, Yuan X, Clifton SW, Roe BA, McLaughlin RE, Proc. national Academic Science. DEER. 98:4658-4663 (2001); "CRISPR RNA maturation by transcoded small RNA and host factor RNase III." E. Deltcheva, K. Chylinski, CM. Sharma, K Gonzales, Y Chao, ZA Pirzada, MR. Eckert, J Vogel, E Charpentier, Nature 471:602-607(2011).
Because RNA-programmable nucleases (e.g. Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins can, in principle, be targeted to any sequence specified by the guide RNA. Methods for using RNA-programmable nucleases such as Cas9 for site-specific cleavage (e.g. to modify a genome) are known in the art (see e.g. Cong, L. et al., Multiplex genome engineering using CRISPR/Cas systems Science 339, 819-823 (2013), Mali, P. et al., RNA-guided human genome engineering via Cas9, Science 339, 823-826 (2013), Hwang, W.Y. et al., Efficient genome editing in zebrafish using a CRISPR-Cas system.natural biotechnology31, 227-229 (2013); Jinek, M. et ah, RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, J.E. et al., Genome engineering inSaccharomyces cerevisiaewith CRISPR-Cas systems.Nucleic Acid Research(2013); Jiang, W. et ah RNA-guided editing of bacterial genomes using CRISPR-Cas systems.natural biotechnology31, 233-239 (2013); the entire contents of which are incorporated herein by reference).
The term "single nucleotide polymorphism (SNP)" is a variation in a single nucleotide occurring at a particular position in the genome, each variation being present at an appreciable level within a population (e.g., >1%). For example, at a given base position in the human genome, the nucleotide C may be present in most individuals, but a minority of individuals will have the position occupied by an A. This means that there is an SNP at that specific position, and the two possible nucleotide variations, C or A, are said to be alleles for that position. SNPs are subject to differences in disease susceptibility. The severity of the condition and the way our bodies respond to treatments are also manifestations of genetic variation. SNPs can fall in coding regions of genes, non-coding regions of genes, or in the intergenic regions (regions between genes). In some embodiments, SNPs within a coding sequence do not necessarily alter the amino acid sequence of the protein produced due to the degeneracy of the genetic code. There are two types of SNPs in the coding region: synonymous and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence while non-synonymous SNPs change the amino acid sequence of the protein. There are two types of non-synonymous SNPs: missense and nonsense. SNPs that are not located in protein-coding regions can still affect gene splicing, binding of transcription factors, degradation of messenger RNA, or the sequence of noncoding RNA. The gene expression affected by this type of SNP is called an eSNP (expression SNP) and it can be upstream or downstream of the gene. A single nucleotide variant (SNV) is a variation of a single nucleotide with no frequency restriction and can occur in somatic cells. A somatic single nucleotide variation can also be referred to as a single nucleotide change.
By "specifically bind" is meant a nucleic acid molecule, polypeptide, or complex thereof (e.g., a nucleic acid-programmable DNA binding domain and guide nucleic acid), compound, or molecule that recognizes and binds a polypeptide and/or nucleic acid molecule of the invention, but the other Essentially does not recognize and bind molecules in a sample, such as a biological sample.
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule encoding a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. Polynucleotides with "substantial identity" to an endogenous sequence are typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule encoding a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. Polynucleotides with "substantial identity" to an endogenous sequence are typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant a pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein) or portions thereof under different stringency conditions. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).
For example, the stringent salt concentration is typically less than about 750mM NaCl and 75mM trisodium citrate, preferably less than about 500mM NaCl and 50mM trisodium citrate, and more preferably less than about 250mM NaCl and 25mM trisodium citrate. Low stringency hybridization can be performed in the absence of an organic solvent, e.g. e.g. formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide and more preferably at least about 50% formamide. Stringent temperature conditions typically include temperatures of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. B. sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA are well known to those skilled in the art. By combining these different conditions as needed, different levels of stringency are achieved. In one embodiment, hybridization takes place at 30°C in 750 mM NaCl, 75 mM trisodium citrate and 1% SDS. Alternatively, hybridization occurs at 37°C in 500mM NaCl, 50mM trisodium citrate, 1% SDS, 35% formamide and 100 µg/ml denatured salmon sperm DNA (ssDNA). In another embodiment, hybridization occurs at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide and 200 µg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
In most applications, the washing steps after hybridization also vary in their stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by reducing the salt concentration or by increasing the temperature. For example, the stringent salt concentration for the wash steps is preferably less than about 30mM NaCl and 3mM trisodium citrate, and most preferably less than about 15mM NaCl and 1.5mM trisodium citrate. Stringent temperature conditions for the washing steps typically include a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C occurs at 25°C in 30mM NaCl, 3mM trisodium citrate and 0. 1% SDS on. In a more preferred embodiment, washing steps take place at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, washing steps take place at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS. Additional variations on these terms will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
By "split" is meant splitting into two or more fragments.
A "split Cas9 protein" or "split Cas9" refers to a Cas9 protein provided as an N-terminal fragment and a C-terminal fragment encoded by two separate nucleotide sequences. The polypeptides corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein can be spliced to form a "reconstituted" Cas9 protein. In certain embodiments, the Cas9 protein is split into two fragments within a disordered region of the protein, e.g. as described in Nishimasu et al., Cell, Vol. 156, Issue 5, pp. 935-949, 2014, or as described in Jiang et al. (2016) Science 351: 867-871. PDB File: 5F9R, each of which is incorporated herein by reference. In some embodiments, the protein is split into two fragments at any C, T, A, or S within a region of SpCas9 between about amino acids A292-G364, F445-K483, or E565-T637, or at corresponding positions in any other Cas9, Cas9 variant (e.g. nCas9, dCas9) or other napDNAbp. In some embodiments, the protein is split into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of splitting the protein into two fragments is referred to as "cleaving" the protein.
In other embodiments, the N-terminal portion of the Cas9 protein comprises amino acids 1-573 or 1-637S. pyogenesCas9 wild-type (SpCas9) (NCBI reference sequence: NC_002737.2, Uniprot reference sequence: Q99ZW2) or a corresponding position/mutation thereof, and the C-terminal part of the Cas9 protein comprises part of amino acids 574-1368 or 638- 1368 from SpCas9 wild type.
The C-terminal portion of cleaved Cas9 can be joined to the N-terminal portion of cleaved Cas9 to form a complete Cas9 protein. In some embodiments, the C-terminal portion of Cas9 protein begins where the N-terminal portion of Cas9 protein ends. As such, in some embodiments, the C-terminal portion of split Cas9 comprises a portion of amino acids (551-651)-1368 of spCas9. "(551-651)-1368" means starting at an amino acid between amino acids 551-651 (inclusive) and ending at amino acid 1368. For example, the C-terminal portion of cleaved Cas9 may include any portion of amino acids 551-1368, 552-1368, 553-1368, 554-1368, 555-1368, 556-1368, 557-1368, 558-1368, 559 -1368, 560-1368, 561-1368, 562-1368, 563-1368, 564-1368, 565-1368, 566-1368, 567-1368, 568-1368, 569-1368, 570-1368, 571-1368 , 572-1368, 573-1368, 574-1368, 576-1368, 577-1368, 578-1368, 580-1368, 581-1368, 583-1368, 584 -1368, 585-1368, 586-1368, 587-1368, 588-1368, 589-1368, 590-1368, 591-1368, 592-1368, 593-1368, 594-1368, 595-1368, 596-1368 , 597-1368, 598-1368, 599-1368, 600 -1368, 601-1368, 602-1368, 603-1368, 604-1368, 606-1368, 608-1368, 609 -1368, 610-1368, 611-1368, 612-1368, 613-1368, 614-1368, 615-1368, 616-1368, 617-1368, 618-1368, 619-1368, 620-1368, 621-1368 , 622-1368, 623-1368, 624-1368, 626-1368, 627-1368, 628-1368, 629-1368, 631-1368, 633-1368, 634 -1368, 635-1368, 636-1368, 637-1368, 638-1368, 639-1368, 640-1368, 641-1368, 642-1368, 643-1368, 644-1368, 645-1368, 646-1368 , 647-1368, 648-1368, 649-1368, 650-1368 or 651-1368 from spCas9. In some embodiments, the C-terminal portion of the split Cas9 protein comprises a portion of amino acids 574-1368 or 638-1368 of SpCas9.
By "Serpin1A polynucleotide" is meant a nucleic acid molecule encoding an A1AT protein or fragment thereof. The sequence of an exemplary Serpin1A polynucleotide disclosed under NCBI Accession NO. NM_000295, is given below:
The PAM sequence is highlighted and the correct sequence after adenine base editing is shown.
By "subject" is meant a mammal, including but not limited to a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline. Subjects include livestock, domesticated animals raised to produce labor and provide commodities such as food, including but not limited to cattle, goats, chickens, horses, pigs, rabbits, and sheep.
By "substantially identical" is meant a polypeptide or nucleic acid molecule that has at least 50% identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or a nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). In one embodiment, such a sequence at the amino acid level or nucleic acid is at least 60%, 80% or 85%, 90%, 95% or even 99% identical to the sequence used for comparison.
Sequence identity is typically determined using sequence analysis software (e.g., Sequence Analysis Software Package from Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs) measured ). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program can be used, with a probability value between e.g−3and e−100indicating a closely related sequence. For example, COBALT is used with the following parameters:
-
- a) Alignment Parameters: Gap Penalties-11, -1 and End Gap Penalties-5, -1,
- b) CDD parameters: Use RPS BLAST on; Explosive E-Value 0.003; Find Conserved columns and recalculate on and
- c) Query clustering parameters: Use query clusters; word size 4; Max cluster distance 0.8; Alphabet regular.
For example, the EMBOSS needle is used with the following parameters: - a) Matrix: BLOSUM62;
- b) GAP OFFEN: 10;
- c) GAP EXTENSION: 0.5;
- d) OUTPUT FORMAT: pair;
- e) END GAP PENALTY: false;
- f) OPEN FINISH: 10; And
- g) END GAP EXTENSION: 0.5.
The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a nucleobase editor. In one embodiment, the target site is deaminated by a deaminase or a fusion protein comprising a deaminase (e.g., adenine deaminase).
As used herein, the terms "treat," "treat," "treatment," and the like refer to reducing or alleviating a disorder and/or symptoms associated therewith, or obtaining a desired pharmacological and/or physiological effect. It is understood that although not excluded, treatment of a disorder or condition does not require that the disorder, condition or associated symptoms be completely eliminated. In some embodiments, the effect is therapeutic, i. i.e., without limitation, the effect reduces, lessens, reverses, alleviates, alleviates, reduces in intensity, or cures, in part or in full, a disease and/or an undesirable symptom attributable to the disease. In some embodiments, the effect is preventive, i. H. the effect protects or prevents the occurrence or recurrence of a disease or condition. To this end, the methods disclosed herein comprise administering a therapeutically effective amount of a composition as described herein. In one embodiment, the disease is alpha-1 antitrypsin deficiency (A1AD).
By "uracil glycosylase inhibitor" or "UGI" is meant an agent that inhibits the uracil excision repair system. In one embodiment, the agent is a protein or fragment thereof that binds a host uracil DNA glycosylase and prevents the removal of uracil residues from the DNA. In one embodiment, a UGI is a protein, fragment thereof, or domain capable of inhibiting a uracil-DNA glycosylase base excision repair enzyme. In some embodiments, a UGI domain comprises a wild-type UGI or a modified version thereof. In some embodiments, a UGI domain comprises a fragment of the exemplary amino acid sequence given below. In some embodiments, a UGI fragment comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% %, at least 98%, at least 99%, or 100% of the example UGI sequence provided below. In some embodiments, a UGI comprises an amino acid sequence homologous to the exemplary UGI amino acid sequence or fragment thereof, as set forth below. In some embodiments, the UGI or a portion thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% 97%, at least 98%, at least 99%, at least 99% .5%, at least 99.9% or 100% identical to a wild-type UGI or a UGI sequence or part thereof as specified below. An exemplary UGI includes an amino acid sequence as follows:
The term "vector" refers to a means of introducing a nucleic acid sequence into a cell resulting in a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes and episomes. "Expression vectors" are nucleic acid sequences comprising the nucleotide sequence to be expressed in the recipient cell. Expression vectors may include additional nucleic acid sequences to enhance and/or facilitate expression of the introduced sequence, such as start, stop, enhancer, promoter, and secretion sequences.
Any composition or method provided herein may be combined with any one or more of the other compositions and methods provided herein.
Reference in the specification to "some embodiments," "an embodiment," "an embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is present in at least some Embodiments are included, but not necessarily all, embodiments of the present disclosures.
Recitation of a chemical group listing in any definition of a variable herein includes definitions of those variables as any single group or combination of listed groups. Citation of an embodiment for a variable or aspect herein encompasses that embodiment as each individual embodiment or in combination with any other embodiment or portion thereof.
DNA editing has emerged as a viable means to modify disease states by correcting pathogenic mutations at the genetic level. Until recently, all DNA editing platforms worked by inducing a DNA double-strand break (DSB) at a specific genomic site and relied on endogenous DNA repair pathways to determine the product outcome in a semi-stochastic manner, resulting in complex populations of genetic products. Although precise, custom repair outcomes can be achieved through the homology-directed repair (HDR) pathway, a number of challenges have prevented highly efficient repair using HDR in therapeutically relevant cell types. In practice, this route is inefficient compared to the competing, error-prone, non-homologous terminating route. In addition, HDR is tightly restricted to the G1 and S phases of the cell cycle, preventing precise repair of DSBs in post-mitotic cells. As a result, in these populations, it has proven difficult or impossible to alter genomic sequences in a user-defined, programmable manner with high efficiency.
DETAILED DESCRIPTION OF THE DISCLOSURE
As described below, the present invention provides compositions and methods for altering mutations associated with alpha-1-antitrypsin deficiency (A1AD). In some embodiments, the editing corrects a deleterious mutation such that the edited polynucleotide is indistinguishable from a wild-type reference polynucleotide sequence. In another embodiment, the editing alters the deleterious mutation such that the edited polynucleotide comprises a benign mutation.
The invention is based, at least in part, on the discovery that a base editor comprising an adenosine deaminase variant can efficiently and precisely edit a deleterious mutation associated with A1AD.
Alpha-1-Antitrypsin-Mangel (A1AD)
Alpha-1-antitrypsin (A1A) is a protease inhibitor encoded by the SERPINA1 gene on chromosome 14. This glycoprotein is primarily synthesized in the liver and with serum concentrations of 1.5–3.0 g/L (20–52 μmon) in healthy adults. It diffuses into the lung interstitium and alveolar lining fluid, where it inactivates neutrophil elastase, thereby protecting lung tissue from protease-mediated damage. Alpha-1 antitrypsin deficiency (A1AD) is inherited in an autosomal codominant manner. Over 100 genetic variants of the SERPINA1 gene have been described, but not all are associated with disease. The alphabetic designation of these variants is based on their migration speed on gel electrophoresis. The most common variant is the M (medium mobility) allele (PiM), and the two most common deficiency alleles are PiS and PiZ (the latter has the slowest migration rate). Several mutations that do not produce a measurable serum protein have been described; these are referred to as "null" alleles. The most common genotype is MM, which produces normal serum levels of alpha-1 antitrypsin. Most people with severe deficiency are homozygous for the Z allele (ZZ). More than 60,000 patients with A1AD in the United States have the severe ZZ phenotype. Z protein is misfolded and polymerized during its production in the endoplasmic reticulum of hepatocytes; These abnormal polymers become trapped in the liver, greatly reducing serum levels of alpha-1-antitrypsin. Deficient or unstable A1AT production causes liver and/or lung pathologies in patients affected by A1AD. The liver disease observed in patients with alpha-1-antitrypsin deficiency is caused by the accumulation of abnormal alpha-1-antitrypsin protein in hepatocytes and the resulting cellular responses, including autophagy, endoplasmic reticulum stress response and apoptosis. Reduced circulating levels of alpha-1-antitrypsin lead to increased neutrophil elastase activity in the lungs; This imbalance of protease and antiprotease leads to the lung disease associated with this condition.
Alpha-1 antitrypsin deficiency ("A1AD") is most common in Caucasians and most commonly affects the lungs and liver. In the lungs, the most common manifestation is an early-onset panacinic emphysema (in patients between the ages of 30 and 40) that is most pronounced in the bases of the lungs. However, diffuse emphysema or upper lobe emphysema can occur, as can bronchiectasis. The most frequently reported symptoms are dyspnoea, wheezing and cough. Lung function tests of affected individuals show findings consistent with COPD; However, a response to bronchodilators can be observed, which can be misdiagnosed as asthma. Liver diseases caused by the ZZ genotype manifest themselves in different ways. Affected infants may present in the neonatal period with cholestatic jaundice, sometimes with acholic stools (pale or clay-colored), and hepatomegaly. Blood levels of conjugated bilirubin, transaminases and gamma-glutamyltransferase are increased. Liver disease in older children and adults may present with an incidental finding of elevated transaminases or with evidence of established cirrhosis, including variceal bleeding or ascites. Alpha-1 antitrypsin deficiency also predisposes patients to hepatocellular carcinoma. Although the homozygous ZZ genotype is required for the development of liver disease, a heterozygous Z mutation can act as a genetic modifier for other diseases by conferring a greater risk of more severe liver diseases, such as liver disease.
The two most common clinical variants of A1AD are the alleles E264V (PiS) and E342K (PiZ). The clinical single nucleotide variant E342K (PiZ) results in an unstable and/or inactive A1AT protein and subsequently causes liver and lung toxicity. Inheritance is autosomal codominant. More than half of A1AD patients carry at least one copy of the E342K mutation.
In some embodiments, the disease or disorder is alpha-1 antitrypsin deficiency (A1AD). In some embodiments, the pathogenic mutation is in the SERPINA1 gene. In some embodiments, the mutation of SERPINA1 is E342K (PiZ allele). In some embodiments, A at position 7 is edited to G to convert the PiZ allele back to a wild-type allele.
Nucleobase-Editor
Disclosed herein is a base editor or a nucleobase editor for editing, modifying, or altering a target nucleotide sequence of a polynucleotide. Described herein is a nucleobase editor or a base editor comprising a polynucleotide-programmable nucleotide binding domain and a nucleobase editing domain (e.g., adenosine deaminase). A polynucleotide-programmable nucleotide binding domain, in conjunction with a bound guide polynucleotide (e.g., gRNA), can specifically bind to a target polynucleotide sequence (i.e., via complementary base pairing between bases of the bound guide nucleic acid and bases of the target polynucleotide). sequence) and thereby to localize the base editor to the target nucleic acid sequence to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid.
Polynucleotide-programmable nucleotide binding domain
It is understood that polynucleotide-programmable nucleotide binding domains can also include nucleic acid-programmable proteins that bind RNA. For example, the polynucleotide-programmable nucleotide-binding domain can be associated with a nucleic acid that directs the polynucleotide-programmable nucleotide-binding domain to an RNA. Other nucleic acid-programmable DNA binding proteins are also within the scope of this disclosure, although not specifically listed in this disclosure.
A polynucleotide-programmable nucleotide binding domain of a base editor may itself comprise one or more domains. For example, a polynucleotide-programmable nucleotide binding domain may include one or more nuclease domains. In some embodiments, the nuclease domain of a polynucleotide-programmable nucleotide binding domain may comprise an endonuclease or an exonuclease. As used herein, the term "exonuclease" refers to a protein or polypeptide capable of digesting a nucleic acid (e.g., RNA or DNA) from free ends, and the term "endonuclease" refers to a protein or polypeptide , which is able to catalyze (e.g. cleave) internally. Regions in a nucleic acid (e.g. DNA or RNA). In some embodiments, an endonuclease can cleave a single strand of a double-stranded nucleic acid. In some embodiments, an endonuclease can cleave both strands of a double-stranded nucleic acid molecule. In some embodiments, a polynucleotide-programmable nucleotide binding domain can be a deoxyribonuclease. In some embodiments, a polynucleotide-programmable nucleotide binding domain can be a ribonuclease.
In some embodiments, a nuclease domain of a polynucleotide-programmable nucleotide binding domain can cleave zero, one, or two strands of a target polynucleotide. In some embodiments, the polynucleotide-programmable nucleotide binding domain may include a nickase domain. As used herein, the term "nickase" refers to a polynucleotide-programmable nucleotide binding domain comprising a nuclease domain capable of cleaving only one strand of the two strands in a double-stranded nucleic acid molecule (e.g., DNA). In some embodiments, a nickase can be derived from a fully catalytically active (e.g., natural) form of a polynucleotide-programmable nucleotide binding domain by introducing one or more mutations into the active polynucleotide-programmable nucleotide binding domain. For example, if a polynucleotide-programmable nucleotide binding domain comprises a Cas9-derived nickase domain, the Cas9-derived nickase domain may contain a D10A mutation and a histidine at position 840. In such embodiments, residue H840 retains catalytic activity and can be cleaved by a single strand of the nucleic acid duplex. In another example, a Cas9-derived nickase domain may comprise an H840A mutation while the amino acid residue at position 10 remains a D. In some embodiments, a nickase may be derived from a fully catalytically active (e.g., natural) form of a polynucleotide-programmable nucleotide binding domain by removing all or part of a nuclease domain that is not required for nickase activity. For example, when a polynucleotide-programmable nucleotide binding domain comprises a Cas9-derived nickase domain, the Cas9-derived nickase domain may comprise a deletion of all or part of the RuvC domain or the HNH domain.
The amino acid sequence of an exemplary catalytically active Cas9 is as follows:
A base editor that includes a polynucleotide-programmable nucleotide binding domain that includes a nickase domain is thus able to create a single-stranded DNA break (nick) at a specific polynucleotide target sequence (e.g., determined by the complementary sequence of a bound leader nucleic acid ) to create. In some embodiments, the strand of a nucleic acid duplex target polynucleotide sequence that is cleaved by a base editor that includes a nickase domain (e.g., a Cas9-derived nickase domain) is the strand that is not cleaved by the base editor is edited (i.e., the strand that is split by the base editor is opposite a strand that includes a base to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain) can cleave the strand of a DNA molecule targeted for editing. In such embodiments, the non-target strand is not cleaved.
Also provided herein are base editors comprising a polynucleotide-programmable nucleotide binding domain that is catalytically dead (i.e., unable to cleave a target polynucleotide sequence). The terms "catalytically dead" and "nuclease dead" are used interchangeably herein to refer to a polynucleotide-programmable nucleotide binding domain that has one or more mutations and/or deletions that result in its inability to cleave a strand of a nucleic acid. In some embodiments, a base editor of a catalytically dead polynucleotide-programmable nucleotide binding domain may lack nuclease activity as a result of specific point mutations in one or more nuclease domains. For example, in the case of a base editor comprising a Cas9 domain, Cas9 may comprise both a D10A mutation and an H840A mutation. Such mutations inactivate both nuclease domains, resulting in loss of nuclease activity. In other embodiments, a catalytically dead polynucleotide-programmable nucleotide binding domain may comprise one or more deletions of all or part of a catalytic domain (e.g., RuvC1 and/or HNH domains). In other embodiments, a catalytically dead polynucleotide-programmable nucleotide binding domain comprises a point mutation (e.g., D10A or H840A) and a deletion of all or part of a nuclease domain.
Also contemplated herein are mutations capable of generating a catalytically dead polynucleotide-programmable nucleotide binding domain from a previously functional version of the polynucleotide-programmable nucleotide binding domain. For example, in the case of catalytically dead Cas9 ("dCas9"), variants are provided with mutations other than D10A and H840A that result in nuclease-inactivated Cas9. Such mutations include, for example, other amino acid substitutions at D10 and H840 or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain). Other suitable nuclease-inactive dCas9 domains will be apparent to those skilled in the art based on this disclosure and knowledge in the art and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A/H840A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains (see, e.g., Prashant et al., CAS9 transcription activators for target specificity screening and paired nickases for cooperative genome engineering.natural biotechnology.2013; 31(9):833-838, the entire contents of which are incorporated herein by reference).
Non-limiting examples of a polynucleotide-programmable nucleotide binding domain that can be incorporated into a base editor include a CRISPR protein-derived domain, a restriction nuclease, a meganuclease, TAL nuclease (TALEN), and a zinc finger nuclease (ZFN). In some embodiments, a base editor comprises a polynucleotide-programmable nucleotide binding domain comprising a natural or modified protein or portion thereof capable of binding to a nucleic acid sequence during CRISPR (i.e., clustered regularly interspaced short palindromic repeats) via a bound guide nucleic acid. . )-mediated modification of a nucleic acid. Such a protein is referred to herein as "CRISPR protein". Accordingly, there is disclosed herein a base editor comprising a polynucleotide-programmable nucleotide binding domain comprising all or part of a CRISPR protein (i.e., a base editor comprising as a domain all or part of a CRISPR protein, also referred to as " CRISPR protein" -derived domain" of the base editor). A CRISPR protein-derived domain that incorporates a base editor can be modified relative to a wild-type or natural version of the CRISPR protein. For example, as described below, a CRISPR protein-derived domain may comprise one or more mutations, insertions, deletions, rearrangements, and/or recombination relative to a wild-type or natural version of the CRISPR protein.
CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to preceding mobile elements, and targeted invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In Type II CRISPR systems, correct processing of pre-crRNA requires a transcoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-assisted processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand that is not complementary to crRNA is first cut endonucleolytically and then trimmed exonucleolytically from 3'-5'. In nature, DNA binding and cleavage typically requires protein and both RNAs. However, single-guide RNAs (“sgRNA” or simply “gNRA”) can be engineered to integrate aspects of both crRNA and tracrRNA into a single RNA species. See e.g. B. Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821 (2012), the entire contents of which are hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeats (the PAM or protospacer-adjacent motif) to help distinguish self from non-self.
In some embodiments, the methods described herein can use an engineered Cas protein. A guide RNA (gRNA) is a short synthetic RNA consisting of a framework sequence required for Cas binding and a custom spacer of approximately 20 nucleotides that defines the genomic target to be modified. Thus, one skilled in the art can change the genomic target of the Cas protein. Specificity is determined in part by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome.
In some embodiments, the gRNA framework sequence is as follows: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAGU GGCACCGAGU CGGUGCUUUU (SEQ ID NO: 11).
In some embodiments, a CRISPR protein-derived domain incorporated into a base editor is an endonuclease (e.g., deoxyribonuclease or ribonuclease) capable of binding a target polynucleotide when in conjunction with a bound guide nucleic acid is. In some embodiments, a CRISPR protein-derived domain incorporated into a base editor is a nickase capable of binding a target polynucleotide when in association with a bound guide nucleic acid. In some embodiments, a CRISPR protein-derived domain incorporated into a base editor is a catalytically dead domain capable of binding a target polynucleotide when in association with a bound guide nucleic acid. In some embodiments, a target polynucleotide bound by a CRISPR protein-derived domain of a base editor is DNA. In some embodiments, a target polynucleotide bound by a CRISPR protein-derived domain of a base editor is RNA.
Cas proteins that can be used herein include class 1 and class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cash, Cas7, Cas8 , Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5 , Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2 , Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h and Cas12i, CARF, DinG, homologues thereof or modified versions of that . An unmodified CRISPR enzyme can exhibit DNA cleavage activity, such as Cas9, which has two functional endonuclease domains: RuvC and HNH. A CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and/or within a complement of a target sequence. For example, a CRISPR enzyme can cause cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
A vector encoding a CRISPR enzyme mutated relative to a corresponding wild-type enzyme such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence can be used . Cas9 can refer to a polypeptide at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% sequence identity and/or sequence homology to an exemplary wild-type Cas9 polypeptide (eg, Cas9 fromS. pyogenes). Cas9 may refer to a polypeptide at no more than or no more than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% sequence identity and/or sequence homology to an exemplary wild-type Cas9 polypeptide (e.gS. pyogenes). Cas9 can refer to the wild-type or a modified form of the Cas9 protein, which can include an amino acid change, such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof
In some embodiments, a CRISPR protein-derived domain of a basic editor may comprise all or part of Cas9Corynebacterium ulcerans(NCBI references: NC_015683.1, NC_017317.1);Corynebacterium Diphtherie(NCBI references: NC_016782.1, NC_016786.1);Spiroplasma syrphidicola(NCBI reference: NC_021284.1);Prevotella intermedia(NCBI reference: NC_017861.1);Spiroplasma taiwanense(NCBI reference: NC_021846.1);Streptococcus iniae(NCBI reference: NC_021314.1);Baltic Warbler(NCBI reference: NC_018010.1);Psychroflexus Twists(NCBI reference: NC_018721.1);Streptococcus thermophilus(NCBI reference: YP_820832.1);harmless listeria(NCBI reference: NP 472073.1);Campylobacter jejuni(NCBI reference: YP_002344900.1);meningococci(NCBI reference: YP_002342100.1),Streptococcus pyogenes, orStaphylococcus aureus.
Cas9 domains of nucleobase editors
Cas9 nuclease sequences and structures are well known to those skilled in the art (see e.g. "Complete genome sequence of an M1stamm ofStreptococcus pyogenes." Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN., Kenton S., Lai H.S., Lin SP, Qian Y. , Jia HG, Najar FZ, Ren Q, Zhu H, Song L, White J, Yuan X, Clifton SW, Roe BA, McLaughlin RE, Proc. national Academic Science. USA 98:4658-4663 (2001); "CRISPR RNA maturation by transcoded small RNA and host factor RNase III." Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E, Nature 471:602-607 (2011); and "A programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E Science 337:816–821 (2012) , the entire contents of which are incorporated herein by reference). Cas9 orthologues have been described in a variety of ways including, but not limited to,S. pyogenesAndS. thermophilus. Other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci described in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immune systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.
In some embodiments, a nucleic acid-programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting example Cas9 domains are provided here. The Cas9 domain can be a nuclease-active Cas9 domain, a nuclease-inactive Cas9 domain (dCas9), or a Cas9 nickase (nCas9). In some embodiments, the Cas9 domain is a nuclease active domain. For example, the Cas9 domain can be a Cas9 domain that cleaves both strands of a double-stranded nucleic acid (eg, both strands of a double-stranded DNA molecule). In some embodiments, the Cas9 domain comprises any of the amino acid sequences set forth herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95%, at least 96%, at least 97% , at least 98%, at least 99% or at least 99.5% identical to any of the amino acid sequences set forth herein. In some embodiments, the Cas9 domain comprises an amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , 46, 47, 48, 49, 50 or more or more mutations compared to any of the amino acid sequences set forth herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that is at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200 , at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100 or at least 1200 identical contiguous amino acid residues compared to any of the amino acid sequences set forth herein.
In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as "Cas9 variants". A Cas9 variant has homology to Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical % identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (eg, a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical % identical is % identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild-type Cas9. In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 , 1200, 1250 or at least 1300 amino acids in length.
In some embodiments, the Cas9 fusion proteins provided herein comprise the complete amino acid sequence of a Cas9 protein, e.g. B. one of the Cas9 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas9 sequence, but only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable Cas9 domain and fragment sequences will be apparent to those skilled in the art.
A Cas9 protein can associate with a guide RNA, which directs the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the polynucleotide-programmable nucleotide binding domain is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Examples of nucleic acid-programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b/C2C1, and Cas12c/C2C3. In some embodiments, wild-type Cas9 corresponds to Cas9 ofStreptococcus pyogenes(NCBI reference sequence: NC_017053.1, nucleotide and amino acid sequences as follows).
In some embodiments, wild-type Cas9 corresponds to or includes the following nucleotide and/or amino acid sequences:
In some embodiments, wild-type Cas9 corresponds to Cas9 ofStreptococcus pyogenes(NCBI reference sequence: NC_002737.2 (nucleotide sequence as follows); and Uniprot reference sequence: Q99ZW2 (amino acid sequence as follows):
In some embodiments, Cas9 refers to Cas9 of:Corynebacterium ulcerans(NCBI references: NC_015683.1, NC_017317.1);Corynebacterium Diphtherie(NCBI references: NC_016782.1, NC_016786.1);Spiroplasma syrphidicola(NCBI reference: NC_021284.1);Prevotella intermedia(NCBI reference: NC_017861.1);Spiroplasma taiwanense(NCBI reference: NC_021846.1);Streptococcus iniae(NCBI reference: NC_021314.1);Baltic Warbler(NCBI reference: NC_018010.1);Psychroflexus torquisl(NCBI reference: NC_018721.1);Streptococcus thermophilus(NCBI reference: YP_820832.1),harmless listeria(NCBI reference: NP 472073.1),Campylobacter jejuni(NCBI Ref: YP_002344900.1) odermeningococci(NCBI Ref: YP_002342100.1) or to a Cas9 from any other organism.
It is understood that additional Cas9 proteins (eg, a nuclease-dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease-active Cas9), including variants and homologues thereof, are within the scope of this disclosure lay. Exemplary Cas9 proteins include, without limitation, those provided below. In some embodiments, the Cas9 protein is a nuclease-dead Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease-active Cas9.
In some embodiments, the Cas9 domain is a nuclease-inactive Cas9 domain (dCas9). For example, the dCas9 domain can bind to a double-stranded nucleic acid molecule (eg, via a gRNA molecule) without cleaving either strand of the double-stranded nucleic acid molecule. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10X mutation and an H840X mutation of the amino acid sequence set forth herein or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid change. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10A mutation and an H840A mutation of the amino acid sequence set forth herein or a corresponding mutation in any of the amino acid sequences provided herein. As an example, a nuclease-inactive Cas9 domain comprises the amino acid sequence presented in the cloning vector pPlatTET-gRNA2 (accession number BAV54124).
The amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows:
Other suitable nuclease-inactive dCas9 domains will be apparent to those skilled in the art based on this disclosure and knowledge in the art and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A/H840A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains (see, e.g., Prashant et al., CAS9 transcription activators for target specificity screening and paired nickases for cooperative genome engineering.natural biotechnology.2013; 31(9):833-838, the entire contents of which are incorporated herein by reference).
In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase referred to as an "nCas9" protein (for "nickase" Cas9). A nuclease-inactivated Cas9 protein may be interchangeably referred to as "dCas9" protein (for nuclease-"dead" Cas9) or catalytically inactive Cas9. Methods for generating a Cas9 protein (or a fragment thereof) with an inactive DNA cleavage domain are known (see, e.g., Jinek et al.,Science.337:816–821 (2012); Qi et al., „Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression“ (2013)Cell.28; 152(5):1173-83, the entire contents of which are incorporated herein by reference). For example, the Cas9 DNA cleavage domain is known to comprise two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the gRNA complementary strand, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence Cas9 nuclease activity. For example, mutations D10A and H840A completely inactivate nuclease activityS. pyogenesCas9 (Jinek et al.,Science.337:816-821 (2012); Qi et al.,Cell.28; 152(5): 1173-83 (2013)).
In some embodiments, the dCas9 domain comprises an amino acid sequence that makes up at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to any of the dCas9 domains provided here. In some embodiments, the Cas9 domain comprises an amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , 46, 47, 48, 49, 50 or more or more mutations compared to any of the amino acid sequences set forth herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that is at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200 , at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100 or at least 1200 identical contiguous amino acid residues compared to any of the amino acid sequences set forth herein.
In some embodiments, dCas9 corresponds to, includes part or all of a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, a dCas9 domain comprises D10A and an H840A mutation or corresponding mutations in another Cas9.
In some embodiments, the dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A):
In some embodiments, the Cas9 domain comprises a D10A mutation while the residue at position 840 remains a histidine in the amino acid sequence provided above or at corresponding positions in any of the amino acid sequences provided herein.
In other embodiments, dCas9 variants are provided with mutations other than D10A and H840A, e.g. B. lead to nuclease-inactivated Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions at D10 and H840 or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain). In some embodiments, variants or homologues of dCas9 are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, about 99 % identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants of dCas9 are provided that have amino acid sequences that are shorter or longer by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.
In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase can be a Cas9 protein that can cleave only one strand of a double-stranded nucleic acid molecule (e.g. a double-stranded DNA molecule). In some embodiments, Cas9 nickase cleaves the target strand of a double-stranded nucleic acid molecule, which means that Cas9 nickase cleaves the strand that is base-paired with (complementary to) a gRNA (e.g., a sgRNA) that is bound to Cas9 . In some embodiments, a Cas9 nickase comprises a D10A mutation and has a histidine at position 840. In some embodiments, the Cas9 nickase cleaves the non-target, non-base-edited strand of a duplexed nucleic acid molecule, meaning that the Cas9 nickase cleaves the Strand that is not base-paired with a gRNA (e.g., an sgRNA) bound to Cas9. In some embodiments, a Cas9 nickase comprises an H840A mutation and has an aspartic acid residue at position 10 or a corresponding mutation. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to any of the Cas9 nickases provided here. Other suitable Cas9 nickases will be apparent to those skilled in the art based on this disclosure and knowledge in the art and are within the scope of this disclosure. The amino acid sequence of an exemplary Cas9 catalytic nickase (nCas9) is as follows:
In some embodiments, Cas9 refers to a Cas9 of archaea (e.g., nanoarchaea), which is a domain and kingdom of unicellular prokaryotic microbes. In some embodiments, the programmable nucleotide binding protein may be a CasX or CasY protein, such as those described in Burstein et al., New CRISPR-Cas systems from uncultivated microbes. cell res. 2017 Feb. 21. doi:10.1038/cr.2017.21, the entire contents of which are hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems have been identified, including the first reported Cas9 in the archaeal domain of life. This aberrant Cas9 protein has been found to be part of an active CRISPR-Cas system in understudied nanoarchaea. Two previously unknown systems have been discovered in bacteria, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems discovered to date. In some embodiments, in a base editor system described herein, Cas9 is replaced with CasX or a variant of CasX. In some embodiments, in a base editor system described herein, Cas9 is replaced with CasY or a variant of CasY. It is understood that RNA-directed DNA-binding proteins other than nucleic acid-programmable DNA-binding protein (napDNAbp) can be used and are within the scope of this disclosure.
In some embodiments, the nucleic acid-programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% or at least 99.5% identical to a naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide-binding protein is a naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide-binding protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98%, at least 99% or at least 99.5% identical to any CasX or CasY protein described herein. It is understood that CasX and CasY from other bacterial species can also be used in accordance with the present disclosure.
An example CasX ((uniprot.org/uniprot/F0NN87; uniprot.org/uniprot/F0NH53) tr|F0NN87|F0NN87_SULIHCRISPR-associatedCasx protein OS=Sulfolobus islandicus(strain HVE10/4) GN=SiH_0402 PE=4 SV=1) amino acid sequence is as follows:
An exemplary CasX (>tr|F0NH53|F0NH53_SULIR CRISPR-associated protein, Casx OS=Sulfolobus islandicus(Strain REY15A) GN=SiRe_0771 PE=4 SV=1) Amino acid sequence is as follows:
Delta proteobacteria CasX
An exemplary amino acid sequence of CasY ((ncbi.nlm.nih.gov/protein/APG80656.1) > APG80656.1 CRISPR-associated protein CasY [uncultured bacterium of Parcubacteria group]) is as follows:
The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Cas9 undergoes a conformational change upon target binding that positions the nuclease domains to cleave opposite strands of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (~3-4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired by one of two general repair pathways: (1) the efficient but error-prone non-homologous end-joining (NHEJ) pathway; or (2) the less efficient but highly accurate homology-directed repair (HDR) pathway.
The "efficiency" of the end non-homologous junction (NHEJ) and/or homology directed repair (HDR) can be calculated by any suitable method. For example, in some embodiments, efficiency may be expressed as a percentage of successful HDR. For example, a Surveyor nuclease assay can be used to generate cleavage products and the ratio of products to substrate can be used to calculate the percentage. For example, a Surveyor nuclease enzyme can be used that directly cleaves DNA containing a newly integrated restriction sequence as a result of a successful HDR. More cleaved substrate will display a greater percentage of HDR (a greater efficiency of HDR). As an illustrative example, a proportion (percentage) of HDR can be calculated using the following equation [(cleavage products)/(substrate plus cleavage products)] (e.g. (b+c)/(a+b+c)), where "a" is the band intensity of the DNA substrate and "b" and "c" are the cleavage products).
In some embodiments, the efficiency can be expressed as a percentage of successful NHEJ. For example, a T7 endonuclease I assay can be used to generate cleavage products and the ratio of products to substrate can be used to calculate the percentage of NHEJ. T7 endonuclease I cleaves mismatched heteroduplex DNA resulting from hybridization of wild-type and mutant DNA strands (NHEJ creates small random insertions or deletions (indels) at the site of the original break). More cleavage indicates a higher percentage of NHEJ (a greater efficiency of NHEJ). As an illustrative example, a fraction (percentage) of NHEJ can be calculated using the following equation: (1−(1−(b+c)/(a+b+c))1/2)×100, where “a” is the band intensity of the DNA substrate and “b” and “c” are the cleavage products (Ran et. al., Cell. 2013 Sep. 12; 154(6):1380-9; and Ran et al., Nat Protoc. 2013 November;8(11):2281-2308).
The NHEJ repair pathway is the most active repair mechanism and often causes small nucleotide insertions or deletions (indels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical implications, since a population of cells expressing Cas9 and a gRNA or leader polynucleotide can give rise to a variety of mutations. In most embodiments, NHEJ results in small indels in the target DNA that result in amino acid deletions, insertions, or frameshift mutations that result in premature stop codons within the target gene's open reading frame (ORF). The ideal end result is a loss-of-function mutation within the target gene.
While NHEJ-mediated DSB repair often disrupts the gene's open reading frame, homology-directed repair (HDR) can be used to generate specific nucleotide changes ranging from a single nucleotide change to large insertions such as the addition of a fluorophore or tag . To use HDR for gene editing, a DNA repair template containing the desired sequence can be introduced into the cell type of interest using the gRNA(s) and Cas9 or Cas9 nickase. The repair template may contain the desired edit as well as additional homologous sequences immediately upstream and downstream of the target (referred to as left and right homology arms). The length of each homology arm can depend on the size of the change introduced, with larger insertions requiring longer homology arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. The efficiency of HDR is generally low (<10% modified alleles) even in cells expressing Cas9, gRNA and an exogenous repair template. The efficiency of HDR can be increased by synchronizing the cells, since HDR takes place during the S and G2 phases of the cell cycle. Chemically or genetically inhibiting genes involved in NHEJ can also increase HDR frequency.
In some embodiments, Cas9 is a modified Cas9. A given gRNA targeting sequence may have additional sites throughout the genome where there is partial homology. These sites are called off-targets and must be considered when designing a gRNA. In addition to optimizing the gRNA design, CRISPR specificity can also be increased by modifications to Cas9. Cas9 generates double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase, a D10A mutant of SpCas9, retains a nuclease domain and generates a DNA nick rather than a DSB. The Nickase system can also be combined with HDR-mediated gene editing for specific gene edits.
In some embodiments, Cas9 is a variant of the Cas9 protein. A variant Cas9 polypeptide has an amino acid sequence that differs by one amino acid (e.g., has a deletion, insertion, substitution, fusion) when compared to the amino acid sequence of a wild-type Cas9 protein. In some cases, the variant Cas9 polypeptide has an amino acid change (e.g., deletion, insertion, or substitution) that decreases the nuclease activity of the Cas9 polypeptide. For example, in some cases, the variant Cas9 polypeptide has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 protein. In some embodiments, the variant Cas9 protein lacks substantial nuclease activity. When an affected Cas9 protein is a variant of the Cas9 protein that lacks significant nuclease activity, it may be referred to as "dCas9".
In some embodiments, a variant of the Cas9 protein has reduced nuclease activity. For example, a variant of the Cas9 protein has less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the endonuclease activity of on Wild-type Cas9 protein, e.g. B. a wild-type Cas9 protein.
In some embodiments, a variant of the Cas9 protein can cleave the complementary strand of a leader target sequence, but has a reduced ability to cleave the non-complementary strand of a double-stranded leader target sequence. For example, the variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, a variant of the Cas9 protein has a D10A (aspartate to alanine at amino acid position 10) and can therefore cleave the complementary strand of a double-stranded leader target sequence, but has a reduced ability to cleave the non- Complementary strand of a double-stranded guide target sequence (resulting in a single-strand break (SSB) instead of a double-strand break (DSB) when the variant Cas9 protein cleaves a double-stranded target nucleic acid) (see, for example, Jinek et al., Science, August 17, 2012, 337(6096 ):816-21).
In some embodiments, a variant of the Cas9 protein can cleave the non-complementary strand of a double-stranded guide target sequence, but has a reduced ability to cleave the complementary strand of the guide target sequence. For example, the variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC/HNH/RuvC domain motifs). As a non-limiting example, in some embodiments the variant Cas9 protein has an H840A mutation (histidine to alanine at amino acid position 840) and can therefore cleave the non-complementary strand of the guide target sequence, but has a reduced ability to cleave the complementary strand of the guide -Targeting sequence (resulting in an SSB instead of a DSB when the variant Cas9 protein cleaves a double-stranded leader targeting sequence). Such a Cas9 protein has a reduced ability to cleave a guide target sequence (e.g., a single-stranded guide target sequence), but retains the ability to bind a guide target sequence (e.g., a single-stranded guide target sequence).
In some embodiments, a variant of the Cas9 protein has a reduced ability to cleave both the complementary and non-complementary strands of a double-stranded target DNA. As a non-limiting example, in some embodiments the variant Cas9 protein harbors both the D10A and H840A mutations such that the polypeptide has a reduced ability to target both the complementary and non-complementary strands of a double-stranded target DNA columns. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g. a single-stranded target DNA) but retains the ability to bind a target DNA (e.g. a single-stranded target DNA). .
As a further non-limiting example, in some embodiments, the variant Cas9 protein harbors W476A and W1126A mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g. a single-stranded target DNA) but retains the ability to bind a target DNA (e.g. a single-stranded target DNA). .
As a further non-limiting example, in some embodiments, the variant Cas9 protein harbors P475A, W476A, N477A, D1125A, W1126A, and D1127A mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g. a single-stranded target DNA) but retains the ability to bind a target DNA (e.g. a single-stranded target DNA). .
As a further non-limiting example, in some embodiments, the variant Cas9 protein harbors H840A, W476A, and W1126A mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g. a single-stranded target DNA) but retains the ability to bind a target DNA (e.g. a single-stranded target DNA). . As a further non-limiting example, in some embodiments, the variant Cas9 protein harbors H840A, D10A, W476A, and W1126A mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g. a single-stranded target DNA) but retains the ability to bind a target DNA (e.g. a single-stranded target DNA). . In some embodiments, the Cas9 variant has restored the His catalytic residue at position 840 in the Cas9 HNH domain (A840H).
As a further non-limiting example, in some embodiments, the variant Cas9 protein harbors H840A, P475A, W476A, N477A, D1125A, W1126A, and D1127A mutations such that the polypeptide has a reduced ability to cleave a target DNA having. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g. a single-stranded target DNA) but retains the ability to bind a target DNA (e.g. a single-stranded target DNA). . As a further non-limiting example, in some embodiments, the variant Cas9 protein harbors D10A, H840A, P475A, W476A, N477A, D1125A, W1126A, and D1127A mutations such that the polypeptide has a reduced ability to cleave a has target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g. a single-stranded target DNA) but retains the ability to bind a target DNA (e.g. a single-stranded target DNA). . In some embodiments, if a variant Cas9 protein harbors W476A and W1126A mutations, or if the variant Cas9 protein harbors P475A, W476A, N477A, D1125A, W1126A, and D1127A mutations, the variant des Cas9 protein does not bind efficiently to a PAM sequence. Therefore, in some such embodiments, when such a variant of the Cas9 protein is used in a binding method, the method does not require a PAM sequence. In other words, when in some embodiments such a variant of the Cas9 protein is used in a binding method, the method may involve a guide RNA, but the method may be performed in the absence of a PAM sequence (and the specificity of the binding is therefore provided by the targeting segment of the guide RNA). Other residues can be mutated to achieve the above effects (i.e. inactivate one or the other nuclease part). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987 may be altered (i.e., substituted). Mutations other than alanine substitutions are also suitable.
In some embodiments, a variant of the Cas9 protein that has reduced catalytic activity (e.g., where a Cas9 protein has a D10, G12, G17, E762, H840, N854, N863, H982 , H983, A984, D986 and/or an A987 mutation, e.g. D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A and/or D986A), the variant can of the Cas9 protein will still bind site-specifically to target DNA (because it is still guided to a target DNA sequence by a guide RNA) as long as it retains the ability to interact with the guide RNA.
In some embodiments, the Cas variant protein may be spCas9, spCas9-VRQR, spCas9-VRER, xCas9(sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.
In some embodiments, a modified SpCas9 with amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E and T1337R (SpCas9-MQKFRAER) and with specificity for the altered PAM 5'-NGC-3' was used.
alternatives toS. pyogenesCas9 may comprise RNA-directed endonucleases from the Cpf1 family that show cleavage activity in mammalian cells. CRISPR byPrevotellaAndFrancisella1 (CRISPR/Cpf1) is a DNA editing technology analogous to the CRISPR/Cas9 system. Cpf1 is an RNA-directed endonuclease of a class II CRISPR/Cas system. This adaptive immune mechanism is found inPrevotellaAndFrancisellaBacteria. Cpf1 genes are associated with the CRISPR locus and encode an endonuclease that uses a guide RNA to locate and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9 that overcomes some of the limitations of the CRISPR/Cas9 system. In contrast to Cas9 nucleases, the result of Cpf1-mediated DNA cleavage is a double-strand break with a short 3' overhang. The staggered cleavage pattern of Cpf1 may open the possibility of directed gene transfer, analogous to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Like the Cas9 variants and orthologues described above, Cpf1 may also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack SpCas9-preferred NGG-PAM sites. The Cpf1 locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. In addition, Cpf1 lacks an HNH endonuclease domain and the N-terminus of Cpf1 lacks the Cas9 alpha-helical recognition lobe. The domain architecture of Cpf1 CRISPR-Cas shows that Cpf1 is functionally unique and classified as a class 2, type V CRISPR system. The Cpf1 loci encode Cas1, Cas2, and Cas4 proteins that are more similar to types I and III than to type II systems. Functional Cpf1 does not require the transactivating CRISPR RNA (tracrRNA), so only CRISPR (crRNA) is required. This is beneficial for genome editing, as not only is Cpf1 smaller than Cas9, but it also has a smaller sgRNA molecule (about half as many nucleotides as Cas9). The Cpf1-crRNA complex cleaves target DNA or RNA by identifying a protospacer-contiguous 5′-YTN-3′ motif in contrast to the G-rich PAM targeted by Cas9. After identifying PAM, Cpf1 introduces a sticky-end-like DNA double-strand break with an overhang of 4 or 5 nucleotides.
Cas12 domains of nucleobase editors
Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while class 2 systems have a single protein effector. For example, Cas9 and Cpf1 are class 2 effectors, albeit different types (type II and type V, respectively). In addition to Cpf1, Class 2, Type V CRISPR-Cas systems also include Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, and Cas12i). See e.g. B. Shmakov et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems", Mol. Cell, November 5, 2015; 60(3): 385-397; Makarova et al., “Classification and nomenclature of CRISPR-Cas systems: where from here?” CRISPR Journal, 2018, 1(5): 325-336; and Yan et al., “Functionally Diverse Type V CRISPR-Cas Systems,” Science, January 4, 2019; 363:88-91; the entire contents of each are hereby incorporated by reference. Type V Cas proteins contain a RuvC (or RuvC-like) endonuclease domain. For example, while production of mature CRISPR RNA (crRNA) is generally tracrRNA-independent, Cas12b/C2c1 requires tracrRNA for production of crRNA. Cas12b/C2c1 relies on both crRNA and tracrRNA for DNA cleavage.
Nucleic acid-programmable DNA binding proteins contemplated by the present invention include Cas proteins classified as Class 2, Type V (Cas12 proteins). Non-limiting examples of Cas class 2, type V proteins include Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h and Cas12i, homologues thereof or modified versions thereof. As used herein, a Cas12 protein may also be referred to as a Cas12 nuclease, a Cas12 domain, or a Cas12 protein domain. In some embodiments, the Cas12 proteins of the present invention comprise an amino acid sequence interrupted by an internally fused protein domain, such as a deaminase domain.
In some embodiments, the Cas12 domain is a nuclease-inactive Cas12 domain or a Cas12 nickase. In some embodiments, the Cas12 domain is a nuclease active domain. For example, the Cas12 domain can be a Cas12 domain that cleaves one strand of a double-stranded nucleic acid (e.g., double-stranded DNA molecule). In some embodiments, the Cas12 domain comprises any of the amino acid sequences set forth herein. In some embodiments, the Cas12 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95%, at least 96%, at least 97% , at least 98%, at least 99% or at least 99.5% identical to any of the amino acid sequences set forth herein. In some embodiments, the Cas12 domain comprises an amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , 46, 47, 48, 49, 50 or more mutations compared to any of the amino acid sequences set forth herein. In some embodiments, the Cas12 domain comprises an amino acid sequence that is at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200 , at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100 or at least 1200 identical contiguous amino acid residues compared to any of the amino acid sequences set forth herein.
In some embodiments, proteins comprising fragments of Cas12 are provided. For example, in some embodiments, a protein comprises one of two Cas12 domains: (1) the gRNA binding domain of Cas12; or (2) the DNA cleavage domain of Cas12. In some embodiments, proteins comprising Cas12 or fragments thereof are referred to as "Cas12 variants". A Cas12 variant shows homology to Cas12 or a fragment thereof. For example, a Cas12 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical % identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas12. In some embodiments, the Cas12 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas12. In some embodiments, the Cas12 variant comprises a fragment of Cas12 (eg, a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical % is identical. identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding one Fragment of wild-type Cas12. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild-type Cas12. In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 , 1200, 1250 or at least 1300 amino acids in length.
In some embodiments, Cas12 corresponds to or includes part or all of a Cas12 amino acid sequence having one or more mutations that alter Cas12 nuclease activity. Such mutations include, for example, amino acid substitutions within the RuvC nuclease domain of Cas12. In some embodiments, variants or homologues of Cas12 are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, or about 99 % identical, at least about 99.5% identical, or at least about 99.9% identical to a wild-type Cas12. In some embodiments, variants of Cas12 are provided that have amino acid sequences that are shorter or longer by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.
In some embodiments, Cas12 fusion proteins as provided herein comprise the complete amino acid sequence of a Cas12 protein, e.g. B. one of the Cas12 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas12 sequence, but only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas12 domains are provided herein, and additional suitable Cas12 domain and fragment sequences will be apparent to those skilled in the art.
In general, the class 2, type V Cas proteins have a single functional RuvC endonuclease domain (see, e.g., Chen et al., “CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity “, Science 360:436-439 (2018 )). In some cases, the Cas12 protein is a variant of the Cas12b protein. (See Strecker et al., Nature Communications, 2019, 10(1): Art. No.: 212). In one embodiment, a variant Cas12 polypeptide has an amino acid sequence that differs from the amino acid sequence by 1, 2, 3, 4, 5, or more amino acids (e.g., has a deletion, insertion, substitution, fusion). a wild-type Cas12 protein. In some cases, the variant Cas12 polypeptide has an amino acid change (e.g., deletion, insertion, or substitution) that reduces the activity of the Cas12 polypeptide. For example, in some cases, variant Cas12 is a Cas12b polypeptide that has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of Nickase activity of the corresponding wild-type Cas12b protein. In some cases, the variant Cas12b protein has no significant nickase activity.
In some cases, a variant of the Cas12b protein has reduced nickase activity. For example, a variant of the Cas12b protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the nickase activity of a wild-type Cas12b protein.
In some embodiments, the Cas12 protein comprises RNA-driven endonucleases from the Cas12a/Cpf1 family that exhibit activity in mammalian cells. CRISPR byPrevotellaAndFrancisella1 (CRISPR/Cpf1) is a DNA editing technology analogous to the CRISPR/Cas9 system. Cpf1 is an RNA-directed endonuclease of a class II CRISPR/Cas system. This adaptive immune mechanism is found inPrevotellaAndFrancisellaBacteria. Cpf1 genes are associated with the CRISPR locus and encode an endonuclease that uses a guide RNA to locate and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9 that overcomes some of the limitations of the CRISPR/Cas9 system. In contrast to Cas9 nucleases, the result of Cpf1-mediated DNA cleavage is a double-strand break with a short 3' overhang. The staggered cleavage pattern of Cpf1 may open the possibility of directed gene transfer, analogous to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Like the Cas9 variants and orthologues described above, Cpf1 may also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack SpCas9-preferred NGG-PAM sites. The Cpf1 locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Furthermore, unlike Cas9, Cpf1 does not have an HNH endonuclease domain, and the N-terminus of Cpf1 does not have the alpha-helical recognition lobe of Cas9. The domain architecture of Cpf1 CRISPR-Cas shows that Cpf1 is functionally unique and classified as a class 2, type V CRISPR system. The Cpf1 loci encode Cas1, Cas2, and Cas4 proteins that are more similar to types I and III than to type II systems. Functional Cpf1 does not require the transactivating CRISPR RNA (tracrRNA), so only CRISPR (crRNA) is required. This is beneficial for genome editing, as not only is Cpf1 smaller than Cas9, but it also has a smaller sgRNA molecule (about half as many nucleotides as Cas9). The Cpf1-crRNA complex cleaves target DNA or RNA by identifying a protospacer-contiguous motif 5′-YTN-3′ or 5′-TTTN-3′ in contrast to the G-rich PAM targeted by Cas9. After identifying PAM, Cpf1 inserts a sticky-end-like DNA double-strand break with an overhang of 4 or 5 nucleotides.
In some aspects of the present invention, a vector encodes a CRISPR enzyme that is mutated relative to a corresponding wild-type enzyme such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence can be used. Cas12 can refer to a polypeptide at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% sequence identity and/or sequence homology with an exemplary wild-type Cas12 polypeptide (eg, Cas12 fromBacillus Hisashii). Cas12 may refer to a polypeptide at no more than or no more than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% sequence identity and/or sequence homology to an exemplary wild-type Cas12 polypeptide (e.gBacillus Hisashii(BhCas12b),Bacillussp. V3-13 (BvCas12b) undAlicyclobacillus acidiphilus(AaCas12b)). Cas12 can refer to the wild-type or a modified form of the Cas12 protein, which can include an amino acid change, such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.
Nucleic acid-programmable DNA-binding proteins
Some aspects of the disclosure provide fusion proteins that include domains that act as nucleic acid-programmable DNA-binding proteins that can be used to convert a protein, such as a base editor, to a specific nucleic acid sequence (e.g., DNA or RNA). In certain embodiments, a fusion protein comprises a nucleic acid-programmable DNA-binding protein domain and a deaminase domain. Non-limiting examples of nucleic acid-programmable DNA binding proteins include Cas9 (eg, dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, and Cas12i. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cash, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Type II Cas Effector Proteins, Type V Cas Effector Proteins, Type VI Cas effector proteins, CARF, DinG, homologues thereof, or modified or engineered versions thereof. Other nucleic acid-programmable DNA-binding proteins are also within the scope of this disclosure, although they may not be specifically listed in this disclosure. See e.g. B. Makarova et al. “Classification and nomenclature of CRISPR-Cas systems: Where from?” CRISPR J. October 2018; 1:325-336. doi: 10.1089/crispr.2018.0033; Yan et al., "Functionally diverse type V CRISPR-Cas systems" Science. January 4, 2019; 363(6422):88-91. doi: 10.1126/science.aav7271, the entire contents are hereby incorporated by reference.
An example of a nucleic acid-programmable DNA-binding protein that has a different PAM specificity than Cas9 is clustered regularly interspaced short palindromic repeats fromPrevotellaAndFrancisella1 (Cpf1). Similar to Cas9, Cpf1 is also a class 2 CRISPR effector. Cpf1 has been shown to mediate robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-directed endonuclease that lacks tracrRNA and uses a T-rich protospacer-contiguous motif (TTN, TTTN, or YTN). In addition, Cpf1 cleaves DNA via a staggered DNA double-strand break. Of the 16 proteins of the Cpf1 family, two enzymes from Acidaminococcus and Lachnospiraceae show efficient genome editing activity in human cells. Cpf1 proteins are known in the art and have been previously described, for example Yamano et al., "Crystal structure of Cpf1 in complex with guide RNA and target DNA". Cell (165) 2016, p. 949-962; the entire contents of which are hereby incorporated by reference.
Useful in the present compositions and methods are nuclease-inactive Cpf1 (dCpf1) variants that can be used as a leader nucleotide sequence-programmable DNA-binding protein domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but lacks an HNH endonuclease domain, and the N-terminus of Cpf1 lacks the Cas9 alpha-helical recognition lobe. It was reported in Zetsche et al. shown,Cell,163, 759-771, 2015 (which is incorporated herein by reference) that the RuvC-like domain of Cpf1 is responsible for cleavage of both DNA strands and inactivation of the RuvC-like domain inactivates Cpf1 nuclease activity. For example, mutations corresponding to D917A, E1006A or D1255A inFrancisella NovizidCpf1 inactivates Cpf1 nuclease activity. In some embodiments, the dCpf1 of the present disclosure includes mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/E1006A/D1255A. It is understood that any mutations, e.g. B. substitution mutations, deletions or insertions that inactivate the RuvC domain of Cpf1 can be used according to the present disclosure.
In some embodiments, the nucleic acid-programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In some embodiments, the Cpf1 protein is a nuclease-inactive Cpf1 (dCpf1). In some embodiments, the Cpf1, nCpf1, or dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% %, at least 98%, at least 99% or at least 99.5% identical to a Cpf1 sequence disclosed herein. In some embodiments, the dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% or at least 99.5% identical to a Cpf1 sequence disclosed herein and includes mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A or D917A/E1006A/D1255A. It should be appreciated that Cpf1 from other bacterial species can also be used in accordance with the present disclosure.
In some embodiments, one of the Cas9 domains present in the fusion protein can be replaced with a leader nucleotide sequence-programmable DNA-binding protein domain that has no PAM sequence requirements.
In some embodiments, the Cas9 domain is a Cas9 domainStaphylococcus aureus(SaCas9). In some embodiments, the SaCas9 domain is a nuclease-active SaCas9, a nuclease-inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, SaCas9 comprises an N579A mutation or a corresponding mutation in any of the amino acid sequences provided herein.
In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having an NNGRRT or an NNGRRT-PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of an E781X, an N967X, and an R1014X mutation or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of an E781K, an N967K, and an R1014H mutation or one or more corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SaCas9 domain comprises an E781K, an N967K, or an R1014H mutation or corresponding mutations in any of the amino acid sequences provided herein.
Exemplary SaCas9 sequence
Residue N579 above, which is underlined and in bold, can be mutated (e.g. to an A579) to yield a SaCas9 nickase.
Exemplary SaCas9n sequence
Residue A579 above, which can be mutated from N579 to give a SaCas9 nickase, is underlined and in bold.
Exemplarisches SaKKH Cas9
Residue A579 above, which can be mutated from N579 to give a SaCas9 nickase, is underlined and in bold. Residues K781, K967 and H1014 above, which can be mutated from E781, N967 and R1014 to give a SaKKH Cas9, are underlined and italicized.
In some embodiments, the napDNAbp is a circular permutant. In the following sequences, the plaintext designates an adenosine deaminase sequence, the boldfaced sequence designates a Cas9-derived sequence, the italicized sequence designates a linker sequence, and the underlined sequence designates a bipartite nuclear localization sequence. CP5 (with MSP "NGC" PID and "D10A" Nickase):
In some embodiments, the nucleic acid-programmable DNA binding protein (napDNAbp) is a single effector of a microbial CRISPR-Cas system. Individual effectors of microbial CRISPR-Cas systems include, without limitation, Cas9, Cpf1, Cas12b/C2c1, and Cas12c/C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while class 2 systems have a single protein effector. For example, Cas9 and Cpf1 are class 2 effectors. In addition to Cas9 and Cpf1, three distinct class 2 CRISPR-Cas systems (Cas12b/C2c1 and Cas12c/C2c3) were identified by Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems”.Mol. cell,Nov 5, 2015; 60(3): 385-397, the entire contents of which are hereby incorporated by reference. Effectors from two of the systems, Cas12b/C2c1 and Cas12c/C2c3, contain RuvC-like endonuclease domains related to Cpf1. A third system contains an effector with two predicted HEPN RNase domains. The production of mature CRISPR RNA is tracrRNA-independent, in contrast to the production of CRISPR RNA by Cas12b/C2c1. Cas12b/C2c1 is dependent on both CRISPR RNA and tracrRNA for DNA cleavage.
The crystal structure ofAlicyclobacillus acidoterrastrisCas12b/C2c1 (AacC2c1) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See e.g. B. Liu et al., "C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism",Mol. cell,January 19, 2017; 65(2):310-322, the entire contents of which are hereby incorporated by reference. The crystal structure was also reported inAlicyclobacillus acidoterrestrisC2c1 is bound to target DNAs as ternary complexes. See e.g. B. Yang et al., "PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease",Cell,Dec 15, 2016; 167(7):1814-1828, the entire contents of which are hereby incorporated by reference. Catalytically competent conformations of AacC2c1, with both target and non-target DNA strands, were independently positioned in a single RuvC catalytic pocket, with Cas12b/C2c1-mediated cleavage resulting in a staggered seven-nucleotide break of the target -DNA led. Structural comparisons between Cas12b/C2c1 ternary complexes and previously identified Cas9 and Cpf1 counterparts reveal the diversity of mechanisms employed by CRISPR-Cas9 systems.
In some embodiments, the nucleic acid-programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a Cas12b/C2c1 or a Cas12c/C2c3 protein. In some embodiments, the napDNAbp is a Cas12b/C2c1 protein. In some embodiments, the napDNAbp is a Cas12c/C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% or at least 99.5% identical to a naturally occurring Cas12b/C2c1 or Cas12c/C2c3 protein. In some embodiments, the napDNAbp is a naturally occurring Cas12b/C2c1 or Cas12c/C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% or at least 99.5% identical to any of the napDNAbp sequences provided herein. It is understood that Cas12b/C2c1 or Cas12c/C2c3 from other bacterial species can also be used in accordance with the present disclosure.
A Cas12b/C2c1 ((uniprot.org/uniprot/TOD7A2#2) sp|T0D7A2|C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acido-terrestris(ATCC 49025/DSM 3922/CIP 106132/NCIMB 13137/GD3B strain) GN=c2c1 PE=1 SV=1) amino acid sequence is as follows:
BhCas12b (Bacillus Hisashii) NCBI reference sequence: WP_095142515
In some embodiments, Cas12b is BvCas12B. In some embodiments, the Cas12b includes the amino acid substitutions S893R, K846R, and E837G as numbered in the exemplary BvCas12b amino acid sequence provided below.
BvCas12b (Bacillussp. V3-13) NCBI reference sequence: WP_101661451.1
Guide Polynucleotides
In one embodiment, the guide polynucleotide is a guide RNA. An RNA/Cas complex can help "guide" the Cas protein to a target DNA. Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand that is not complementary to the crRNA is first cut endonucleolytically and then trimmed exonucleolytically from 3'-5'. In nature, DNA binding and cleavage typically requires protein and both RNAs. However, single-guide RNAs (“sgRNA” or simply “gNRA”) can be engineered to integrate aspects of both crRNA and tracrRNA into a single RNA species. See e.g. B. Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeats (the PAM or protospacer-adjacent motif) to help distinguish self from non-self. Cas9 nuclease sequences and structures are well known to those skilled in the art (see e.g. "Complete genome sequence of an M1stamm ofStreptococcus pyogenes." Ferretti, J.J. et al., Natl. Academic Science. USA 98:4658-4663 (2001); "CRISPR RNA maturation by transcoded small RNA and host factor RNase III." Deltcheva E et al., Nature 471:602-607 (2011); and "Programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity". Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference). Cas9 orthologues have been described in a variety of ways including, but not limited to,S. pyogenesAndS. thermophilus. Other suitable Cas9 nucleases and sequences may be apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci described in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immune systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase.
In some embodiments, the guide polynucleotide is at least a single guide RNA ("sgRNA" or "gNRA"). In some embodiments, the lead polynucleotide is at least one tracrRNA. In some embodiments, the guide polynucleotide does not require a PAM sequence to guide the polynucleotide-programmable DNA-binding domain (e.g., Cas9 or Cpf1) to the target nucleotide sequence.
The polynucleotide-programmable nucleotide binding domain (e.g., a CRISPR-derived domain) of the base editors disclosed herein can recognize a target polynucleotide sequence by associating it with a guide polynucleotide. A guide polynucleotide (e.g. gRNA) is typically single-stranded and can be programmed to bind site-specifically (i.e. via complementary base pairing) to a target sequence of a polynucleotide, thereby controlling a base editor associated with the guide nucleic acid to the target sequence . A lead polynucleotide can be DNA. A lead polynucleotide can be RNA. In some embodiments, the leader polynucleotide includes natural nucleotides (e.g., adenosine). In some embodiments, the leader polynucleotide includes non-natural (or unnatural) nucleotides (e.g., peptide nucleic acid or nucleotide analogs). In some embodiments, the target region of a lead nucleic acid sequence may be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. A target region of a guide nucleic acid may be between 10-30 nucleotides in length, or between 15-25 nucleotides in length, or between 15-20 nucleotides in length.
In some embodiments, a leader polynucleotide comprises two or more individual polynucleotides that can interact with one another, for example via complementary base pairing (e.g., a dual leader polynucleotide). For example, a lead polynucleotide may include a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA). For example, a lead polynucleotide may include one or more transactivating CRISPR RNA (tracrRNA).
In Type II CRISPR systems, targeting of a nucleic acid by a CRISPR protein (e.g. Cas9) typically requires complementary base pairing between a first RNA molecule (crRNA), which comprises a sequence that recognizes the target sequence, and a second RNA molecule (trRNA), which comprises repeat sequences that form a framework region that stabilizes the guide RNA-CRISPR protein complex. Such dual guide RNA systems can be used as a guide polynucleotide to direct the base editors disclosed herein to a target polynucleotide sequence.
In some embodiments, the base editor provided herein uses a single leader polynucleotide (e.g., gRNA). In some embodiments, the base editor provided herein uses a dual leader polynucleotide (eg, dual gRNAs). In some embodiments, the base editor provided herein uses one or more leader polynucleotides (e.g., multiple gRNA). In some embodiments, a single leader polynucleotide is used for different base editors described herein. For example, a single guide polynucleotide can be used for an adenosine base editor or an adenosine base editor and a cytidine base editor, e.g. as described in PCT/US19/44935.
In other embodiments, a guide polynucleotide may comprise both the polynucleotide targeting portion of the nucleic acid and the backbone portion of the nucleic acid in a single molecule (i.e., a single molecule guide nucleic acid). For example, a single molecule guide polynucleotide can be a single guide RNA (sgRNA or gRNA). As used herein, the term guide polynucleotide sequence encompasses any single, double, or multi-molecule nucleic acid capable of interacting with and directing a base editor to a target polynucleotide sequence.
Typically, a leader polynucleotide (eg, crRNA/trRNA complex or a gRNA) includes a "polynucleotide-targeting segment" that includes a sequence that can recognize and bind to a target polynucleotide sequence, and a "protein-binding segment" that the sequence stabilizes leader polynucleotide within a polynucleotide-programmable nucleotide binding domain component of a base editor. In some embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to a DNA polynucleotide, thereby facilitating editing of a base in the DNA. In other embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to an RNA polynucleotide, thereby facilitating editing of a base in RNA. Here, a "segment" refers to a portion or region of a molecule, e.g. B. a contiguous stretch of nucleotides in the leader polynucleotide. A segment can also refer to a region/section of a complex, such that a segment can encompass regions of more than one molecule. For example, when a leader polynucleotide comprises multiple nucleic acid molecules, the protein-binding segment may comprise all or part of multiple separate molecules hybridized, for example, along a region of complementarity. In some embodiments, a protein-binding segment of a DNA-targeting RNA comprising two separate molecules may comprise (i) base pairs 40-75 of a first RNA molecule that is 100 base pairs in length; and (ii) base pairs 10-25 of a second RNA molecule that is 50 base pairs long. The definition of "segment", unless otherwise defined in a particular context, is not limited to any specific number of base pairs in total, is not limited to any specific number of base pairs from a given RNA molecule, is not limited to any specific number separate molecules within a complex and may include regions of RNA molecules of any overall length and may include regions of complementarity to other molecules.
A leader RNA or leader polynucleotide may comprise two or more RNAs, e.g. B. CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). A guide RNA or guide polynucleotide can sometimes comprise a single-chain RNA or a single guide RNA (sgRNA) formed by fusing a portion (e.g., a functional portion) of crRNA and tracrRNA. A guide RNA or guide polynucleotide can also be a dual RNA comprising a crRNA and a tracrRNA. In addition, a crRNA can hybridize with a target DNA.
As discussed above, a leader RNA or leader polynucleotide can be an expression product. For example, a DNA encoding a guide RNA can be a vector comprising a sequence encoding the guide RNA. A guide RNA or guide polynucleotide can be transferred into a cell by transfecting the cell with an isolated guide RNA or plasmid DNA comprising a sequence encoding the guide RNA and a promoter. A guide RNA or guide polynucleotide can also be delivered into a cell in other ways, for example using virus-mediated gene delivery.
A guide RNA or guide polynucleotide can be isolated. For example, a guide RNA can be transfected into a cell or organism in the form of an isolated RNA. A guide RNA can be produced by in vitro transcription using any in vitro transcription system known in the art. A guide RNA may be transferred into a cell in the form of an isolated RNA rather than in the form of a plasmid comprising a coding sequence for a guide RNA.
A leader RNA or leader polynucleotide may comprise three regions: a first region at the 5' end, which may be complementary to a target site in a chromosomal sequence, a second inner region, which may form a stem loop structure, and a third 3' Region capable of forming a stem loop structure. Region that may be single-stranded. A first region of each guide RNA can also be different, such that each guide RNA guides a fusion protein to a specific target site. Furthermore, second and third regions of each guide RNA can be identical in all guide RNAs.
A first region of a guide RNA or guide polynucleotide can be complementary to a sequence at a target site in a chromosomal sequence such that the first region of the guide RNA can base pair with the target site. In some embodiments, a first region of a guide RNA may comprise from or from about 10 nucleotides to 25 nucleotides (ie, from 10 nucleotides to nucleotides; or from about 10 nucleotides to about 25 nucleotides; or from 10 nucleotides to about 25 nucleotides; or from about 10 nucleotides to 25 nucleotides) or more. For example, a region of base pairing between a first region of a guide RNA and a target site in a chromosomal sequence may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20, 22, 23, 24, 25 or more nucleotides in length. Sometimes a first region of a guide RNA may be about 19, 20, or 21 nucleotides in length.
A leader RNA or leader polynucleotide may also include a second region that forms secondary structure. For example, a secondary structure formed by a guide RNA may include a stem (or hairpin) and a loop. A length of a loop and a stem can vary. For example, a loop may be in the range of or about 3 to 10 nucleotides in length and a stem may be in the range of or about 6 to 20 base pairs in length. A stem may comprise one or more bulges of 1 to 10 or about 10 nucleotides. The total length of a second region can range from about 16 to 60 nucleotides. For example, a loop may be about 4 nucleotides in length and a stem may be about 12 base pairs in length.
A leader RNA or leader polynucleotide may also include a third region at the 3' end, which may be substantially single-stranded. For example, a third region is sometimes non-complementary to any chromosomal sequence in a cell of interest and sometimes non-complementary to the remainder of a leader RNA. Furthermore, the length of a third area can vary. A third region can be greater than or greater than about 4 nucleotides in length. For example, a third region may range in length from about 5 to 60 nucleotides.
A guide RNA or guide polynucleotide can be targeted to any exon or intron of a target gene. In some embodiments, a guide can target exon 1 or 2 of a gene; in other embodiments, a guide may target exon 3 or 4 of a gene. A composition can include multiple guide RNAs that all target the same exon, or in some embodiments, multiple guide RNAs that can target different exons. An exon and an intron of a gene can be targeted.
A guide RNA or guide polynucleotide can be targeted to a nucleic acid sequence of about 20 nucleotides. A target nucleic acid can be less than or less than about 20 nucleotides. A target nucleic acid can be at least or at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or anywhere from 1-100 nucleotides in length. A target nucleic acid can be at most or at most about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50 or anywhere between 1-100 nucleotides in length. A target nucleic acid sequence may or may not be about 20 bases immediately 5' of the first nucleotide of the PAM. A target RNA can target a nucleic acid sequence. A target nucleic acid can be at least or at least about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, or 1-100 nucleotides.
A guide polynucleotide, such as a guide RNA, may refer to a nucleic acid that can hybridize to another nucleic acid, such as the target nucleic acid or the protospacer in a genome of a cell. A lead polynucleotide can be RNA. A lead polynucleotide can be DNA. The guide polynucleotide can be programmed or designed to bind site-specifically to a nucleic acid sequence. A leader polynucleotide may comprise a chain of polynucleotides and may be referred to as a single leader polynucleotide. A leader polynucleotide may comprise two polynucleotide chains and may be referred to as a double leader polynucleotide. A guide RNA can be introduced into a cell or embryo as an RNA molecule. For example, an RNA molecule can be transcribed in vitro and/or chemically synthesized. An RNA can be derived from a synthetic DNA molecule, e.g. B. a gBlocks® gene fragment, are transcribed. A guide RNA can then be introduced into a cell or embryo as an RNA molecule. A guide RNA can also be in the form of a non-RNA nucleic acid molecule, e.g. a DNA molecule, into a cell or embryo. For example, a DNA encoding a guide RNA can be operably linked to a promoter control sequence for expression of the guide RNA in a cell or embryo of interest. An RNA coding sequence may be operably linked to a promoter sequence recognized by RNA polymerase III (Pol III). Plasmid vectors that can be used to express guide RNA include, but are not limited to, px330 vectors and px333 vectors. In some embodiments, a plasmid vector (e.g., a px333 vector) may include at least two leader RNA-encoding DNA sequences.
Methods for selecting, designing and validating lead polynucleotides, e.g. B. guide RNAs and targeting sequences are described herein and are known to those skilled in the art. For example, to minimize the impact of potential substrate promiscuity of a deaminase domain in the nucleobase editor system (e.g., an AID domain), the number of residues that might be inadvertently targeted for deamination (e.g., off-target C- Residues potentially located on ssDNA within the target nucleic acid locus) can be minimized. In addition, software tools can be used to optimize the gRNAs corresponding to a target nucleic acid sequence, e.g. B. to minimize overall off-target activity across the genome. For example, use for any targeting domain choiceS. pyogenesCas9, all off-target sequences (before selected PAMs, e.g. NAG or NGG) across the genome can be identified, up to a certain number (e.g. 1, 2, 3, 4, 5, 6 , 7, 8, 9) or 10) of mismatched base pairs. First regions of gRNAs that are complementary to a target site can be identified, and each first region (e.g., crRNAs) can be ranked according to their total predicted off-target score; The top targeting domains represent those likely to have the most on-target and least off-target activity. Candidate targeting gRNAs can be functionally evaluated using methods known in the art and/or as set forth herein.
As a non-limiting example, target DNA hybridization sequences in crRNAs of a guide RNA for use with Cas9s can be identified using a DNA sequence search algorithm. gRNA design can be performed using custom gRNA design software based on the public tool cas-offinder as described in Bae S., Park J., & Kim J.-S. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-driven endonucleases. Bioinformatics 30, 1473-1475 (2014). This software scores guides after calculating their genome-wide off-target propensity. Typically, for guides that are 17 to 24 in length, matches ranging from perfect matches to 7 mismatches are considered. Once the off-target sites are computed, a total score is calculated for each guideline and presented in a tabular output using a web interface. In addition to identifying potential target sites adjacent to PAM sequences, the software also identifies all neighboring PAM sequences that differ by 1, 2, 3, or more than 3 nucleotides from the selected target sites. Genomic DNA sequences for a target nucleic acid sequence, e.g. B. a target gene, can be obtained and repeat elements can be identified using publicly available tools, e.g. B. the RepeatMasker program, are screened. RepeatMasker searches input DNA sequences for repeated elements and regions of low complexity. The output is a detailed annotation of the repetitions that are present in a given query sequence.
Once identified, first regions of guide RNAs, e.g. B. crRNAs based on their distance to the target site, their orthogonality and the presence of 5' nucleotides for close matches to relevant PAM sequences (e.g. a 5' G based on the identification of close matches in the human genome, that contain a relevant PAM, such as NGG PAM forS. pyogenes, NNGRRT or NNGRRV PAM forS aureus). As used herein, orthogonality refers to the number of sequences in the human genome that contain a minimal number of mismatches with the target sequence. For example, a "high degree of orthogonality" or "good orthogonality" may refer to 20-mer targeting domains that do not share identical sequences in the human genome adjacent to the intended target, nor any sequences that contain one or two mismatches in the target contain order. Targeting domains with good orthogonality can be chosen to minimize off-target DNA cleavage.
In some embodiments, a reporter system can be used to detect base editing activities and to test candidate guide polynucleotides. In some embodiments, a reporter system can include a reporter gene-based assay in which base editing activity results in expression of the reporter gene. For example, a reporter system may include a reporter gene that includes a deactivated start codon, e.g. B. a mutation on the template strand from 3'-TAC-5' to 3'-CAC-5'. After successful deamination of target C, the corresponding mRNA is transcribed as 5′-AUG-3′ instead of 5′-GUG-3′, allowing translation of the reporter gene. Suitable reporter genes will be known to those skilled in the art. Non-limiting examples of reporter genes include genes encoding green fluorescent protein (GFP), red fluorescent protein (RFP), luciferase, secreted alkaline phosphatase (SEAP), or any other gene whose expression is detectable and obvious to one skilled in the art. The reporter system can be used to test many different gRNAs, e.g. B. to determine which residue(s) relative to the target DNA sequence is targeted by the particular deaminase. sgRNAs targeting non-template strands can also be tested to detect off-target effects of a particular base-editing protein, e.g. B. a Cas9 deaminase fusion protein to evaluate. In some embodiments, such gRNAs can be designed such that the mutated start codon is not base paired with the gRNA. The leader polynucleotides may include standard ribonucleotides, modified ribonucleotides (e.g., pseudouridine), ribonucleotide isomers, and/or ribonucleotide analogs. In some embodiments, the leader polynucleotide may include at least one detectable label. The detectable label can be a fluorophore (e.g. FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluors, Halo tags or a suitable fluorescent dye), a detection tag (e.g. biotin, digoxigenin and the like). , quantum dots or gold particles.
The leader polynucleotides can be chemically synthesized, enzymatically synthesized, or a combination thereof. For example, the guide RNA can be synthesized using standard phosphoramidite-based solid-phase synthesis methods. Alternatively, the guide RNA can be synthesized in vitro by operatively linking DNA encoding the guide RNA to a promoter control sequence recognized by a phage RNA polymerase. Examples of suitable phage promoter sequences include T7, T3, SP6 promoter sequences or variations thereof. In embodiments where the guide RNA comprises two separate molecules (e.g., crRNA and tracrRNA), the crRNA can be synthesized chemically and the tracrRNA can be synthesized enzymatically.
In some embodiments, a base editor system may include multiple leader polynucleotides, e.g. gRNAs. For example, the gRNAs can target one or more target loci (eg, at least 1 gRNA, at least 2 gRNA, at least 5 gRNA, at least 10 gRNA, at least 20 gRNA, at least 30 g RNA, at least 50 gRNA) contained in a basic editor system are. The multiple gRNA sequences can be arranged in tandem and are preferably separated by a direct repeat.
A DNA sequence encoding a leader RNA or leader polynucleotide can also be part of a vector. In addition, a vector may contain additional expression control sequences (eg, enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selectable marker sequences (eg, GFP or antibiotic resistance genes such as puromycin), origins of replication, and the like. A DNA molecule encoding a guide RNA can also be linear. A DNA molecule encoding a guide RNA or guide polynucleotide can also be circular.
In some embodiments, one or more components of a base editor system can be encoded by DNA sequences. Such DNA sequences can be incorporated together or separately into an expression system, e.g. B. a cell are introduced. For example, DNA sequences encoding a polynucleotide-programmable nucleotide binding domain and a guide RNA can be introduced into a cell, each DNA sequence can be part of a separate molecule (e.g., a vector encoding the polynucleotide programmable nucleotide binding domain, and a second vector containing the coding sequence of the guide RNA) or both may be part of the same molecule (e.g. a vector containing the coding (and regulatory) sequence for both the polynucleotide-programmable nucleotide binding domain as well as the guide RNA).
A lead polynucleotide may include one or more modifications to provide a nucleic acid with a new or improved characteristic. A lead polynucleotide may include a nucleic acid affinity tag. A leader polynucleotide can include a synthetic nucleotide, a synthetic nucleotide analog, nucleotide derivatives, and/or modified nucleotides.
In some embodiments, a gRNA or leader polynucleotide may include modifications. Modification can be made at any site of a gRNA or leader polynucleotide. More than one modification can be made to a single gRNA or leader polynucleotide. A gRNA or lead polynucleotide can be subjected to quality control after modification. In some embodiments, quality control may include PAGE, HPLC, MS, or any combination thereof
A modification of a gRNA or a leader polynucleotide can be a substitution, insertion, deletion, chemical modification, physical modification, stabilization, purification, or any combination thereof.
A gRNA or lead polynucleotide can also be labeled by 5'adenylate, 5'guanosine triphosphate cap, 5'N7-methylguanosine triphosphate cap, 5'triphosphate cap, 3'phosphate, 3'thiophosphate, 5'phosphate, 5' be modified. Thiophosphate, cis-syn thymidine dimer, trimers, C12 spacer, C3 spacer, C6 spacer, dspacer, PC spacer, rSpacer, spacer 18, spacer 9, 3′-3′ modifications, 5′-5 ′-Modifications, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, dual-biotin, PC-biotin, psoralen C2, psoralen C6 , TINA, 3′DABCYL, Black Hole Quencher 1, Black Hole Quencer 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker , thiol linker, 2′-deoxyribonucleoside analog purine, 2′-deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2′-O-methyl ribonucleoside analog, sugar-modified analogs, wobble/universal bases, fluorescent dye label, 2′- Fluorine RNA, 2'-O-methyl RNA, methyl phosphonate, phosphodiester DNA, phosphodiester RNA, phosphorothioate DNA, phosphorothioate RNA, UNA, pseudouridine 5'-triphosphate, 5'-methylcytidine-5'-triphosphate or a any combination of these.
In some embodiments, a modification is permanent. In other embodiments, a modification is temporary. In some embodiments, multiple modifications are made to a gRNA or lead polynucleotide. A gRNA or lead polynucleotide modification can alter physiochemical properties of a nucleotide, such as its conformation, polarity, hydrophobicity, chemical reactivity, base pair interactions, or any combination thereof.
The PAM sequence can be any PAM sequence known in the art. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NGN, NGT, NGCG, NGG, NGAN, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNNGATT, TATV or NAAAAC. Y is a pyrimidine; N is any nucleotide base; W is A or T
A modification can also be a phosphorothioate replacement. In some embodiments, a natural phosphodiester bond may be susceptible to rapid degradation by cellular nucleases and; modification of the internucleotide bond using phosphorothioate (PS) bond substitutes may be more stable to hydrolysis by cellular degradation. Modification can increase stability in a gRNA or lead polynucleotide. Modification can also enhance biological activity. In some embodiments, a phosphorothioate-enhanced RNA-gRNA can inhibit RNase A, RNase T1, calf serum nucleases, or any combination thereof. These properties may allow the use of PS-RNA-gRNAs in applications where exposure to nucleases in vivo or in vitro is likely. For example, phosphorothioate (PS) linkages can be introduced between the last 3-5 nucleotides at the 5' or '' end of a gRNA, which can inhibit exonuclease degradation. In some embodiments, phosphorothioate linkages can be added throughout an entire gRNA to reduce attack by endonucleases.
Protospacer Adjacent Motiv
The term "protospacer adant motif (PAM)" or PAM-like motif refers to a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the bacterial adaptive CRISPR immune system targets. In some embodiments, the PAM may be a 5' PAM (i.e. located upstream of the 5' end of the protospacer). In other embodiments, the PAM may be a 3' PAM (i.e. located downstream of the 5' end of the protospacer).
The PAM sequence is essential for target binding, but the exact sequence depends on a type of Cas protein.
A base editor provided herein may comprise a CRISPR protein-derived domain capable of binding a nucleotide sequence containing a canonical or non-canonical protospacer minor motif (PAM) sequence. A PAM site is a nucleotide sequence near a target polynucleotide sequence. Some aspects of the disclosure provide for base editors that include all or part of CRISPR proteins that have different PAM specificities. For example Cas9 proteins, such as Cas9 fromS. pyogenes(spCas9), typically require a canonical NGG-PAM sequence to bind a specific nucleic acid region, where the "N" in "NGG" is adenine (A), thymine (T), guanine (G), or cytosine (C). , and G is guanine. A PAM may be CRISPR protein specific and may differ between different base editors comprising different CRISPR protein derived domains. A PAM can be 5' or 3' to a target sequence. A PAM can precede or follow a target sequence. A PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. A PAM is often between 2-6 nucleotides long. Several PAM variants are described in Table 1 below.
In some embodiments, the PAM is NGC. In some embodiments, the NGC-PAM is recognized by a Cas9 variant. In some embodiments, the NGC-PAM variant comprises one or more amino acid substitutions selected from D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E and T1337R (collectively referred to as "MQKFRAER").
In some embodiments, the PAM is NGT. In some embodiments, the NGT-PAM is recognized by a Cas9 variant. In some embodiments, the NGT-PAM variant is generated by targeted mutations at one or more residues 1335, 1337, 1135, 1136, 1218 and/or 1219. In some embodiments, the NGT-PAM variant is generated by targeted mutations at one or more residues 1219, 1335, 1337, 1218. In some embodiments, the NGT-PAM variant is generated by targeted mutations at one or more residues 1135, 1136, 1218 , 1219 and 1335 generates targeted mutation sets provided in Table 2 and Table 3 below.
In some embodiments, the NGT-PAM variant is selected from variant 5, 7, 28, 31 or 36 in Tables 2 and 3. In some embodiments, the variants have improved NGT-PAM detection.
In some embodiments, the NGT-PAM variants have mutations at residues 1219, 1335, 1337, and/or 1218. In some embodiments, the NGT-PAM variant with mutations for enhanced recognition is selected from the variants provided in Table 4 below.
In some embodiments, base editors can be created with specificity for NGT-PAM, as provided in Table 5A below.
In some embodiments, the NGTN variant is variant 1. In some embodiments, the NGTN variant is variant 2. In some embodiments, the NGTN variant is variant 3. In some embodiments, the NGTN variant is variant 4. In some embodiments, the NGTN -Variant Variant 5 is. In some embodiments, the NGTN variant is variant 6.
In some embodiments, the Cas9 domain is a Cas9 domainStreptococcus pyogenes(SpCas9). In some embodiments, the SpCas9 domain is a nuclease-active SpCas9, a nuclease-inactive SpCas9 (SpCas9d), or a SpCas9 nickase (SpCas9n). In some embodiments, the SpCas9 comprises a D10X mutation or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid except D. In some embodiments, the SpCas9 comprises a D10A mutation or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence having an NGG, an NGA, or an NGCG PAM sequence. In some embodiments, the SpCas9 domain comprises one or more of a D1135X, an R1335X, and a T1337X mutation or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1135E, R1335Q, and T1337R mutation or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises a D1135E, an R1335Q, and a T1337R mutation or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises one or more of a D1135X, an R1335X, and a T1337X mutation or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1135V, an R1335Q, and a T1337R mutation or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises a D1135V, an R1335Q, and a T1337R mutation or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises one or more of a D1135X, a G1218X, an R1335X, and a T1337X mutation or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1135V, a G1218R, an R1335Q, and a T1337R mutation or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises a D1135V, a G1218R, an R1335Q, and a T1337R mutation or corresponding mutations in any of the amino acid sequences provided herein.
In some embodiments, Cas9 is a Cas9 variant with specificity for an altered PAM sequence. In some embodiments, the additional Cas9 variants and PAM sequences are described in Miller et al., Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat Biotechnology (2020). doi.org/10.1038/s41587-020-0412-8, the entirety of which is incorporated herein by reference. in some embodiments, a Cas9 variant has no specific PAM requirements. In some embodiments, a Cas9 variant, e.g. a SpCas9 variant has specificity for an NRNH-PAM where R is A or G and H is A, C or T. In some embodiments, the SpCas9 variant has specificity for a PAM sequence AAA, TAA, CAA, GAA, TAT, GAT, or CAC. In some embodiments, the SpCas9 variant includes an amino acid substitution at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1218, 1219, 1221, 1249, 1256, 1264, 1290, 1318, 13207, 1321, 1323, 1332, 1333, 1335, 1337 or 1339 as numbered in SEQ ID NO: 1, or a corresponding position thereof. In some embodiments, the SpCas9 variant includes an amino acid substitution at position 1114, 1135, 1218, 1219, 1221, 1249, 1320, 1321, 1323, 1332, 1333, 1335 or 1337 as numbered in SEQ ID NO: 1 or a corresponding position of that. In some embodiments, the SpCas9 variant includes an amino acid substitution at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1219, 1221, 1256, 1264, 1290, 1318, 1317, 1320, 1333, as numbered in SEQ ID NO: 1, or a corresponding position thereof. In some embodiments, the SpCas9 variant includes an amino acid substitution at position 1114, 1131, 1135, 1150, 1156, 1180, 1191, 1218, 1219, 1221, 1227, 1249, 1253, 1286, 1293, 1320, 1321, 13352, 1339 as numbered in SEQ ID NO: 1, or a corresponding position thereof. In some embodiments, the SpCas9 variant includes an amino acid substitution at position 1114, 1127, 1135, 1180, 1207, 1219, 1234, 1286, 1301, 1332, 1335, 1337, 1338, 1349 as in SEQ ID NO: 1 or numbered one corresponding position of it. Exemplary amino acid substitutions and PAM specificity of SpCas9 variants are shown in Tables 5B, 5C, 5D and 5E below.
In some embodiments, the Cas9 domains of any of the fusion proteins provided herein comprise an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% %, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to a Cas9 polypeptide described herein. In some embodiments, the Cas9 domains of any of the fusion proteins provided herein comprise the amino acid sequence of any Cas9 polypeptide described herein. In some embodiments, the Cas9 domains of any of the fusion proteins provided herein consist of the amino acid sequence of any Cas9 polypeptide described herein.
In some examples, a PAM recognized by a CRISPR protein-derived domain of a base editor disclosed herein may be provided to a cell on a separate oligonucleotide to an insert (e.g., an AAV insert) encoding the base editor become. In such embodiments, providing PAM on a separate oligonucleotide may allow for cleavage of a target sequence that otherwise could not be cleaved because no adjacent PAM is present on the same polynucleotide as the target sequence.
In one embodimentS. pyogenesCas9 (SpCas9) can be used as a CRISPR endonuclease for genomic engineering. However, others can also be used. In some embodiments, a different endonuclease can be used to target specific genomic targets. In some embodiments, synthetic SpCas9-derived variants with non-NGG PAM sequences can be used. In addition, other Cas9 orthologues from different species have been identified, and these "non-SpCas9s" can bind a variety of PAM sequences that may also be useful for the present disclosure. For example, the relatively large size of SpCas9 (approximately 4 kb coding sequence) may result in plasmids carrying the SpCas9 cDNA that cannot be efficiently expressed in a cell. Conversely, the coding sequence forStaphylococcus aureusCas9 (SaCas9) is approximately 1 kilobase shorter than SpCas9, potentially allowing efficient expression in a cell. Similar to SpCas9, the SaCas9 endonuclease is able to modify target genes in mammalian cells in vitro and in mice in vivo. In some embodiments, a Cas protein can target a different PAM sequence. For example, in some embodiments, a target gene may be adjacent to a Cas9 PAM, 5'-NGG. In other embodiments, other Cas9 orthologues may have different PAM requirements. For example, other PAMs like those ofS. thermophilus(5'-NNAGAA for CRISPR1 and 5'-NGGNG for CRISPR3) andNeisseria-Meningitis(5′-NNNNGATT) can also be found adjacent to a target gene.
In some embodiments for aS. pyogenesSystem, a target gene sequence can precede (i.e., be 5' of) a 5' NGG PAM, and a 20 nt leader RNA sequence can base-pair with an opposite strand to mediate Cas9 cleavage adjacent to a PAM. In some embodiments, a contiguous cut may be about 3 base pairs upstream from a PAM. In some embodiments, a contiguous cut may be about 10 base pairs upstream of a PAM. In some embodiments, a contiguous cut may be about 0-20 base pairs upstream of a PAM. For example, an adjacent cut may be next to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, or 30 base pairs upstream of a PAM. An adjacent cut can also be 1 to 30 base pairs downstream from a PAM. The sequences of exemplary SpCas9 proteins capable of binding a PAM sequence follow:
The amino acid sequence of an exemplary PAM-binding SpCas9 is as follows:
The amino acid sequence of an exemplary PAM-binding SpCas9n is as follows:
The amino acid sequence of an exemplary PAM-binding SpEQR Cas9 is as follows:
In the above sequence, residues E1134, Q1334 and R1336, which can be mutated from D1134, R1335 and T1336 to give a SpEQR Cas9, are underlined and in bold.
The amino acid sequence of an exemplary PAM-binding SpVQR-Cas9 is as follows:
In the above sequence, residues V1134, Q1334 and R1336, which can be mutated from D1134, R1335 and T1336 to give a SpVQR-Cas9, are underlined and in bold.
The amino acid sequence of an exemplary PAM-binding SpVRER Cas9 is as follows:
In the above sequence, residues V1134, R1217, Q1334 and R1336, which can be mutated from D1134, G1217, R1335 and T1336 to give a SpVRER-Cas9, are underlined and in bold.
In some embodiments, engineered SpCas9 variants are able to recognize protospacer adjacent motif (PAM) sequences flanked by a 3'-H (non-G-PAM) (see
In some embodiments, the Cas9 domain is a recombinant Cas9 domain. In some embodiments, the recombinant Cas9 domain is a SpyMacCas9 domain. In some embodiments, the SpyMacCas9 domain is a nuclease-active SpyMacCas9, a nuclease-inactive SpyMacCas9 (SpyMacCas9d), or a SpyMacCas9 nickase (SpyMacCas9n). In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SpyMacCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence having a NAA-PAM sequence.
The sequence of an exemplary Cas9 A homologue of Spy Cas9 inStreptococcus macacaewith native 5'-NAAN-3'-PAM specificity is known in the art and is described, for example, by Jakimo et al., (www.biorxiv.org/content/biorxiv/early/2018/09/27/429654.full . pdf) and is provided below. SpyMacCas9
In some embodiments, a variant Cas9 protein harbors H840A, P475A, W476A, N477A, D1125A, W1126A, and D1218A mutations such that the polypeptide has a reduced ability to cleave a target DNA or RNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g. a single-stranded target DNA) but retains the ability to bind a target DNA (e.g. a single-stranded target DNA). . As a further non-limiting example, in some embodiments, the variant Cas9 protein harbors D10A, H840A, P475A, W476A, N477A, D1125A, W1126A, and D1218A mutations such that the polypeptide has a reduced ability to cleave a has target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g. a single-stranded target DNA) but retains the ability to bind a target DNA (e.g. a single-stranded target DNA). . In some embodiments, if a variant Cas9 protein harbors W476A and W1126A mutations, or if the variant Cas9 protein harbors P475A, W476A, N477A, D1125A, W1126A, and D1218A mutations, the variant des Cas9 protein does not bind efficiently to a PAM sequence. Therefore, in some such cases, when such a variant of the Cas9 protein is used in a binding procedure, the method does not require a PAM sequence. In other words, when in some embodiments such a variant of the Cas9 protein is used in a binding method, the method may involve a guide RNA, but the method may be performed in the absence of a PAM sequence (and the specificity of the binding is therefore provided by the targeting segment of the guide RNA). Other residues can be mutated to achieve the above effects (i.e. inactivate one or the other nuclease part). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987 may be altered (i.e., substituted). Mutations other than alanine substitutions are also suitable.
In some embodiments, a CRISPR protein-derived domain of a base editor may comprise all or part of a Cas9 protein having a PAM canonical sequence (NGG). In other embodiments, a Cas9-derived domain of a base editor may use a non-canonical PAM sequence. Such sequences have been described in the prior art and would be apparent to those skilled in the art. For example, Cas9 domains that bind non-canonical PAM sequences have been identified in Kleinstiver, B.P., et al., "Engineered CRISPR-Cas9 nucleases with altered PAM specificities" Nature 523, 481-485 (2015); and Kleinstiver, B.P., et al., "Broadening the targeting range ofStaphylococcus aureusCRISPR-Cas9 by modifying PAM detection” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference.
Cas9 domains with reduced PAM exclusivity
Typically, Cas9 proteins, such as Cas9, are offS. pyogenes(spCas9), require a canonical NGG-PAM sequence to bind a specific nucleic acid region, where the "N" in "NGG" is adenosine (A), thymidine (T), or cytosine (C) and the G is guanosine. This can limit the ability to edit desired bases within a genome. In some embodiments, the base-editing fusion proteins provided herein may need to be placed in a precise location, such as in a region that includes a target base that is upstream of the PAM. See e.g. B. Komor, AC, et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference. Accordingly, in some embodiments, each of the fusion proteins provided herein may contain a Cas9 domain capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the prior art and would be apparent to those skilled in the art. For example, Cas9 domains that bind non-canonical PAM sequences have been identified in Kleinstiver, B.P., et al., "Engineered CRISPR-Cas9 nucleases with altered PAM specificities" Nature 523, 481-485 (2015); and Kleinstiver, B.P., et al., "Broadening the targeting range ofStaphylococcus aureusCRISPR-Cas9 by modifying PAM detection”natural biotechnology33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference.
High fidelity Cas9 domains
Some aspects of the disclosure provide high fidelity Cas9 domains. In some embodiments, high fidelity Cas9 domains are engineered Cas9 domains comprising one or more mutations that alter electrostatic interactions between the Cas9 domain and a sugar-phosphate backbone of a DNA compared to a corresponding wild-type Cas9 domain reduce. Without wishing to be bound by any particular theory, high-fidelity Cas9 domains that exhibit reduced electrostatic interactions with a sugar-phosphate backbone of DNA may have fewer off-target effects. In some embodiments, a Cas9 domain (e.g., a wild-type Cas9 domain) comprises one or more mutations that decrease the association between the Cas9 domain and a sugar-phosphate backbone of a DNA. In some embodiments, a Cas9 domain comprises one or more mutations that reduce the association between the Cas9 domain and a sugar-phosphate backbone of a DNA by at least 1%, at least 2%, at least 3%, at least 4%. at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% % or at least 70%.
In some embodiments, each of the Cas9 fusion proteins provided herein comprises one or more of an N497X, an R661X, a Q695X and/or a Q926X mutation or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid . In some embodiments, each of the Cas9 fusion proteins provided herein comprises one or more of an N497A, an R661A, a Q695A and/or a Q926A mutation or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the Cas9 domain comprises a D10A mutation or a corresponding mutation in any of the amino acid sequences provided herein. High fidelity Cas9 domains are known in the art and are obvious to those skilled in the art. For example, high fidelity Cas9 domains were described in Kleinstiver, B.P., et al. “High-fidelity CRISPR-Cas9 nucleases with no detectable off-target genome-wide effects.” Nature 529, 490-495 (2016); and Slaymaker, I.M., et al. "Rationally designed Cas9 nucleases with improved specificity."Science351, 84-88 (2015); the entire content of each is incorporated herein by reference.
In some embodiments, the modified Cas9 is a high fidelity Cas9 enzyme. In some embodiments, the high fidelity Cas9 enzyme is SpCas9(K855A), eSpCas9(1.1), SpCas9-HF1, or a hyperprecise Cas9 variant (HypaCas9). The modified Cas9 eSpCas9(1.1) contains alanine substitutions that weaken the interactions between the HNH/RuvC groove and the non-target DNA strand, preventing strand separation and cutting at off-target sites. Similarly, SpCas9-HF1 decreases off-target editing by alanine substitutions that disrupt Cas9's interactions with the DNA phosphate backbone. HypaCas9 contains mutations (SpCas9 N692A/M694A/Q695A/H698A) in the REC3 domain that enhance Cas9 proofreading and target discrimination. All three high-fidelity enzymes produce less off-target editing than wild-type Cas9.
An example high fidelity Cas9 is provided below.
High-fidelity Cas9 domain mutations relative to Cas9 are shown in bold and underlined.
Fusion proteins comprising a nuclear localization sequence (NLS).
In some embodiments, the fusion proteins provided herein further comprise one or more (e.g. 2, 3, 4, 5) nuclear targeting sequences, such as a nuclear localization sequence (NLS). In one embodiment, a two-part NLS is used. In some embodiments, an NLS comprises an amino acid sequence that facilitates import of a protein comprising an NLS into the nucleus (e.g., by nuclear transport). In some embodiments, each of the fusion proteins provided herein further comprises a nuclear localization sequence (NLS). In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the Cas9 domain. In some embodiments, the NLS is fused to the C-terminus of an nCas9 domain or a dCas9 domain. In some embodiments, the NLS is fused to the N-terminus of the deaminase. In some embodiments, the NLS is fused to the C-terminus of the deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises an amino acid sequence of any of the NLS sequences provided or referenced herein. Additional nuclear localization sequences are known in the art and would be apparent to those skilled in the art. For example, NLS sequences are described in Plank et al., PCT/EP2000/011690, the content of which is incorporated herein by reference for its disclosure of exemplary nuclear localization sequences. In some embodiments, an NLS comprises the amino acid sequence PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 95), KRTADGSEFESPKKKRKV (SEQ ID NO: 64), KRPAATKKAGQAKKKK (SEQ ID NO: 65), KKTELQTTNAENKTKKL (SEQ ID NO: 66), KRGINDRNFWRGENGRKTR (SEQ ID NO: 64). ID NO: 67), RKSGKIAAIVVKRPRKPKKKRKV (SEQ ID NO: 96) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 70).
In some embodiments, the NLS is present in a linker or the NLS is flanked by linkers, such as the linkers described herein. In some embodiments, the N-terminus or C-terminus NLS is a two-part NLS. A two-part NLS comprises two basic amino acid clusters separated by a relatively short spacer sequence (hence two-part - 2 parts, while one-part NLS are not). The NLS of nucleoplasmin, KR[PAATKKAGQA]KKKK (SEQ ID NO:65), is the prototype of the ubiquitous bipartite signal: two clusters of basic amino acids separated by a spacer of about 10 amino acids. The flow of an example two-part NLS follows:
In some embodiments, the fusion proteins of the invention do not include a linker sequence. In some embodiments, linker sequences are present between one or more of the domains or proteins.
It is understood that the fusion proteins of the present disclosure may include one or more additional features. For example, in some embodiments, the fusion protein may include inhibitors, cytoplasmic localization sequences, export sequences such as nuclear export sequences or other localization sequences, and sequence tags useful for solubilizing, purifying, or detecting the fusion proteins. Suitable protein tags provided herein include biotin carboxylase carrier protein (BCCP) tags, myc tags, calmodulin tags, FLAG tags, hemagglutinin (HA) tags, polyhistidine tags, also known as histidine tags denotes, but are not limited to, or His tags, Maltose Binding Protein (MBP) tags, Nus tags, Glutathione-S-Transferase (GST) tags, Green Fluorescent Protein (GFP) tags, Thioredoxin- Tags, S tags, soft tags (e.g. Softag 1, Softag 3), strep tags, biotin ligase tags, FlAsH tags, V5 tags and SBP tags. Other suitable sequences will be apparent to those skilled in the art. In some embodiments, the fusion protein includes one or more His tags.
A vector encoding a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs) can be used. For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs can be used. A CRISPR enzyme can include the NLSs at or near the amino terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs at or near the carboxy terminus, or any combination of these ( e.g., one or more NLS at the amino terminus and one or more NLS at the carboxy terminus). When more than one NLS is present, each can be selected independently of others, such that a single NLS can be present in more than one copy and/or in combination with one or more other NLS present in one or more copies.
CRISPR enzymes used in the methods can include about 6 NLS. An NLS is considered near the N- or C-terminus if the nearest amino acid to the NLS is within about 50 amino acids along a polypeptide chain from the N- or C-terminus, e.g. B. within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40 or 50 amino acids.
Nucleobase edit domain
Described herein are base editors comprising a fusion protein containing a polynucleotide-programmable nucleotide binding domain and a nucleobase editing domain (e.g., a deaminase domain). The base editor can be programmed to edit one or more bases in a target polynucleotide sequence by interacting with a guide polynucleotide capable of recognizing the target sequence. Once the target sequence has been recognized, the base editor is anchored to the polynucleotide where editing is to occur, and the deaminase domain components of the base editor can then edit a target base.
In some embodiments, the nucleobase editing domain includes a deaminase domain. As specifically described herein, the deaminase domain includes an adenosine deaminase. In some embodiments, the terms "adenine deaminase" and "adenosine deaminase" can be used interchangeably. Details of nucleobase-editing proteins are described in International PCT Application Nos. PCT/2017/045381 (WO2018/027078) and PCT/US2016/058344 (WO2017/070632), each of which is incorporated herein by reference in its entirety. See also Komor, A.C., et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, N.M., et al., "Programmable base editing of A⋅T to G⋅C in genomic DNA without DNA cleavage" Nature 551, 464-471 (2017); and Komor, A.C., et al., "Improved inhibition of base excision repair and bacteriophage Mu-Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity" Science Advances 3:eaao4774 (2017), the The entirety of which is hereby incorporated by reference.
Processing from A to G
In some embodiments, a base editor described herein may include a deaminase domain that includes an adenosine deaminase. Such a base editor adenosine deaminase domain may facilitate editing of an adenine (A) nucleobase to a guanine (G) nucleobase by deaminating the A to form inosine (I) which has G base-pairing properties. Adenosine deaminase is capable of deaminating (i.e. removing an amine group) from adenine a deoxyadenosine residue in deoxyribonucleic acid (DNA).
In some embodiments, the nucleobase editors provided herein can be made by fusing one or more protein domains together, creating a fusion protein. In certain embodiments, the fusion proteins provided herein include one or more features that enhance the base-editing activity (e.g., efficiency, selectivity, and specificity) of the fusion proteins. For example, the fusion proteins provided herein may include a Cas9 domain that exhibits reduced nuclease activity. In some embodiments, the fusion proteins provided herein may have a Cas9 domain that lacks nuclease activity (dCas9) or a Cas9 domain that cleaves one strand of a double-stranded DNA molecule termed Cas9 nickase (nCas9). Without wishing to be bound by any particular theory, the presence of the catalytic moiety (e.g. H840) maintains the activity of Cas9 to cleave the non-edited (e.g. non-deaminated) strand containing a T which faces Target-A. Mutation of the Cas9 catalytic residue (eg, D10 to A10) prevents cleavage of the edited strand containing the target A residue. Such Cas9 variants are capable of generating a single-strand DNA break (nick) at a specific site based on the gRNA-defined target sequence, resulting in repair of the unprocessed strand, ultimately resulting in a T to C change on the non-strand leads -processed strand. In some embodiments, an A-to-G base editor further comprises an inosine base excision repair inhibitor, such as a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine-specific nuclease. Without wishing to be bound by any particular theory, the UGI domain or catalytically inactive inosine-specific nuclease can inhibit or prevent base excision repair of a deaminated adenosine residue (e.g., inosine), which can improve the activity or efficiency of the base editor.
A base editor that includes an adenosine deaminase can act on any polynucleotide, including DNA, RNA, and DNA-RNA hybrids. In certain embodiments, a base editor comprising an adenosine deaminase can deaminate a target A of a polynucleotide comprising RNA. For example, the base editor may comprise an adenosine deaminase domain capable of deaminating a target A of an RNA polynucleotide and/or a DNA-RNA hybrid polynucleotide. In one embodiment, an adenosine deaminase incorporated into a base editor comprises all or part of the adenosine deaminase acting on RNA (ADAR, e.g., ADAR1 or ADAR2). In another embodiment, an adenosine deaminase incorporated into a base editor comprises all or part of tRNA-acting adenosine deaminase (ADAT). A base editor comprising an adenosine deaminase domain may also be capable of deamination of an A nucleobase of a DNA polynucleotide. In one embodiment, an adenosine deaminase domain of a base editor comprises all or part of an ADAT comprising one or more mutations that enable the ADAT to deaminate a target A in DNA. For example, the base editor may include all or part of an ADATEscherichia coli(EcTadA) comprising one or more of the following mutations: D108N, A106V, D147Y, E155V, L84F, H123Y, I157F or a corresponding mutation in another adenosine deaminase.
The adenosine deaminase can be obtained from any suitable organism (e.g.E coli). In some embodiments, the adenine deaminase is a naturally occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). The corresponding residue in each homologous protein can e.g. B. be identified by sequence alignment and determination of homologous residues. The mutations in any naturally occurring adenosine deaminase (e.g. with homology to ecTadA) corresponding to one of the mutations described herein (e.g. one of the mutations identified in ecTadA) can be generated accordingly.
Adenosin-Deaminasen
In some embodiments, a base editor described herein may include a deaminase domain that includes an adenosine deaminase. Such a base editor adenosine deaminase domain may facilitate editing of an adenine (A) nucleobase to a guanine (G) nucleobase by deaminating the A to form inosine (I) which has G base-pairing properties. Adenosine deaminase is capable of deaminating (i.e. removing an amine group) from adenine a deoxyadenosine residue in deoxyribonucleic acid (DNA).
In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine in a deoxyadenosine residue of DNA. In some embodiments, the adenine deaminase is a naturally occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). One skilled in the art will be able to identify the corresponding residue in any homologous protein, e.g. B. by sequence alignment and determination of homologous residues. Accordingly, one skilled in the art would be able to generate mutations in any naturally occurring adenosine deaminase (e.g. having homology to ecTadA) corresponding to any of the mutations described herein, e.g. B. any of the mutations identified in ecTadA. In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, adenosine deaminase is offEscherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, orBacillus subtilis. In some embodiments, adenosine deaminase is offE coli.
The invention provides adenosine deaminase variants that have increased efficiency (>50-60%) and specificity. In particular, the adenosine deaminase variants described herein are more likely to edit a desired base within a polynucleotide and less likely to edit bases that are not intended to be altered (i.e., "spectators").
In certain embodiments, the TadA is one of the TadA described in PCT/US2017/045381 (WO 2018/027078), which is incorporated herein by reference in its entirety.
In some embodiments, the nucleobase editors of the invention are adenosine deaminase variants comprising an alteration in the following sequence:
In certain embodiments, the fusion proteins comprise a single (e.g. provided as a monomer) TadA*8 variant. In some embodiments, the TadA*8 is linked to a Cas9 nickase. In some embodiments, the fusion proteins of the invention comprise, as a heterodimer, a wild-type TadA (TadA(wt)) linked to a TadA*8 variant. In other embodiments, the fusion proteins of the invention comprise, as a heterodimer, a TadA*7.10 linked to a TadA*8 variant. In some embodiments, the base editor is ABE8 comprising a TadA*8 variant monomer. In some embodiments, the base editor is ABE8 comprising a heterodimer of a TadA*8 variant and a TadA(wt). In some embodiments, the base editor is ABE8 comprising a heterodimer of a TadA*8 variant and TadA*7.10. In some embodiments, the base editor is ABE8 comprising a heterodimer of a TadA*8 variant. In some embodiments, the TadA*8 variant from Table 7 is selected. In some embodiments, the ABE8 is selected from Table 7. The relevant sequences follow:
Wild-type TadA (TadA(Wt)) or "the TadA reference sequence"
In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% %, at least 98%, at least 99% or at least 99.5% identical to any of the amino acid sequences given in any of the adenosine deaminases provided herein. It is understood that the adenosine deaminases provided herein may comprise one or more mutations (e.g. any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identity plus any of the mutations described herein or combinations thereof. In some embodiments, the adenosine deaminase comprises an amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50 or more mutations compared to a reference sequence or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues compared to any of the amino acid sequences known in the art or described herein.
In some embodiments, the TadA deaminase is full-lengthE coliTadA deaminase. For example, in certain embodiments, adenosine deaminase comprises the amino acid sequence:
However, it should be noted that additional adenosine deaminases useful in the present application will be apparent to those skilled in the art and are within the scope of this disclosure. For example, adenosine deaminase may be a homologue of adenosine deaminase acting on tRNA (ADAT). Without limitation, the amino acid sequences of exemplary AD AT homologues include the following:
An embodiment ofE coliTadA (ecTadA) includes the following:
In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, adenosine deaminase is offEscherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, orBacillus subtilis. In some embodiments, adenosine deaminase is offE coli.
In one embodiment, a fusion protein of the invention comprises a wild-type TadA linked to TadA7.10 linked to Cas9 nickase. In certain embodiments, the fusion proteins comprise a single TadA7.10 domain (e.g. provided as a monomer). In other embodiments, the ABE7.10 editor includes TadA7.10 and TadA(wt), which can form heterodimers.
In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% %, at least 98%, at least 99% or at least 99.5% identical to any of the amino acid sequences given in any of the adenosine deaminases provided herein. It is understood that the adenosine deaminases provided herein may comprise one or more mutations (e.g. any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identity plus any of the mutations described herein or combinations thereof. In some embodiments, the adenosine deaminase comprises an amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50 or more mutations compared to a reference sequence or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues compared to any of the amino acid sequences known in the art or described herein.
It is understood that any of the mutations provided herein (e.g. based on the TadA reference sequence) can be introduced into other adenosine deaminases, such as e.gE coliTadA (ecTadA),S aureusTadA (saTadA) or other adenosine deaminases (e.g. bacterial adenosine deaminases). Those skilled in the art will appreciate that other deaminases can be similarly targeted to identify homologous amino acid residues, which can be mutated as provided herein. Thus, any of the mutations identified in the TadA reference sequence can be made in other adenosine deaminases (e.g., ecTada) that have homologous amino acid residues. It should also be noted that any of the mutations provided herein can be made individually or in any combination in the TadA reference sequence or another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises a D108X mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises a D108G, D108N, D108V, D108A, or D108Y mutation or a corresponding mutation in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises an A106X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises an A106V mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, wild-type TadA or ecTadA).
In some embodiments, the adenosine deaminase comprises an E155X mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where the presence of X indicates any amino acid other than the corresponding wild-type amino acid adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E155D, E155G, or E155V mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises a D147X mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where the presence of X indicates any amino acid other than the corresponding wild-type amino acid adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D147Y mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA).
In some embodiments, the adenosine deaminase comprises an A106X, E155X, or D147X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X is any amino acid other than the corresponding amino acid in indicates wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E155D, E155G, or E155V mutation. In some embodiments, the adenosine deaminase comprises a D147Y.
For example, an adenosine deaminase may contain a D108N, an A106V, an E155V, and/or a D147Y mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). In some embodiments, an adenosine deaminase comprises the following group of mutations (groups of mutations are separated by a ";") in the TadA reference sequence or corresponding mutations in another adenosine deaminase (e.g., ecTadA): D108N and A106V ; D108N and E155V; D108N and D147Y; A106V and E155V; A106V and D147Y; E155V and D147Y; D108N, A106V and E155V; D108N, A106V and D147Y; D108N, E155V and D147Y; A106V, E155V and D147Y; and D108N, A106V, E155V and D147Y. However, it should be noted that any combination of corresponding mutations provided herein in an adenosine deaminase (e.g. ecTadA) can be made.
In some embodiments, the adenosine deaminase comprises one or more of H8X, T17X, L18X, W23X, L34X, W45X, R51X, A56X, E59X, E85X, M94X, I95X, V102X, F104X, A106X, R107X, D108X, K110X, M118X, N127X, A138X, F149X, M151X, R153X, Q154X, I156X and/or K157X mutation in the TadA reference sequence or one or more corresponding mutations in another adenosine deaminase (e.g. ecTadA), wherein the presence of X indicates any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H8Y, T17S, L18E, W23L, L34S, W45L, R51H, A56E or A56S, E59G, E85K or E85G, M94L, I95L, V102A, F104L, A106V, R107C, or R107H or R107P, D108G or D108N or D108V or D108A or D108Y, K110I, M118K, N127S, A138V, F149Y, M151V, R153C, Q154L, I156D and/or K157R mutation in the TadA reference sequence or one or more corresponding mutations in another adenosine deaminase (e.g. ecTadA).
In some embodiments, the adenosine deaminase comprises one or more of an H8X, D108X, and/or N127X mutation in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid. In some embodiments, the adenosine deaminase comprises one or more of an H8Y, D108N, and/or N127S mutation in the TadA reference sequence or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises one or more of the mutations H8X, R26X, M61X, L68X, M70X, A106X, D108X, A109X, N127X, D147X, R152X, Q154X, E155X, K161X, Q163X and/or T166X in the reference TadA sequence , or one or more corresponding mutations in another adenosine deaminase (e.g. ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H8Y, R26W, M61I, L68Q, M70V, A106T, D108N, A109T, N127S, D147Y, R152C, Q154H or Q154R, E155G or E155V or E155D, K161Q, Q163H and/or or T166P - Mutation in the TadA reference sequence or one or more corresponding mutations in another adenosine deaminase (e.g. ecTadA).
In some embodiments, the adenosine deaminase comprises one, two, three, four, five or six mutations selected from the group consisting of H8X, D108X, N127X, D147X, R152X and Q154X in the TadA reference sequence or a corresponding mutation or mutations in another adenosine - deaminase (e.g. ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven or eight mutations selected from the group consisting of H8X, M61X, M70X, D108X, N127X, Q154X, E155X and Q163X in the TadA reference - Sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g. ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four or five mutations selected from the group consisting of H8X, D108X, N127X, E155X and T166X in the TadA reference sequence or a corresponding mutation or mutations in another adenosine deaminase (e.g. B. ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.
In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8X, A106X, D108X mutation, or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8X, R26X, L68X, D108X, N127X, D147X, and E155X, or a corresponding mutation or mutations in another Adenosine deaminase, wherein X indicates the presence of any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four or five mutations selected from the group consisting of H8X, D108X, A109X, N127X and E155X in the TadA reference sequence or a corresponding mutation or mutations in another adenosine deaminase (e.g. B. ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.
In some embodiments, the adenosine deaminase comprises one, two, three, four, five or six mutations selected from the group consisting of H8Y, D108N, N127S, D147Y, R152C and Q154H in the TadA reference sequence or a corresponding mutation or mutations in another adenosine deaminase (e.g. ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven or eight mutations selected from the group consisting of H8Y, M61I, M70V, D108N, N127S, Q154R, E155G and Q163H in the TadA reference sequence or one corresponding mutation or mutations in another adenosine deaminase (e.g. ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four or five mutations selected from the group consisting of H8Y, D108N, N127S, E155V and T166P in the TadA reference sequence or a corresponding mutation or mutations in another adenosine deaminase (e.g. B. ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, five or six mutations selected from the group consisting of H8Y, A106T, D108N, N127S, E155D and K161Q in the TadA reference sequence or a corresponding mutation or mutations in another adenosine deaminase (e.g. ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8Y, R26W, L68Q, D108N, N127S, D147Y, and E155V in the TadA reference sequence. or a corresponding mutation or mutations in another adenosine deaminase (e.g. ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8Y, D108N, A109T, N127S, and E155G in the TadA reference sequence or a corresponding mutation or mutations in another adenosine deaminase (e.g. B. ecTadA).
Any of the mutations provided herein and any additional mutations (e.g. based on the ecTadA amino acid sequence) may be introduced into any other adenosine deaminase. Any of the mutations provided herein can be made individually or in any combination in the TadA reference sequence or another adenosine deaminase (e.g. ecTadA).
Details on A to G nucleobase editing proteins are given in PCT International Application No. PCT/2017/045381 (WO2018/027078) and Gaudelli, N.M., et al., "Programmable base editing of A⋅T to G ⋅C in genomic DNA without DNA cleavage” Nature, 551, 464-471 (2017), the entire contents of which are hereby incorporated by reference.
In some embodiments, the adenosine deaminase comprises one or more corresponding mutations in another adenosine deaminase (eg, ecTadA). In some embodiments, the adenosine deaminase comprises a D108N, D108G, or D108V mutation in the TadA reference sequence, or corresponding mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises an A106V and D108N mutation in the TadA reference sequence or corresponding mutations in another adenosine deaminase (eg, ecTadA). In some embodiments, the adenosine deaminase comprises R107C and D108N mutations in the TadA reference sequence or corresponding mutations in another adenosine deaminase (eg, ecTadA). In some embodiments, the adenosine deaminase comprises an H8Y, D108N, N127S, D147Y, and Q154H mutation in the TadA reference sequence or corresponding mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises an H8Y, D108N, N127S, D147Y, and E155V mutation in the TadA reference sequence or corresponding mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises a D108N, D147Y, and E155V mutation in the TadA reference sequence or corresponding mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises an H8Y, D108N, and N127S mutation in the TadA reference sequence, or corresponding mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises an A106V, D108N, D147Y, and E155V mutation in the TadA reference sequence or corresponding mutations in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises one or more of a S2X, H8X, I49X, L84X, H123X, N127X, I156X, and/or K160X mutation in the TadA reference sequence or one or more corresponding mutations in another Adenosine deaminase, where the presence of X denotes any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of S2A, H8Y, I49F, L84F, H123Y, N127S, I156F and/or K160S mutations in the TadA reference sequence or one or more corresponding mutations in another adenosine deaminase (e.g. ecTadA). .
In some embodiments, the adenosine deaminase comprises an L84X mutant adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an L84F mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA).
In some embodiments, the adenosine deaminase comprises an H123X mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises an H123Y mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA).
In some embodiments, the adenosine deaminase comprises an I156X mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises an I156F mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA).
In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six or seven mutations selected from the group consisting of L84X, A106X, D108X, H123X, D147X, E155X and I156X in the TadA reference sequence or a corresponding mutation or mutations in another adenosine deaminase (e.g. ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of S2X, I49X, A106X, D108X, D147X, and E155X in the TadA reference sequence or a corresponding mutation or mutations in another adenosine - deaminase (e.g. ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four or five mutations selected from the group consisting of H8X, A106X, D108X, N127X and K160X in the TadA reference sequence or a corresponding mutation or mutations in another adenosine deaminase (e.g. B. ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.
In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six or seven mutations selected from the group consisting of L84F, A106V, D108N, H123Y, D147Y, E155V and I156F in the TadA reference sequence or a corresponding mutation or mutations in another adenosine deaminase (e.g. ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of S2A, I49F, A106V, D108N, D147Y, and E155V in the TadA reference sequence.
In some embodiments, the adenosine deaminase comprises one, two, three, four or five mutations selected from the group consisting of H8Y, A106T, D108N, N127S and K160S in the TadA reference sequence or a corresponding mutation or mutations in another adenosine deaminase (e.g. B. ecTadA).
In some embodiments, the adenosine deaminase comprises one or more of an E25X, R26X, R107X, A142X, and/or A143X mutation in the TadA reference sequence or one or more corresponding mutations in another adenosine deaminase (e.g .ecTadA), where the presence of X denotes any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of E25M, E25D, E25A, E25R, E25V, E25S, E25Y, R26G, R26N, R26Q, R26C, R26L, R26K, R107P, R107K, R107A, R107N, R107W, R107H, R107S- , A142N, A142D, A142G, A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q and/or A143R mutation in the TadA reference sequence or one or more corresponding mutations in another adenosine deaminase (e.g. ecTadA). In some embodiments, the adenosine deaminase comprises one or more mutations described herein that correspond to the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises an E25X mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises an E25M, E25D, E25A, E25R, E25V, E25S, or E25Y mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (e.g. ecTadA).
In some embodiments, the adenosine deaminase comprises an R26X mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises an R26G, R26N, R26Q, R26C, R26L, or R26K mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises an R107X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises an R107P, R107K, R107A, R107N, R107W, R107H, or R107S mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises an A142X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises an A142N, A142D, A142G mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA).
In some embodiments, the adenosine deaminase comprises an A143X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises an A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q, and/or A143R mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase ( e.g. ecTadA).
In some embodiments, the adenosine deaminase comprises one or more of an H36X, N37X, P48X, I49X, R51X, M70X, N72X, D77X, E134X, S146X, Q154X, K157X, and/or K161X Mutation in the TadA reference sequence or one or more corresponding mutations in another adenosine deaminase (e.g. ecTadA), where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the mutations H36L, N37T, N37S, P48T, P48L, I49V, R51H, R51L, M70L, N72S, D77G, E134G, S146R, S146C, Q154H, K157N and/or K161T TadA reference sequence or one or more corresponding mutations in another adenosine deaminase (e.g. ecTadA).
In some embodiments, the adenosine deaminase comprises an H36X mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises an H36L mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA).
In some embodiments, the adenosine deaminase comprises an N37X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises an N37T or N37S mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises a P48X mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises a P48T or P48L mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (eg, ecTadA).
In some embodiments, the adenosine deaminase comprises an R51X mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase, where X indicates a different amino acid than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R51H or R51L mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises an S146X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises an S146R or S146C mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises a K157X mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises a K157N mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA).
In some embodiments, the adenosine deaminase comprises a P48X mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises a P48S, P48T, or P48A mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises an A142X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises an A142N mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA).
In some embodiments, the adenosine deaminase comprises a W23X mutation in the TadA reference sequence or a corresponding mutation in another adenosine deaminase (eg, ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises a W23R or W23L mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises an R152X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X is any amino acid other than the corresponding amino acid in the wild-type adenosine indicates deaminase. In some embodiments, the adenosine deaminase comprises an R152P or R52H mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In one embodiment, the adenosine deaminase may include mutations H36L, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N. In some embodiments, the adenosine deaminase comprises the following combination of mutations relative to the TadA reference sequence, where each mutation of a combination is separated by a "_" and each combination of mutations is enclosed in parentheses:
(A106V_D108N), (R107C_D108N), (H8Y_D108N_N127S_D147Y_Q154H), (H8Y_D108N_N127S_D147Y_E155V), (D108N_D147Y_E155V), (H8Y_D108N_N127S), (H8Y_D108N_N127S_D147Y_Q154H), (A106V_D108N_D147Y_E155V), (D108Q_D147Y_E155V), (D108M_D147Y_E155V), (D108L_D147Y_E155V), (D108K_D147Y_E155V), (D108I_D147Y_E155V), (D108F_D147Y_E155V), (A106V_D108N_D147Y), (A106V_D108M_D147Y_E155V), (E59A_A106V_D108N_D147Y_E155V),
(E59A cat dead A106V_D108N_D147Y_E155V),
(L84F_A106V_D108N_H123Y_D147Y_E155V_I156Y), (L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), (E25G_R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), (E25D_R26G_L84F_A106V_R107K_D108N_H123Y_A142N_A143G_D147Y_E155V_I156F), (R26Q_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), (E25M_R26G_L84F_A106V_R107P_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), (R26C_L84F_A106V_R107H_D108N_H123Y_A142N_D147Y_E155V_I156F), (L84F_A106V_D108N_H123Y_A142N_A143L_D147Y_E155V_I156F), (R26G_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), (E25A_R26G_L84F_A106V_R107N_D108N_H123Y_A142N_A143E_D147Y_E155V_I156F), (R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), (A106V_D108N_A142N_D147Y_E155V), (R26G_A106V_D108N_A142N_D147Y_E155V), (E25D_R26G_A106V_R107K_D108N_A142N_A143G_D147Y_E155V), (R26G_A106V_D108N_R107H_A142N_A143D_D147Y_E155V), (E25D_R26G_A106V_D108N_A142N_D147Y_E155V), (A106V_R107K_D108N_A142N_D147Y_E155V), (A106V_D108N_A142N_A143G_D147Y_E155V), (A106V_D108N_A142N_A143L_D147Y_E155V), (H36L_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), (N37T_P48T_M70L_L84F_A106V_D108N_H123Y_D147Y_I49V_E155V_I156F), (N37S_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K161T), (H36L_L84F_A106V_D108N_H123Y_D147Y_Q154H_E155V_I156F), (N72S_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F), (H36L_P48L_L84F_A106V_D108N_H123Y_E134G_D147Y_E155V_I156F), (H36L_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K157N), (H36L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F), (L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T), (N37S_R51H_D77G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (R51L_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K157N), (D24G_Q71R_L84F_H96L_A106V_D108N_H123Y_D147Y_E155V_I156F_K160E), (H36L_G67V_L84F_A106V_D108N_H123Y_S146T_D147Y_E155V_I156F), (Q71L_L84F_A106V_D108N_H123Y_L137M_A143E_D147Y_E155V_I156F), (E25G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L), (L84F_A91T_F104I_A106V_D108N_H123Y_D147Y_E155V_I156F), (N72D_L84F_A106V_D108N_H123Y_G125A_D147Y_E155V_I156F), (P48S_L84F_S97C_A106V_D108N_H123Y_D147Y_E155V_I156F), (W23G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (D24G_P48L_Q71R_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L), (L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), (H36L_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N), (N37S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156TF_K161), (L84F_A106V_D108N_D147Y_E155V_I156F), (R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K161T), (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K161T), (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K160E_K161T), (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K160E), (R74Q_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (R74A_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (R74Q_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (L84F_R98Q_A106V_D108N_H123Y_D147Y_E155V_I156F), (L84F_A106V_D108N_H123Y_R129Q_D147Y_E155V_I156F), (P48S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), (P48S_A142N), (P48T_I49V_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F_L157N), (P48T_I49V_A142N), (H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), (H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F (H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), (H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152H_E155V_I156F_K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_R152P_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T), (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155V_I156F_K157N).
In certain embodiments, the fusion proteins provided herein include one or more features that enhance the base-editing activity of the fusion proteins. For example, any of the fusion proteins provided herein may include a Cas9 domain that exhibits reduced nuclease activity. In some embodiments, each of the fusion proteins provided herein may have a Cas9 domain without nuclease activity (dCas9) or a Cas9 domain that cleaves one strand of a double-stranded DNA molecule termed Cas9 nickase (nCas9).
In some embodiments, the adenosine deaminase is TadA*7.10. In some embodiments, TadA*7.10 includes at least one change. In certain embodiments, TadA*7.10 includes one or more of the following changes or additional changes to TadA*7.10: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R. The change Y123H is also referred to herein as H123H (the change H123Y in TadA*7.10 reverted to Y123H (wt)). In other embodiments, the TadA*7.10 includes a combination of changes selected from the group of: Y147T + Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. In certain embodiments, an adenosine deaminase variant comprises a deletion of the C-terminus beginning at residue 149, 150, 151, 152, 153, 154, 155, 156 and 157.
In other embodiments, a base editor of the invention is a monomer comprising an adenosine deaminase variant (e.g. TadA*8) comprising one or more of the following changes: Y147T, Y147R, Q154S, Y123H, V82S, T166R and/or Q154R relative to TadA7.10 or the TadA reference sequence. In other embodiments, the adenosine deaminase variant (TadA*8) is a monomer comprising a combination of changes selected from the group of: Y147T + Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. In other embodiments, a base editor is a heterodimer comprising a wild-type adenosine deaminase and a variant adenosine deaminase (eg, TadA*8) comprising one or more of the following alterations Y147T, Y147R, Q154S, Y123H, V82S, T166R and/or Q154R. In other embodiments, the base editor is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (eg, TadA*8) comprising a combination of changes selected from the group consisting of: Y147T + Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.
In one embodiment, an adenosine deaminase is a TadA*8 comprising or consisting essentially of the following sequence or a fragment thereof having adenosine deaminase activity:
In some embodiments, the TadA*8 is an abbreviated one. In some embodiments, the truncated TadA*8 is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N- terminal amino acid residues relative to full-length TadA*8. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C- terminal amino acid residues relative to full-length TadA*8. In some embodiments, the adenosine deaminase variant is a full-length TadA*8.
In einigen Erzätigungen ist TadA*8 TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA* 8.11 , TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, TadA *8.24.
In one embodiment, a fusion protein of the invention comprises a wild-type TadA linked to a variant adenosine deaminase described herein (e.g., TadA*8) linked to Cas9 nickase. In certain embodiments, the fusion proteins comprise a single TadA*8 domain (e.g. provided as a monomer). In other embodiments, the base editor includes TadA*8 and TadA(wt), which can form heterodimers. Sample sequences are as follows:
In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% %, at least 98%, at least 99% or at least 99.5% identical to any of the amino acid sequences given in any of the adenosine deaminases provided herein. It is understood that the adenosine deaminases provided herein may comprise one or more mutations (e.g. any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identity plus any of the mutations described herein or combinations thereof. In some embodiments, the adenosine deaminase comprises an amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50 or more mutations compared to a reference sequence or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues compared to any of the amino acid sequences known in the art or described herein.
In certain embodiments, a TadA*8 comprises one or more mutations at any of the following boldfaced positions. In other embodiments, a TadA*8 comprises one or more mutations at any of the positions shown underlined:
For example, TadA*8 includes changes at amino acid position 82 and/or 166 (e.g. V82S, T166R) alone or in combination with one or more of the following Y147T, Y147R, Q154S, Y123H and/or Q154R. In certain embodiments, a combination of changes is selected from the group consisting of: Y147T + Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.
In some embodiments, the adenosine deaminase is TadA*8, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:
In some embodiments, TadA*8 is truncated. In some embodiments, the truncated TadA*8 is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N- terminal amino acid residues relative to full-length TadA*8. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C- terminal amino acid residues relative to full-length TadA*8. In some embodiments, the adenosine deaminase variant is a full-length TadA*8.
In one embodiment, a fusion protein of the invention comprises a wild-type TadA linked to a variant adenosine deaminase described herein (e.g., TadA*8) linked to Cas9 nickase. In certain embodiments, the fusion proteins comprise a single TadA*8 domain (e.g. provided as a monomer). In other embodiments, the base editor includes TadA*8 and TadA(wt), which can form heterodimers.
Additional domains
A base editor as described herein may include any domain that helps facilitate nucleobase editing, modification, or alternation of a nucleobase of a polynucleotide. In some embodiments, a base editor includes a polynucleotide-programmable nucleotide binding domain (e.g., Cas9), a nucleobase editing domain (e.g., deaminase domain), and one or more additional domains. In some embodiments, the additional domain may facilitate enzymatic or catalytic functions of the base editor, binding functions of the base editor, or be inhibitors of cellular machinery (e.g., enzymes) that might interfere with the desired base editing outcome. In some embodiments, a base editor may include a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain.
In some embodiments, a base editor may include a uracil glycosylase inhibitor (UGI) domain. In some embodiments, the cellular DNA repair response to the presence of U:G heteroduplex DNA may be responsible for a decrease in nucleobase editing efficiency in cells. In such embodiments, uracil-DNA glycosylase (UDG) can catalyze the removal of U from DNA in cells, which can initiate a base excision repair (BER), mostly resulting in reversion of the U:G pair to a C:G pair . In such embodiments, BER can be inhibited in base editors comprising one or more domains that bind the single strand, block the edited base, inhibit UGI, inhibit BER, protect the edited base, and/or promote repair of the unedited strand. Thus, this disclosure contemplates a base editor fusion protein comprising a UGI domain.
In some embodiments, a base editor comprises as a domain all or part of a double-strand break (DSB)-binding protein. For example, a DSB-binding protein may include a bacteriophage Mu Gam protein that can bind to the ends of DSBs and protect them from degradation. See Komor, A.C., et al., "Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity", Science Advances 3:eaao4774 (2017), in full the contents of which are hereby incorporated by reference.
Additionally, in some embodiments, a Gam protein can be fused to an N-terminus of a base editor. In some embodiments, a Gam protein can be fused to a C-terminus of a base editor. Bacteriophage Mu's Gam protein can bind to the ends of double-strand breaks (DSBs) and protect them from degradation. In some embodiments, using Gam to bind the free ends of DSBs may reduce indel formation during the base editing process. In some embodiments, the 174 residue Gam protein is fused to the N-terminus of the base editor. See. Komor, A.C., et al., "Improved inhibition of base excision repair and bacteriophage Mu-Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity" Science Advances 3:eaao4774 (2017). In some embodiments, a mutation or mutations can change the length of a basic editor domain relative to a wild-type domain. For example, a deletion of at least one amino acid in at least one domain can reduce the length of the base editor. In another case, a mutation or mutations do not change the length of a domain relative to a wild-type domain. For example, substitution(s) in any domain does not change the length of the base editor.
In some embodiments, a base editor may comprise all or part of a nucleic acid polymerase (NAP) as a domain. For example, a basic editor may include all or part of a eukaryotic NAP. In some embodiments, a NAP or portion thereof incorporated into a base editor is a DNA polymerase. In some embodiments, a NAP or portion thereof incorporated into a base editor has translation polymerase activity. In some embodiments, a NAP or portion thereof incorporated into a base editor is a translesional DNA polymerase. In some embodiments, a NAP or portion thereof incorporated into a base editor is a Rev7, Rev1 complex, polymerase iota, polymerase kappa, or polymerase eta. In some embodiments, a NAP or portion thereof built into a base editor is an alpha, beta, gamma, delta, epsilon, gamma, eta, iota, kappa, lambda, mu or Nu component of a eukaryotic polymerase. In some embodiments, a NAP or portion thereof incorporated into a base editor comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a nucleic acid polymerase (eg, a translesional DNA polymerase).
Basis-Editor-System
Use of the base editor system provided herein comprises the steps of: (a) contacting a target nucleotide sequence of a polynucleotide (e.g. double or single stranded DNA or RNA) of a subject with a base editor system comprising a nucleobase editor (e.g., an adenosine base editor) and a guide polynucleic acid (e.g., gRNA), wherein the target nucleotide sequence comprises a target nucleobase pair; (b) inducing strand separation of the target region; (c) converting a first nucleobase of the target nucleobase pair in a single strand of the target region to a second nucleobase; and (d) cleaving no more than one strand of the target region, replacing a third nucleobase complementary to the first nucleobase base with a fourth nucleobase complementary to the second nucleobase. It is understood that in some embodiments step (b) is omitted. In some embodiments, the target nucleobase pair is a plurality of nucleobase pairs in one or more genes. In some embodiments, the base editing system provided herein is capable of multiplex editing of multiple nucleobase pairs in one or more genes. In some embodiments, the plurality of nucleobase pairs are located in the same gene. In some embodiments, the plurality of nucleobase pairs are located in one or more genes, with at least one gene located at a different locus.
In some embodiments, the cut single strand (nicked strand) is hybridized to the lead nucleic acid. In some embodiments, the nicked single strand is opposite the strand comprising the first nucleobase. In some embodiments, the base editor includes a Cas9 domain. In some embodiments, the first base is adenine and the second base is not G, C, A, or T. In some embodiments, the second base is inosine.
The base editing system provided herein provides a new approach to genome editing using a fusion protein containing a catalytically defective oneStreptococcus pyogenesCas9, an adenosine deaminase and a base excision repair inhibitor, to induce programmable single nucleotide changes (C→T or A→G) in DNA without creating double-stranded DNA breaks, without the need for a donor DNA template, and without a to induce excess of stochastic insertions and deletions.
Provided herein are systems, compositions, and methods for editing a nucleobase using a base editor system. In some embodiments, the base editor system comprises (1) a base editor (BE) comprising a polynucleotide-programmable nucleotide binding domain and a nucleobase editing domain (e.g., a deaminase domain) for editing the nucleobase; and (2) a guide polynucleotide (e.g., guide RNA) in association with the polynucleotide-programmable nucleotide binding domain. In some embodiments, the base editor system includes an adenosine base editor (ABE). In some embodiments, the polynucleotide-programmable nucleotide binding domain is a polynucleotide-programmable DNA binding domain. In some embodiments, the polynucleotide-programmable nucleotide binding domain is a polynucleotide-programmable RNA binding domain. In some embodiments, the nucleobase editing domain is a deaminase domain. In some embodiments, a deaminase domain can be an adenine deaminase or an adenosine deaminase. In some embodiments, the adenosine base editor is capable of deaminating adenine in DNA. In some embodiments, ABE includes an evolved TadA variant.
Details of nucleobase-editing proteins are described in International PCT Application Nos. PCT/2017/045381 (WO2018/027078) and PCT/US2016/058344 (WO2017/070632), each of which is incorporated herein by reference in its entirety. See also Komor, A.C., et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, N.M., et al., "Programmable base editing of A⋅T to G⋅C in genomic DNA without DNA cleavage" Nature 551, 464-471 (2017); and Komor, A.C., et al., "Improved inhibition of base excision repair and bacteriophage Mu-Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity" Science Advances 3:eaao4774 (2017), the The entirety of which is hereby incorporated by reference.
In some embodiments, a single guide polynucleotide can be used to direct a deaminase to a target nucleic acid sequence. In some embodiments, a single pair of guide polynucleotides can be used to direct different deaminases to a target nucleic acid sequence.
The nucleobase components and the polynucleotide-programmable nucleotide binding component of a base editor system can be covalently or non-covalently associated with one another. For example, in some embodiments, the deaminase domain can be targeted to a target nucleotide sequence by a polynucleotide-programmable nucleotide-binding domain. In some embodiments, a polynucleotide-programmable nucleotide binding domain can be fused or linked to a deaminase domain. In some embodiments, a polynucleotide-programmable nucleotide binding domain can target a deaminase domain to a target nucleotide sequence through non-covalent interaction with or association with the deaminase domain. For example, in some embodiments, the nucleobase editing component, e.g. the deaminase component, an additional heterologous part or domain, capable of interacting with, associating with, or forming a complex with an additional heterologous part or domain, as part of a polynucleotide -programmable nucleotide binding domain. In some embodiments, the additional heterologous portion may be capable of binding, interacting, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a leader polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous part can be a protein domain. In some embodiments, the additional heterologous portion may be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu-Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif, and a Ku -protein be B. a telomerase Sm7 binding motif and Sm7 protein or an RNA recognition motif.
A base editor system may further comprise a lead polynucleotide component. It is understood that components of the base editor system may be linked to each other via covalent bonds, non-covalent interactions, or any combination of associations and interactions thereof. In some embodiments, a deaminase domain can be targeted to a target nucleotide sequence by a target polynucleotide. For example, in some embodiments, the nucleobase editing component of the base editor system, e.g. the deaminase component, an additional heterologous portion or domain (e.g. a polynucleotide binding domain such as an RNA or DNA binding protein) capable of doing so with a portion or segment (e.g., a polynucleotide motif) of a lead polynucleotide can interact with, associate with, or form a complex with. In some embodiments, the additional heterologous portion or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or linked to the deaminase domain. In some embodiments, the additional heterologous portion may be capable of binding, interacting, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a leader polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous part can be a protein domain. In some embodiments, the additional heterologous portion may be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu-Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif, and a Ku -protein be B. a telomerase Sm7 binding motif and Sm7 protein or an RNA recognition motif.
In some embodiments, a base editor system may further comprise a base excision repair (BER) component inhibitor. It is understood that components of the base editor system may be linked to each other via covalent bonds, non-covalent interactions, or any combination of associations and interactions thereof. The inhibitor of the BER component may include a base excision repair inhibitor. In some embodiments, the base excision repair inhibitor may be a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the base excision repair inhibitor may be an inosine base excision repair inhibitor. In some embodiments, the base excision repair inhibitor can be targeted to the target nucleotide sequence by the polynucleotide-programmable nucleotide binding domain. In some embodiments, a polynucleotide-programmable nucleotide binding domain may be fused or linked to a base excision repair inhibitor. In some embodiments, a polynucleotide-programmable nucleotide binding domain may be fused or linked to a deaminase domain and a base excision repair inhibitor. In some embodiments, a polynucleotide-programmable nucleotide binding domain can target a base excision repair inhibitor to a target nucleotide sequence through non-covalent interaction with or association with the base excision repair inhibitor. For example, in some embodiments, the base excision repair component inhibitor may comprise an additional heterologous portion or domain that is capable of interacting with, or associating with, an additional heterologous portion or domain that is part of a or to form a complex with a polynucleotide programmable nucleotide binding domain.
In some embodiments, the base excision repair inhibitor can be targeted to the target nucleotide sequence by the leader polynucleotide. For example, in some embodiments, the base excision repair inhibitor may comprise an additional heterologous portion or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) capable of interacting with, associating with, it or to form a complex with a portion or segment (e.g., a polynucleotide motif) of a leader polynucleotide. In some embodiments, the additional heterologous portion or domain of the leader polynucleotide (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) may be fused or linked to the base excision repair inhibitor. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a leader polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous part can be a protein domain. In some embodiments, the additional heterologous portion may be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu-Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif, and a Ku -protein be B. a telomerase Sm7 binding motif and Sm7 protein or an RNA recognition motif.
In some embodiments, the base editor inhibits base excision repair (BER) of the edited strand. In some embodiments, the base editor protects or binds the unedited strand. In some embodiments, the base editor includes UGI activity. In some embodiments, the base editor comprises a catalytically inactive inosine-specific nuclease. In some embodiments, the base editor includes nickase activity. In some embodiments, the intended base pair editing is upstream of a PAM site. In some embodiments, the intended base pair editing is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream the PAM site. In some embodiments, the intended base pair processing is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream of the PAM site.
In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor includes a linker or a spacer. In some embodiments, the linker or spacer is 1-25 amino acids in length. In some embodiments, the linker or spacer is 5-20 amino acids in length. In some embodiments, the linker or spacer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.
In some embodiments, the base-editing fusion proteins provided herein must be positioned at a precise location, such as where a target base will be placed within a defined region (e.g., a "deamination window"). In some embodiments, a target can be within a 4-base region. In some embodiments, such a defined target region may be approximately 15 bases upstream from the PAM. See Komor, A.C., et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, N.M., et al., "Programmable base editing of A⋅T to G⋅C in genomic DNA without DNA cleavage" Nature 551, 464-471 (2017); and Komor, A.C., et al., "Improved inhibition of base excision repair and bacteriophage Mu-Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity" Science Advances 3:eaao4774 (2017), the The entirety of which is hereby incorporated by reference.
In some embodiments, the targeting region includes a targeting window, where the targeting window includes the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended processing of the base pair is within the target window. In some embodiments, the target window includes the intended editing of the base pair. In some embodiments, the method is performed using one of the basic editors provided herein. In some embodiments, a target window is a deamination window. A deamination window can be the defined region in which a base editor acts on and deaminates a target nucleotide. In some embodiments, the deamination window is within a 2, 3, 4, 5, 6, 7, 8, 9, or 10 base region. In some embodiments, the deamination window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases upstream of the PAM.
The base editors of the present disclosure may include any domain, feature, or amino acid sequence that facilitates editing of a target polynucleotide sequence. For example, in some embodiments, the base editor includes a nuclear localization sequence (NLS). In some embodiments, a base editor NLS is located between a deaminase domain and a polynucleotide-programmable nucleotide binding domain. In some embodiments, a base editor NLS is located C-terminal to a polynucleotide-programmable nucleotide binding domain.
Other exemplary features that may be present in a base editor as disclosed herein are localization sequences, such as cytoplasmic localization sequences, export sequences, such as nuclear export sequences or other localization sequences, and sequence tags useful for solubilization, purification, or detection of the fusion proteins. Suitable protein tags provided herein include biotin carboxylase carrier protein (BCCP) tags, myc tags, calmodulin tags, FLAG tags, hemagglutinin (HA) tags, polyhistidine tags, also known as histidine tags denotes, but are not limited to, or His tags, Maltose Binding Protein (MBP) tags, Nus tags, Glutathione-S-Transferase (GST) tags, Green Fluorescent Protein (GFP) tags, Thioredoxin- Tags, S tags, soft tags (e.g. Softag 1, Softag 3), strep tags, biotin ligase tags, FlAsH tags, V5 tags and SBP tags. Other suitable sequences will be apparent to those skilled in the art. In some embodiments, the fusion protein includes one or more His tags.
Non-limiting examples of protein domains that can be included in the fusion protein include deaminase domains (e.g., adenosine deaminase), a uracil glycosylase inhibitor (UGI) domain, epitope tags, and reporter gene sequences.
Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed B. DsRed , cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). Additional protein sequences may include amino acid sequences that bind DNA molecules or bind other cellular molecules including, but not limited to, maltose binding protein (MBP), S-Tag, Lex A DNA binding domain (DBD) fusions, GAL4 -DNA-binding domain fusions and herpes simplex virus (HSV)-BP16 protein fusions.
In some embodiments, the adenosine base editor (ABE) can deaminate adenine in DNA. In some embodiments, ABE is generated by replacing the APOBEC1 component of BE3 with natural or genetically engineered onesE coliTadA, human ADAR2, mouse ADA or human ADAT2. In some embodiments, ABE includes an evolved TadA variant. In some embodiments, the ABE is ABE 1.2 (TadA*-XTEN-nCas9-NLS). In some embodiments, TadA* includes A106V and D108N mutations.
In some embodiments, the ABE is a second generation ABE. In some embodiments, the ABE is ABE2.1, which includes additional mutations D147Y and E155V in TadA* (TadA*2.1). In some embodiments, the ABE is ABE2.2, ABE2.1 fused to a catalytically inactivated version of human alkyladenine DNA glycosylase (AAG with E125Q mutation). In some embodiments, the ABE is ABE2.3, ABE2.1 fused to the catalytically inactivated version ofE coliEndo V (inactivated with D35A mutation). In some embodiments, the ABE is ABE2.6, which has a linker twice as long (32 amino acids, (SGGS)2-XTEN-(SGGS)2(„(SGGS)2” disclosed as SEQ ID NO: 99)) as the linker in ABE2.1. In some embodiments, the ABE is ABE2.7, which is ABE2.1 linked to an additional wild-type TadA monomer. In some embodiments, the ABE is ABE2.8, which is ABE2.1 attached with an additional TadA*2.1 monomer. In some embodiments, the ABE is ABE2.9, which is a direct fusion of evolved TadA (TadA*2.1) to the N-terminus of ABE2.1. In some embodiments, the ABE is ABE2.10, which is a direct fusion of wild-type TadA to the N-terminus of ABE2.1. In some embodiments, the ABE is ABE2.11, which is ABE2.9 with an E59A inactivating mutation at the N-terminus of the TadA* monomer. In some embodiments, the ABE is ABE2.12, which is ABE2.9 with an E59A inactivating mutation in the internal TadA* monomer.
In some embodiments, the ABE is a third generation ABE. In some embodiments, the ABE is ABE3.1, which is ABE2.3 with three additional TadA mutations (L84F, H123Y, and I157F).
In some embodiments, the ABE is a fourth generation ABE. In some embodiments, the ABE is ABE4.3, which is ABE3.1 with an additional TadA mutation A142N (TadA*4.3).
In some embodiments, the ABE is a fifth generation ABE. In some embodiments, the ABE is ABE5.1, which is generated by importing a consensus set of mutations from surviving clones (H36L, R51L, S146C, and K157N) into ABE3.1. In some embodiments, the ABE is ABE5.3, which has a heterodimeric construct containing wild-typeE coliTadA merges with an internally developed TadA*. In some embodiments, the ABE is ABE5.2, ABE5.4, ABE5.5, ABE5.6, ABE5.7, ABE5.8, ABE5.9, ABE5.10, ABE5.11, ABE5.12, ABE5.13 or ABE5.14 as shown in Table 6 below. In some embodiments, the ABE is a sixth generation ABE. In some embodiments, the ABE is ABE6.1, ABE6.2, ABE6.3, ABE6.4, ABE6.5, or ABE6.6 as shown in Table 6 below. In some embodiments, the ABE is a seventh generation ABE. In some embodiments, the ABE is ABE7.1, ABE7.2, ABE7.3, ABE7.4, ABE7.5, ABE7.6, ABE7.7, ABE7.8, ABE 7.9, or ABE7.10, as shown in Table 6 below.
In some embodiments, the base editor is an eighth generation ABE (ABE8). In some embodiments, the ABE8 includes a TadA*8 variant. In some embodiments, the ABE8 has a monomeric construct containing a TadA*8 variant ("ABE8.x-m"). In some embodiments, ABE8 is ABE8.1-m having a monomeric construct containing TadA*7.10 with a Y147T mutation (TadA*8.1). In some embodiments, ABE8 is ABE8.2-m having a monomeric construct containing TadA*7.10 with a Y147R mutation (TadA*8.2). In some embodiments, ABE8 is ABE8.3-m having a monomeric construct containing TadA*7.10 with a Q154S mutation (TadA*8.3). In some embodiments, ABE8 is ABE8.4-m having a monomeric construct containing TadA*7.10 with a Y123H mutation (TadA*8.4). In some embodiments, ABE8 is ABE8.5-m having a monomeric construct containing TadA*7.10 with a V82S mutation (TadA*8.5). In some embodiments, ABE8 is ABE8.6-m having a monomeric construct containing TadA*7.10 with a T166R mutation (TadA*8.6). In some embodiments, ABE8 is ABE8.7-m having a monomeric construct containing TadA*7.10 with a Q154R mutation (TadA*8.7). In some embodiments, ABE8 is ABE8.8-m having a monomeric construct containing TadA*7.10 with Y147R, Q154R and Y123H mutations (TadA*8.8). In some embodiments, ABE8 is ABE8.9-m having a monomeric construct containing TadA*7.10 with Y147R, Q154R and I76Y mutations (TadA*8.9). In some embodiments, ABE8 is ABE8.10-m having a monomeric construct containing TadA*7.10 with Y147R, Q154R and T166R mutations (TadA*8.10). In some embodiments, ABE8 is ABE8.11-m having a monomeric construct containing TadA*7.10 with Y147T and Q154R mutations (TadA*8.11). In some embodiments, ABE8 is ABE8.12-m, which has a monomeric construct containing TadA*7.10 with Y147T and Q154S mutations (TadA*8.12). In some embodiments, ABE8 is ABE8.13-m having a monomeric construct containing TadA*7.10 with Y123H (Y123H reverted from H123Y), Y147R, Q154R and I76Y mutations (TadA*8.13). In some embodiments, ABE8 is ABE8.14-m, which has a monomeric construct containing TadA*7.10 with I76Y and V82S mutations (TadA*8.14). In some embodiments, ABE8 is ABE8.15-m, which has a monomeric construct containing TadA*7.10 with V82S and Y147R mutations (TadA*8.15). In some embodiments, the ABE8 is ABE8.16-m, which has a monomeric construct containing TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), and Y147R (TadA*8.16) mutations. In some embodiments, ABE8 is ABE8.17-m, which has a monomeric construct containing TadA*7.10 with V82S and Q154R mutations (TadA*8.17). In some embodiments, ABE8 is ABE8.18-m having a monomeric construct containing TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), and Q154R (TadA*8.18) mutations. In some embodiments, the ABE8 is ABE8.19-m, which has a monomeric construct containing TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), Y147R, and Q154R mutations (TadA*8.19). In some embodiments, ABE8 is ABE8.20-m having a monomeric construct containing TadA*7.10 with I76Y, V82S, Y123H (Y123H reverted from H123Y), Y147R and Q154R mutations (TadA*8.20). . In some embodiments, ABE8 is ABE8.21-m, which has a monomeric construct containing TadA*7.10 with Y147R and Q154S mutations (TadA*8.21). In some embodiments, ABE8 is ABE8.22-m, which has a monomeric construct containing TadA*7.10 with V82S and Q154S mutations (TadA*8.22). In some embodiments, ABE8 is ABE8.23-m having a monomeric construct containing TadA*7.10 with V82S and Y123H mutations (TadA*8.23) (Y123H reverted from H123Y). In some embodiments, the ABE8 is ABE8.24-m, which has a monomeric construct containing TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), and Y147T (TadA*8.24) mutations.
In some embodiments, the ABE8 has a heterodimeric construct containing wild-typeE coliTadA merged with a TadA*8 variant ("ABE8.x-d"). In some embodiments, ABE8 is ABE8.1-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with a Y147T mutation (TadA*8.1). In some embodiments, ABE8 is ABE8.2-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with a Y147R mutation (TadA*8.2). In some embodiments, ABE8 is ABE8.3-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with a Q154S mutation (TadA*8.3). In some embodiments, ABE8 is ABE8.4-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with a Y123H mutation (TadA*8.4). In some embodiments, ABE8 is ABE8.5-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with a V82S mutation (TadA*8.5). In some embodiments, ABE8 is ABE8.6-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with a T166R mutation (TadA*8.6). In some embodiments, ABE8 is ABE8.7-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with a Q154R mutation (TadA*8.7). In some embodiments, ABE8 is ABE8.8-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with Y147R, Q154R and Y123H mutations (TadA*8.8). In some embodiments, ABE8 is ABE8.9-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with Y147R, Q154R and I76Y mutations (TadA*8.9). In some embodiments, ABE8 is ABE8.10-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with Y147R, Q154R and T166R mutations (TadA*8.10). In some embodiments, ABE8 is ABE8.11-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with Y147T and Q154R mutations (TadA*8.11). In some embodiments, ABE8 is ABE8.12-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with Y147T and Q154S mutations (TadA*8.12). In some embodiments, ABE8 is ABE8.13-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with Y123H (Y123H reverted from H123Y), Y147R, Q154R and I76Y mutations (TadA*8.13). In some embodiments, ABE8 is ABE8.14-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with I76Y and V82S mutations (TadA*8.14). In some embodiments, ABE8 is ABE8.15-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with V82S and Y147R mutations (TadA*8.15). In some embodiments, ABE8 is ABE8.16-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with V82S, Y123H (Y123H reversed from H123Y) and Y147R mutations (TadA*8.16). In some embodiments, ABE8 is ABE8.17-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with V82S and Q154R mutations (TadA*8.17). In some embodiments, ABE8 is ABE8.18-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with V82S, Y123H (Y123H reversed from H123Y) and Q154R mutations (TadA*8.18). In some embodiments, ABE8 is ABE8.19-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with V82S, Y123H (Y123H reversed from H123Y), Y147R and Q154R mutations (TadA*8.19). In some embodiments, ABE8 is ABE8.20-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with I76Y, V82S, Y123H (Y123H reversed from H123Y), Y147R and Q154R mutations (TadA*8.20). In some embodiments, ABE8 is ABE8.21-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with Y147R and Q154S mutations (TadA*8.21). In some embodiments, ABE8 is ABE8.22-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with V82S and Q154S mutations (TadA*8.22). In some embodiments, ABE8 is ABE8.23-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with V82S and Y123H mutations (Y123H reversed from H123Y) (TadA*8.23). In some embodiments, ABE8 is ABE8.24-d, which has a heterodimeric construct containing wild-typeE coliTadA fused to TadA*7.10 with V82S, Y123H (Y123H reversed from H123Y) and Y147T mutations (TadA*8.24).
In some embodiments, the ABE8 has a heterodimeric construct containing TadA*7.10 fused to a TadA*8 variant ("ABE8.x-7"). In some embodiments, ABE8 is ABE8.1-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a Y147T mutation (TadA*8.1). In some embodiments, ABE8 is ABE8.2-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a Y147R mutation (TadA*8.2). In some embodiments, ABE8 is ABE8.3-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a Q154S mutation (TadA*8.3). In some embodiments, ABE8 is ABE8.4-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a Y123H mutation (TadA*8.4). In some embodiments, ABE8 is ABE8.5-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 having a V82S mutation (TadA*8.5). In some embodiments, ABE8 is ABE8.6-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 having a T166R mutation (TadA*8.6). In some embodiments, ABE8 is ABE8.7-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 having a Q154R mutation (TadA*8.7). In some embodiments, ABE8 is ABE8.8-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147R, Q154R, and Y123H mutations (TadA*8.8). In some embodiments, ABE8 is ABE8.9-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147R, Q154R, and I76Y mutations (TadA*8.9). In some embodiments, ABE8 is ABE8.10-7 having a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147R, Q154R and T166R mutations (TadA*8.10). In some embodiments, ABE8 is ABE8.11-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147T and Q154R mutations (TadA*8.11). In some embodiments, ABE8 is ABE8.12-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147T and Q154S mutations (TadA*8.12). In some embodiments, ABE8 is ABE8.13-7 containing a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y123H (Y123H reverted from H123Y), Y147R, Q154R and I76Y mutations (TadA*8.13). In some embodiments, ABE8 is ABE8.14-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with I76Y and V82S mutations (TadA*8.14). In some embodiments, ABE8 is ABE8.15-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S and Y147R mutations (TadA*8.15). In some embodiments, the ABE8 is ABE8.16-7, which has a heterodimeric construct containing TadA*7.10 reverted to TadA*7.10 with V82S, Y123H (Y123H from H123Y), and Y147R mutations (TadA*8.16) is merged. In some embodiments, ABE8 is ABE8.17-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S and Q154R mutations (TadA*8.17). In some embodiments, the ABE8 is ABE8.18-7, which has a heterodimeric construct containing TadA*7.10 reverted to TadA*7.10 with V82S, Y123H (Y123H from H123Y), and Q154R mutations (TadA*8.18) is merged. In some embodiments, ABE8 is ABE8.19-7 containing a heterodimeric construct containing TadA*7.10 mutated at TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), Y147R and Q154R mutations (TadA* 8.19) is merged. In some embodiments, the ABE8 is ABE8.20-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with I76Y-, V82S-, Y123H- (Y123H-reverted from H123Y), Y147R - and Q154R mutations (TadA*8.20). In some embodiments, ABE8 is ABE8.21-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147R and Q154S mutations (TadA*8.21). In some embodiments, ABE8 is ABE8.22-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S and Q154S mutations (TadA*8.22). In some embodiments, ABE8 is ABE8.23-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S and Y123H mutations (Y123H reverted from H123Y) (TadA*8.23). In some embodiments, ABE8 is ABE8.24-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S, Y123H (Y123H reversed from H123Y), and Y147T (TadA*8.24) mutations.
In some embodiments, the ABE is ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, ABE8.5-m, ABE8.6-m, ABE8.7-m, ABE8. 8m, ABE8.9m, ABE8.10m, ABE8.11m, ABE8.12m, ABE8.13m, ABE8.14m, ABE8.15m, ABE8.16 - m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8.23-m, ABE8.24-m , ABE8.1-d, ABE8.2-d, ABE8.3-d, ABE8.4-d, ABE8.5-d, ABE8.6-d, ABE8.7-d, ABE8.8-d, ABE8 . 9-d, ABE8.10-d, ABE8.11-d, ABE8.12-d, ABE8.13-d, ABE8.14-d, ABE8.15-d, ABE8.16-d, ABE8.17 - d, ABE8.18-d, ABE8.19-d, ABE8.20-d, ABE8.21-d, ABE8.22-d, ABE8.23-d or ABE8.24-d as shown in Table 7 below.
In some embodiments, base editors (e.g. ABE8) are generated by cloning an adenosine deaminase variant (e.g. TadA*8) into a scaffold containing a circular permutant Cas9 (e.g. CP5 or CP6) and a two-part nuclear localization sequence. In some embodiments, the base editor (e.g. ABE7.9, ABE7.10 or ABE8) is an NGC PAM CP5 variant (S. PyrogeneCas9 or spVRQR Cas9). In some embodiments, the base editor (e.g. ABE7.9, ABE7.10 or ABE8) is an AGA PAM CP5 variant (S. PyrogeneCas9 or spVRQR Cas9). In some embodiments, the base editor (e.g. ABE7.9, ABE7.10 or ABE8) is an NGC PAM CP6 variant (S. PyrogeneCas9 or spVRQR Cas9). In some embodiments, the base editor (e.g. ABE7.9, ABE7.10 or ABE8) is an AGA PAM CP6 variant (S. PyrogeneCas9 or spVRQR Cas9).
In some embodiments, the ABE has a genotype as shown in Table 8 below.
As shown in Table 9 below, genotypes of 40 ABE8s are described. residual positions in the evolvedE coliTadA share of ABE are given. Mutational changes in ABE8 are shown if they differ from ABE7.10 mutations. In some embodiments, the ABE has a genotype of one of the ABEs, as shown in Table 9.
In some embodiments, the base editor is ABE8.1, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:
In the above sequence, the plaintext designates an adenosine deaminase sequence, the boldfaced sequence designates a Cas9-derived sequence, the italicized sequence designates a linker sequence, and the underlined sequence designates a bipartite nuclear localization sequence.
In some embodiments, the base editor is ABE8.1, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:
In the above sequence, the plaintext designates an adenosine deaminase sequence, the boldfaced sequence designates a Cas9-derived sequence, the italicized sequence designates a linker sequence, and the underlined sequence designates a bipartite nuclear localization sequence.
In some embodiments, the base editor is ABE8.14, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:
In the above sequence, the plaintext designates an adenosine deaminase sequence, the boldfaced sequence designates a Cas9-derived sequence, the italicized sequence designates a linker sequence, and the underlined sequence designates a bipartite nuclear localization sequence.
In some embodiments, the base editor is ABE8.8-m, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:
In the above sequence, the plaintext designates an adenosine deaminase sequence, the boldfaced sequence designates a Cas9-derived sequence, the italicized sequence designates a linker sequence, the underlined sequence designates a bipartite nuclear localization sequence, and the double-underlined sequence designates mutations.
In some embodiments, the base editor is ABE8.8-d, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:
In the above sequence, the plaintext designates an adenosine deaminase sequence, the boldfaced sequence designates a Cas9-derived sequence, the italicized sequence designates a linker sequence, the underlined sequence designates a bipartite nuclear localization sequence, and the double-underlined sequence designates mutations.
In some embodiments, the base editor is ABE8.13-m, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:
In the above sequence, the plaintext designates an adenosine deaminase sequence, the boldfaced sequence designates a Cas9-derived sequence, the italicized sequence designates a linker sequence, the underlined sequence designates a bipartite nuclear localization sequence, and the double-underlined sequence designates mutations.
In some embodiments, the base editor is ABE8.13-d, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:
In the above sequence, the plaintext designates an adenosine deaminase sequence, the boldfaced sequence designates a Cas9-derived sequence, the italicized sequence designates a linker sequence, the underlined sequence designates a bipartite nuclear localization sequence, and the double-underlined sequence designates mutations.
In some embodiments, the base editor is ABE8.17-m, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:
In the above sequence, the plaintext designates an adenosine deaminase sequence, the boldfaced sequence designates a Cas9-derived sequence, the italicized sequence designates a linker sequence, the underlined sequence designates a bipartite nuclear localization sequence, and the double-underlined sequence designates mutations.
In some embodiments, the base editor is ABE8.17-d, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:
In the above sequence, the plaintext designates an adenosine deaminase sequence, the boldfaced sequence designates a Cas9-derived sequence, the italicized sequence designates a linker sequence, the underlined sequence designates a bipartite nuclear localization sequence, and the double-underlined sequence designates mutations.
In some embodiments, the base editor is ABE8.20-m, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:
In the above sequence, the plaintext designates an adenosine deaminase sequence, the boldfaced sequence designates a Cas9-derived sequence, the italicized sequence designates a linker sequence, the underlined sequence designates a bipartite nuclear localization sequence, and the double-underlined sequence designates mutations.
In some embodiments, the base editor is ABE8.20-d, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:
In the above sequence, the plaintext designates an adenosine deaminase sequence, the boldfaced sequence designates a Cas9-derived sequence, the italicized sequence designates a linker sequence, the underlined sequence designates a bipartite nuclear localization sequence, and the double-underlined sequence designates mutations.
In some embodiments, an ABE8 of the invention is selected from the following sequences:
In some embodiments, the base editor is a fusion protein comprising a polynucleotide-programmable nucleotide binding domain (e.g., a Cas9-derived domain) fused to a nucleobase editing domain (e.g., all or part of a deaminase domain). In certain embodiments, the fusion proteins provided herein include one or more features that enhance the base-editing activity of the fusion proteins. For example, any of the fusion proteins provided herein may include a Cas9 domain that exhibits reduced nuclease activity. In some embodiments, each of the fusion proteins provided herein may have a Cas9 domain without nuclease activity (dCas9) or a Cas9 domain that cleaves one strand of a double-stranded DNA molecule termed Cas9 nickase (nCas9).
In some embodiments, the base editor further comprises a domain comprising all or part of a uracil glycosylase inhibitor (UGI). In some embodiments, the base editor comprises a domain comprising all or part of a uracil binding protein (UBP), such as a uracil DNA glycosylase (UDG). In some embodiments, the base editor comprises a domain comprising all or part of a nucleic acid polymerase. In some embodiments, a nucleic acid polymerase or portion thereof incorporated into a base editor is a translesional DNA polymerase.
In some embodiments, a domain of the base editor may include multiple domains. For example, the base editor comprising a Cas9-derived polynucleotide-programmable nucleotide binding domain may comprise a REC lobe and a NUC lobe corresponding to the REC lobe and NUC lobe of a wild-type or natural Cas9. In another example, the base editor may be one or more of a RuvCI domain, BH domain, REC1 domain, REC2 domain, RuvCII domain, L1 domain, HNH domain, L2 domain, RuvCIII domain, WED domain , TOPO domain or CTD domain. In some embodiments, one or more domains of the base editor comprises a mutation (e.g., substitution, insertion, deletion) relative to a wild-type version of a polypeptide comprising the domain. For example, an HNH domain of a polynucleotide-programmable DNA binding domain may include an H840A substitution. In another example, a RuvCI domain of a polynucleotide-programmable DNA binding domain may include a D10A substitution.
Different domains (e.g., contiguous domains) of the basic editor disclosed herein may be linked together with or without using one or more linker domains (e.g., an XTEN linker domain). In some embodiments, a linker domain can be a bond (e.g., covalent bond), chemical group, or molecule that connects two molecules or entities, e.g. B. two domains of a fusion protein, such as. B. a first domain (e.g., Cas9-derived domain) and a second domain (e.g. an adenosine deaminase domain). In some embodiments, a linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, a linker is a carbon-nitrogen bond of an amide bond. In certain embodiments, a linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched, aliphatic or heteroaliphatic linker. In certain embodiments, a linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, a linker comprises a monomer, dimer, or polymer of amino alkanoic acid. In some embodiments, a linker comprises an amino alkanoic acid (e.g., glycine, acetic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In some embodiments, a linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, a linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, a linker comprises a polyethylene glycol (PEG) moiety. In certain embodiments, a linker includes an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. A linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile can be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates. In some embodiments, a linker connects a gRNA-binding domain of an RNA-programmable nuclease, including a Cas9 nuclease domain, and the catalytic domain of a nucleic acid-editing protein. In some embodiments, a linker connects a dCas9 and a second domain (eg, UGI, etc.).
Typically, a linker is positioned between or flanked by two groups, molecules, or other entities and is joined together by a covalent bond, thereby joining the two. In some embodiments, a linker is one or more amino acids (e.g., a peptide or protein). In some embodiments, a linker is an organic molecule, group, polymer, or chemical entity. In some embodiments, a linker is 2-100 amino acids long, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150 or 150-200 amino acids long. In some embodiments, the linker is from about 3 to about 104 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100) amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, a linker domain comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 51), which may also be referred to as an XTEN linker. Any method of joining the fusion protein domains can be used (e.g. ranging from very flexible linkers of the form (SGGS)n (SEQ ID NO: 124), (GGGS)n (SEQ ID NO: 125), (GGGGS)n ( SEQ ID NO: 126) and (G)n, to more rigid linkers of the form (EAAAK)n (SEQ ID NO: 127), (GGS)n, SGSETPGTSESATPES (SEQ ID NO: 51) (see e.g. , Guilinger J.P., Thompson DB, Liu DR, Fusion ofcatalyally inactive Cas9 to FokI nuclease Improves the specificity of genome modification, Nat Biotechnol., 2014, 32(6): 577-82 (the entire content is incorporated herein by reference) , or (XP)NMotive to achieve the optimal length of activity for the nucleobase editor. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)NMotif (SEQ ID NO: 128) wherein n is 1, 3 or 7. In some embodiments, the Cas9 domain of the fusion proteins provided herein is fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO:51). . In some embodiments, a linker comprises multiple proline residues and is 5-21, 5-14, 5-9, 5-7 amino acids long, e.g. B. PAPAP (SEQ ID NO: 129), PAPAPA (SEQ ID NO: 129). : 130), PAPAPAP (SEQ ID NO: 131), PAPAPAPA (SEQ ID NO: 132), P(AP)4(SEQ ID NO: 133), P(AP)7(SEQ ID NO: 134), P(AP)10(SEQ ID NO: 135) (see e.g. Tan J, Zhang F, Karcher D, Bock R. Engineering of high-precision base editors for site-specific single nucleotide replacement. Nat Commun. 2019 Jan. 25; 10( 1 ):439; the entire content is incorporated herein by reference). Such proline-rich linkers are also referred to as "rigid" linkers.
A fusion protein of the invention comprises a nucleic acid editing domain. In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is a vertebrate deaminase. In some embodiments, the deaminase is an invertebrate deaminase. In some embodiments, the deaminase is a human, chimpanzee, gorilla, monkey, cow, canine, rat, or mouse deaminase. In some embodiments, the deaminase is a human deaminase. In some embodiments, the deaminase is a rat deaminase.
Linker
In certain embodiments, linkers can be used to join any of the peptides or peptide domains of the invention. The linker can be as simple as a covalent bond, or it can be a polymeric linker many atoms in length. In certain embodiments, the linker is a polypeptide or is based on amino acids. In other embodiments, the linker is non-peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide bond. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched, aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of amino alkanoic acid. In certain embodiments, the linker comprises an amino alkanoic acid (e.g., glycine, acetic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol (PEG) moiety. In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile can be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.
In some embodiments, the linker is one or more amino acids (e.g., a peptide or protein). In some embodiments, the linker is a bond (e.g., a covalent bond), an organic molecule, a group, a polymer, or a chemical entity. In some embodiments, the linker is from about 3 to about 104 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100) amino acids in length.
In some embodiments, the adenosine deaminase and the napDNAbp are fused via a linker that is 4, 16, 32, or 104 amino acids in length. In some embodiments, the linker is about 3 to about 104 amino acids in length. In some embodiments, each of the fusion proteins provided herein comprises an adenosine deaminase and a Cas9 domain fused together via a linker. Various linker lengths and flexibilities can be used between the deaminase domain (e.g. an engineered ecTadA) and the Cas9 domain (e.g. ranging from very flexible linkers of the form (GGGS)N(SEQ ID NO: 125), (GGGGS)N(SEQ ID NO: 126) and (G)Nto more rigid linkers of the form (EAAAK)N(SEQ ID NO: 127), (SGGS)N(SEQ ID NO: 124), SGSETPGTSESATPES (SEQ ID NO: 51) (see e.g. Guilinger JP, Thompson DB, Liu DR, Fusion ofcatalyally inactive Cas9 to FokI nuclease improves specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference) and (XP)N) to achieve the optimal activity length for the nucleobase editor. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)NMotif (SEQ ID NO: 128, where n is 1, 3 or 7). In some embodiments, the adenosine deaminase and Cas9 domain of each of the fusion proteins provided herein are fused via a linker (e.g., an XTEN linker) comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 51).
Cas9 complexes with guide RNAs
Some aspects of this disclosure provide for complexes comprising any of the fusion proteins provided herein and a guide RNA (e.g., a guide RNA targeting an A\mutation) linked to a CAS9 domain (e.g., B. a dCas9, a nuclease-active Cas9 or a Cas9) nickase) of the fusion protein is bound. Any method of joining the fusion protein domains can be used (e.g. ranging from very flexible linkers of the form (GGGS)N(SEQ ID NO: 125), (GGGGS)N(SEQ ID NO: 126) and (G)Nto more rigid linkers of the form (EAAAK)N(SEQ ID NO: 127), (SGGS)N(SEQ ID NO: 124), SGSETPGTSESATPES (SEQ ID NO: 51) (see e.g. Guilinger JP, Thompson DB, Liu DR, Fusion ofcatalyally inactive Cas9 to FokI nuclease improves specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference) and (XP)N) to achieve the optimal activity length for the nucleobase editor. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS) motif (SEQ ID NO: 128) where n is 1, 3 or 7. In some embodiments, the Cas9 domain of the fusion proteins provided herein is fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO:51).
In some embodiments, the guide nucleic acid (e.g., guide RNA) is 15-100 nucleotides in length and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In some embodiments, the guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 B. 35, 36, 37, 38, 39 or 40 contiguous nucleotides complementary to a target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is a sequence in the genome of a bacterium, yeast, fungus, insect, plant, or animal. In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3' end of the target sequence is contiguous with a canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is immediately adjacent to a non-canonical PAM sequence (e.g., a sequence listed in Table 1 or 5'-NAA-3'). In some embodiments, the guide nucleic acid (e.g., guide RNA) is complementary to a sequence in a gene of interest (e.g., a gene associated with a disease or disorder). In some embodiments, the guide nucleic acid (e.g., guide RNA) is complementary to a sequence associated with alpha-1-antitrypsin deficiency (A1AD).
Some aspects of this disclosure provide methods of using the fusion proteins or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins provided herein and with at least one guide RNA, the guide RNA being about 15-100 nucleotides in length and having a sequence of at least 10 contiguous nucleotides complementary to a target sequence. In some embodiments, the 3' end of the target sequence is contiguous with an AGC, GAG, TTT, GTG, or CAA sequence. In some embodiments, the 3' end of the target sequence is immediately adjacent to an NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, NGTN, NGTN, or 5'(TTTV) Sequence.
It is understood that the numbering of the specific positions or residues in the particular sequences depends on the particular protein and numbering scheme used. The numbering can be different, e.g. B. in precursors of a mature protein and the mature protein itself, and differences in the sequences from species to species can affect the numbering. One skilled in the art will be able to identify the particular residue in each homologous protein and in the particular encoding nucleic acid by methods well known in the art, e.g. B. by sequence alignment and determination of homologous residues.
Those skilled in the art will recognize that in order to direct any of the fusion proteins disclosed herein to a target site, e.g. B. a site containing a mutation to be edited Protein together with a guide RNA. As discussed in more detail elsewhere herein, a leader RNA typically includes a tracrRNA backbone that allows Cas9 binding and a leader sequence that confers sequence specificity on the Cas9:nucleic acid-editing enzyme/domain fusion protein. Alternatively, the guide RNA and the tracrRNA can be provided separately as two nucleic acid molecules. In some embodiments, the guide RNA comprises a structure wherein the guide sequence comprises a sequence complementary to the target sequence. The leader sequence is typically 20 nucleotides long. The sequences of appropriate guide RNAs for targeting Cas9:nucleic acid editing enzyme/domain fusion proteins to specific genomic target sites will be apparent to those skilled in the art based on the present disclosure. Such suitable guide RNA sequences typically include guide sequences complementary to a nucleotide sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting each of the provided fusion proteins to specific target sequences are provided herein.
Cas12 complexes with guide RNAs
Some aspects of this disclosure provide for complexes comprising any of the fusion proteins provided herein and a guide RNA (e.g., a guide RNA that targets a target polynucleotide for editing).
In some embodiments, the guide nucleic acid (e.g., guide RNA) is 15-100 nucleotides in length and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In some embodiments, the guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 B. 35, 36, 37, 38, 39 or 40 contiguous nucleotides complementary to a target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is a sequence in the genome of a bacterium, yeast, fungus, insect, plant, or animal. In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3' end of the target sequence is contiguous with a canonical PAM sequence. In some embodiments, the 3' end of the target sequence is immediately adjacent to a non-canonical PAM sequence.
Some aspects of this disclosure provide methods of using the fusion proteins or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins provided herein and with at least one guide RNA, the guide RNA being about 15-100 nucleotides in length and having a sequence of at least 10 contiguous nucleotides complementary to a target sequence. In some embodiments, the 3' end of the target sequence is immediately adjacent to a z. B. TTN, DTTN, GTTN, ATTN, ATTC, DTTNT, WTTN, HATY, TTTN, TTTV, TTTC, TG, RTR or YTN PAM site.
It is understood that the numbering of the specific positions or residues in the particular sequences depends on the particular protein and numbering scheme used. The numbering can be different, e.g. B. in precursors of a mature protein and the mature protein itself, and differences in the sequences from species to species can affect the numbering. One skilled in the art will be able to identify the particular residue in each homologous protein and in the particular encoding nucleic acid by methods well known in the art, e.g. B. by sequence alignment and determination of homologous residues.
Those skilled in the art will recognize that in order to direct any of the fusion proteins disclosed herein to a target site, e.g. B. a site containing a mutation to be edited Protein together with a guide RNA. As discussed in more detail elsewhere herein, a guide RNA typically includes a tracrRNA backbone that allows Cas12 binding and a guide sequence that confers sequence specificity on the Cas12:nucleic acid-editing enzyme/domain fusion protein. Alternatively, the guide RNA and the tracrRNA can be provided separately as two nucleic acid molecules. In some embodiments, the guide RNA comprises a structure wherein the guide sequence comprises a sequence complementary to the target sequence. The leader sequence is typically 20 nucleotides long. The sequences of appropriate guide RNAs for targeting Cas12:nucleic acid editing enzyme/domain fusion proteins to specific genomic target sites will be apparent to those skilled in the art based on the present disclosure. Such suitable guide RNA sequences typically include guide sequences complementary to a nucleotide sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting each of the provided fusion proteins to specific target sequences are provided herein.
The domains of the base editor disclosed herein can be arranged in any order as long as the deaminase domain is internalized into the Cas12 protein. Non-limiting examples of a base editor comprising a fusion protein e.g. B. comprises a Cas12 domain and a deaminase domain can be arranged as follows:
NH2-[Cas12 domain]-Linker1-[ABE8]-Linker2-[Cas12 domain]-COOH;
NH2-[Cas12 domain]-Linker1-[ABE8]-[Cas12 domain]-COOH;
NH2-[Cas12 domain]-[ABE8]-Linker2-[Cas12 domain]-COOH;
NH2-[Cas12 domain]-[ABE8]-[Cas12 domain]-COOH;
NH2-[Cas12 domain]-linker1-[ABE8]-linker2-[Cas12 domain]-[inosine BER inhibitor]-COOH;
NH2-[Cas12 domain]-Linker1-[ABE8]-[Cas12 domain]-[inosine BER inhibitor]-COOH;
NH2-[Cas12 domain]-[ABE8]-Linker2-[Cas12 domain]-[inosine BER inhibitor]-COOH;
NH2-[Cas12 domain]-[ABE8]-[Cas12 domain]-[inosine BER inhibitor]-COOH;
NH2-[inosine BER inhibitor]-[Cas12 domain]-linker1-[ABE8]-linker2-[Cas12 domain]-COOH;
NH2-[inosine BER inhibitor]-[Cas12 domain]-Linker1-[ABE8]-[Cas12 domain]-COOH;
NH2-[inosine BER inhibitor]-[Cas12 domain]-[ABE8]-Linker2-[Cas12 domain]-COOH;
NH2-[inosine BER inhibitor]NH2-[Cas12 domain]-[ABE8]-[Cas12 domain]-COOH;
In addition, in some cases, a Gam protein can be fused to an N-terminus of a base editor. In some cases, a Gam protein can be fused to a C-terminus of a base editor. Bacteriophage Mu's Gam protein can bind to the ends of double-strand breaks (DSBs) and protect them from degradation. In some embodiments, using Gam to bind the free ends of DSBs may reduce indel formation during the base editing process. In some embodiments, the 174 residue Gam protein is fused to the N-terminus of the base editor. See. Komor, A.C., et al., "Improved inhibition of base excision repair and bacteriophage Mu-Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity" Science Advances 3:eaao4774 (2017). In some cases, a mutation or mutations can change the length of a basic editor domain relative to a wild-type domain. For example, a deletion of at least one amino acid in at least one domain can reduce the length of the base editor. In another case, a mutation or mutations do not change the length of a domain relative to a wild-type domain. For example, substitution(s) in any domain does not change the length of the base editor
In some embodiments, the base-editing fusion proteins provided herein must be positioned at a precise location, such as where a target base will be placed within a defined region (e.g., a "deamination window"). In some cases a target may be within a 4 base region. In some cases, such a defined target region may be about 15 bases upstream from the PAM. See Komor, A.C., et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, N.M., et al., "Programmable base editing of A⋅T to G⋅C in genomic DNA without DNA cleavage" Nature 551, 464-471 (2017); and Komor, A.C., et al., "Improved inhibition of base excision repair and bacteriophage Mu-Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity" Science Advances 3:eaao4774 (2017), the The entirety of which is hereby incorporated by reference.
A defined target area can be a deamination window. A deamination window can be the defined region in which a base editor acts on and deaminates a target nucleotide. In some embodiments, the deamination window is within a 2, 3, 4, 5, 6, 7, 8, 9, or 10 base region. In some embodiments, the deamination window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases upstream of the PAM.
The base editors of the present disclosure may include any domain, feature, or amino acid sequence that facilitates editing of a target polynucleotide sequence. For example, in some embodiments, the base editor includes a nuclear localization sequence (NLS). In some embodiments, a base editor NLS is located between a deaminase domain and a napDNAbp domain. In some embodiments, a base editor NLS is located C-terminal to a napDNAbp domain.
Protein domains contained in the fusion protein can be a heterologous functional domain. Non-limiting examples of protein domains that can be included in the fusion protein include a deaminase domain (e.g., adenosine deaminase), a uracil glycosylase inhibitor (UGI) domain, epitope tags, and reporter gene sequences. Protein domains can be a heterologous functional domain that has, for example, one or more of the following activities: transcription activation activity, transcription repression activity, transcription releasing factor activity, gene silencing activity, chromatin modifying activity, epigenetic modification activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Such heterologous functional domains can confer functional activity, such as B. Modification of a target polypeptide associated with target DNA (e.g., a histone, a DNA-binding protein, etc.), resulting in, for example, histone methylation, histone acetylation, histone ubiquitination, and the like. Other conferred functions and/or activities may be transposase activity, integrase activity, recombinase activity, ligase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, or deSUMOylation activity include any combination of the above.
A domain can be detected or tagged with an epitope tag, a reporter protein, or other binding domains. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed B. DsRed , cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). Additional protein sequences may include amino acid sequences that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-Tag, Lex A DNA binding domain (DBD) fusions, GAL4 fusions -DNA binding domains, and herpes simplex virus (HSV)-BP16 protein fusions.
In some embodiments, the BhCas12b leader polynucleotide has the following sequence:
In some embodiments, BvCas12b and AaCas12b leader polynucleotides have the following sequences:
Methods of using fusion proteins comprising an adenosine deaminase variant and a Cas9 domain
Some aspects of this disclosure provide methods of using the fusion proteins or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule encoding a mutant form of a protein with one of the fusion proteins provided herein and with at least one guide RNA, wherein the guide RNA is about 15- 100 nucleotides in length and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the 3' end of the target sequence is contiguous with a canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is not immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is contiguous with an AGC, GAG, TTT, GTG, or CAA sequence. In some embodiments, the 3' end of the target sequence is immediately adjacent to an NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, NGTN, NGTN, or 5'(TTTV) Sequence.
It is understood that the numbering of the specific positions or residues in the particular sequences depends on the particular protein and numbering scheme used. The numbering can be different, e.g. B. in precursors of a mature protein and the mature protein itself, and differences in the sequences from species to species can affect the numbering. One skilled in the art will be able to identify the particular residue in each homologous protein and in the particular encoding nucleic acid by methods well known in the art, e.g. B. by sequence alignment and determination of homologous residues.
It will be appreciated by those skilled in the art that in order to target one of the fusion proteins comprising a Cas9 domain and an adenosine deaminase variant (e.g. ABE8) as disclosed herein to a target site, e.g. a mutation to be engineered, it is typically necessary to deliver the fusion protein together with a guide RNA, e.g. B. an sgRNA to co-express. As discussed in more detail elsewhere herein, a leader RNA typically includes a tracrRNA backbone that allows Cas9 binding and a leader sequence that confers sequence specificity on the Cas9:nucleic acid-editing enzyme/domain fusion protein. Alternatively, the guide RNA and the tracrRNA can be provided separately as two nucleic acid molecules. In some embodiments, the guide RNA comprises a structure wherein the guide sequence comprises a sequence complementary to the target sequence. The leader sequence is typically 20 nucleotides long. The sequences of appropriate guide RNAs for targeting Cas9:nucleic acid editing enzyme/domain fusion proteins to specific genomic target sites will be apparent to those skilled in the art based on the present disclosure. Such suitable guide RNA sequences typically include guide sequences complementary to a nucleotide sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting each of the provided fusion proteins to specific target sequences are provided herein.
Basic editor efficiency
CRISPR-Cas9 nucleases are widely used to mediate targeted genome editing. In most genome editing applications, Cas9 forms a complex with a guide polynucleotide (eg, single guide RNA (sgRNA)) and induces a double-stranded DNA break (DSB) at the target site specified by the sgRNA sequence. Cells respond to this DSB primarily through the NHEJ (Non Homologous End Joining) repair pathway, which results in stochastic insertions or deletions (indels) that can cause frameshift mutations that disrupt the gene. In the presence of a donor DNA template with a high degree of homology to the sequences flanking the DSB, gene correction can be achieved through an alternative pathway known as homology-directed repair (HDR). Unfortunately, under most non-interfering conditions, HDR is inefficient, dependent on cell state and cell type, and dominated by a greater abundance of indels. Because most of the known genetic variations associated with human disease are point mutations, methods that can more efficiently and cleanly generate precise point mutations are needed. Base editing systems as provided herein provide a new way to provide genome editing without creating double-stranded DNA breaks, without requiring a donor DNA template, and without inducing excess stochastic insertions and deletions.
The fusion proteins of the invention advantageously modify a specific nucleotide base encoding a protein comprising a mutation without generating a significant proportion of indels. An "indel" as used herein refers to the insertion or deletion of a nucleotide base within a nucleic acid. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. In some embodiments, it is desirable to create base editors that efficiently modify (e.g., mutate) a specific nucleotide within a nucleic acid without creating a large number of insertions or deletions (i.e., indels) in the nucleic acid. In certain embodiments, each of the base editors provided herein is capable of generating a greater proportion of intended modifications (e.g., mutations) compared to indels.
In some embodiments, each of the basic editor systems provided herein results in less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0, 6% Less than 0.5% Less than 0.4% Less than 0.3% Less than 0.2% Less than 0.1% Less than 0.09% Less than 0.08 %, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02% or less than 0.01% Indel formation in the target polynucleotide sequence.
Some aspects of the disclosure are based on the recognition that each of the base editors provided herein is capable of efficiently generating a intended mutation, such as a point mutation, in a nucleic acid (e.g., a nucleic acid within a subject's genome). without generating a significant number of unintended mutations, such as unintended point mutations. In some embodiments, each of the base editors provided herein is capable of generating at least 0.01% of intended mutations (i.e., at least 0.01% base editing efficiency). In some embodiments, each of the basic editors provided herein is capable of at least 0.01%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40 %, 45%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of intended mutations.
In some embodiments, the basic editors provided herein are capable of generating a ratio of intended mutations to indels that is greater than 1:1. In some embodiments, the basic editors provided herein are capable of generating a ratio of intended mutations to indels that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3, 5 is: 1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5 :1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100 :1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1 or more.
The number of intended mutations and indels can be determined using any suitable method, for example as described in PCT International Application Nos. PCT/2017/045381 (WO2018/027078) and PCT/US2016/058344 (WO2017/070632); Komor, A.C., et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, N.M., et al., "Programmable base editing of A⋅T to G⋅C in genomic DNA without DNA cleavage" Nature 551, 464-471 (2017); and Komor, AC, et al., "Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity" Science Advances 3:eaao4774 (2017); the entire contents of which are hereby incorporated by reference.
In some embodiments, to calculate indel frequencies, sequencing reads are scanned for exact matches with two 10 bp sequences flanking either side of a window where indels can occur. If no exact matches are found, the read is excluded from the analysis. If the length of this indel window exactly matches the reference sequence, the read is classified as containing no indel. If the indel window is two or more bases longer or shorter than the reference sequence, the sequencing read is classified as an insertion or deletion, respectively. In some embodiments, the base editors provided herein can limit the formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor.
The number of indels formed at a target nucleotide region may depend on the length of time a nucleic acid (e.g., a nucleic acid within a cell's genome) is exposed to a base editor. In some embodiments, the number or proportion of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, 3 days, at least 4 days, at least 5 days , at least 7 days, at least 10 days, or at least 14 days of exposure of the target nucleotide sequence (e.g., a nucleic acid within a cell's genome) to a base editor. It is understood that the base editor properties described herein can be applied to any of the fusion proteins or methods of using the fusion proteins provided herein.
In some embodiments, the base editors provided herein are capable of limiting the formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor. In some embodiments, each of the base editors provided herein is capable of reducing the formation of indels in a region of a nucleic acid to less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3 to limit %, less than 3.5%, less than 4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15% or less than 20%. The number of indels formed in a nucleic acid region may depend on the length of time that a nucleic acid (e.g., a nucleic acid within a cell's genome) is exposed to a base editor. In some embodiments, each number or proportion of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, 3 days, at least 4 days, at least 5 days , at least 7 days, at least 10 days, or at least 14 days of exposure of a nucleic acid (e.g., a nucleic acid within the genome of a cell) to a base editor.
Some aspects of the disclosure are based on the recognition that each of the base editors provided herein is capable of efficiently generating a deliberate mutation in a nucleic acid (e.g., a nucleic acid within a subject's genome) without introducing a significant number of unintended mutations generate . In some embodiments, a deliberate mutation is a mutation generated by a specific base editor linked to a gRNA specifically designed to alter or correct an HBG mutation.
In some embodiments, each of the basic editors provided herein is capable of generating a ratio of intended mutations to unintended mutations (e.g., intended mutations:unintended mutations) that is greater than 1:1. In some embodiments, each of the basic editors provided herein is capable of generating a ratio of intended mutations to unintended mutations that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7, 5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least 500:1 or at least 1000:1 or more. It should be noted that the properties of the base editors described herein can be applied to any of the fusion proteins or methods of using the fusion proteins provided herein.
multiplex processing
In some embodiments, the base editing system provided herein is capable of multiplex editing of multiple nucleobase pairs in one or more genes. In some embodiments, the plurality of nucleobase pairs are located in the same gene. In some embodiments, the plurality of nucleobase pairs are located in one or more genes, with at least one gene located at a different locus. In some embodiments, the multiplex editing may include one or more lead polynucleotides. In some embodiments, multiplexed editing may include one or more base editor systems. In some embodiments, the multiplexed editing may involve one or more base editor systems with a single leader polynucleotide. In some embodiments, the multiplexed editing may involve one or more base editor systems with multiple guide polynucleotides. In some embodiments, the multiplex editing may include one or more lead polynucleotides with a single base editing system. In some embodiments, the multiplex editing may include at least one target polynucleotide that does not require a PAM sequence to target binding to a target polynucleotide sequence. In some embodiments, the multiplex editing may include at least one target polynucleotide that requires a PAM sequence to target binding to a target polynucleotide sequence. In some embodiments, the multiplex editing can be a mixture of at least one leader polynucleotide that does not require a PAM sequence to achieve target binding to a target polynucleotide sequence and at least one leader polynucleotide that requires a PAM sequence to target binding to a target polynucleotide reach order. It is understood that the properties of multiplex editing using any of the basic editors as described herein can be applied to any combination of the methods of using any of the basic editors provided herein. It should also be noted that multiplex editing using any of the base editors described herein may involve sequential editing of a plurality of nucleobase pairs.
In some embodiments, the plurality of nucleobase pairs are located in one or more genes. In some embodiments, the plurality of nucleobase pairs are located in the same gene. In some embodiments, at least one gene is located at a different location in the one or more genes.
In some embodiments, editing is editing of the plurality of nucleobase pairs in at least one protein coding region. In some embodiments, editing is editing of the plurality of nucleobase pairs in at least one protein non-coding region. In some embodiments, editing is editing of the plurality of nucleobase pairs in at least one protein coding region and at least one protein non-coding region.
In some embodiments, processing occurs in conjunction with one or more lead polynucleotides. In some embodiments, the base editor system may include one or more base editor systems. In some embodiments, the base editor system may comprise one or more base editor systems associated with a single lead polynucleotide. In some embodiments, the base editor system may comprise one or more base editor systems in conjunction with a plurality of leader polynucleotides. In some embodiments, the editing is done in conjunction with one or more lead polynucleotides with a single base editing system. In some embodiments, the processing is done in conjunction with at least one target polynucleotide that does not require a PAM sequence to direct binding to a target polynucleotide sequence. In some embodiments, the processing is done in conjunction with at least one target polynucleotide that requires a PAM sequence to target binding to a target polynucleotide sequence. In some embodiments, processing occurs in conjunction with a mixture of at least one leader polynucleotide that does not require a PAM sequence to achieve target binding to a target polynucleotide sequence and at least one leader polynucleotide that requires a PAM sequence to achieve target binding to a target to achieve polynucleotide sequence. It should be understood that the properties of multiplexed editing using any of the basic editors described herein can be applied to any combination of the methods of using any of the basic editors provided herein. It should also be noted that editing may involve sequential editing of multiple nucleobase pairs.
Methods for processing nucleic acids
Some aspects of the disclosure provide methods for editing a nucleic acid. In some embodiments, the method is a method of editing a nucleobase of a nucleic acid molecule that encodes a protein (e.g., a base pair of a double-stranded DNA sequence). In some embodiments, the method comprises the steps of: a) contacting a target region of a nucleic acid (eg, a double-stranded DNA sequence) with a complex comprising a base editor and a guide nucleic acid (eg, gRNA), b) inducing a Strand separation of the target region, c) converting a first nucleobase of the target nucleobase pair in a single strand of the target region into a second nucleobase, and d) cleaving no more than one strand of the target region using nCas9, cutting a third nucleobase complementary to the first nucleobase base a fourth nucleobase complementary to the second nucleobase is replaced. In some embodiments, the method results in less than 20% indel formation in the nucleic acid. It is understood that in some embodiments step b is omitted. In some embodiments, the method results in less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2 % or less than 0.1% indel formation. In some embodiments, the method further comprises replacing the second nucleobase with a fifth nucleobase that is complementary to the fourth nucleobase, thereby creating an intended edited base pair (e.g., G⋅C to A⋅T). In some embodiments, at least 5% of the intended base pairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended base pairs are edited.
In some embodiments, the ratio of intended products to unintended products in the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1 , 80:1, 90:1, 100:1 or 200:1 or more. In some embodiments, the ratio of intended mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1 or more. In some embodiments, the cut single strand (nicked strand) is hybridized to the lead nucleic acid. In some embodiments, the nicked single strand is opposite the strand comprising the first nucleobase. In some embodiments, the base editor includes a dCas9 domain. In some embodiments, the base editor protects or binds the unedited strand. In some embodiments, the intended edited base pair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited base pair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor includes a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In one embodiment, the linker is 32 amino acids in length. In another embodiment, a "long linker" is at least about 60 amino acids in length. In other embodiments, the linker is between about 3-100 amino acids in length. In some embodiments, the targeting region includes a targeting window, where the targeting window includes the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended base pair being processed is within the target window. In some embodiments, the target window includes the intended base pair being processed. In some embodiments, the method is performed using one of the basic editors provided herein.
In some embodiments, the disclosure provides methods for editing a nucleotide (e.g., SNP in a gene encoding a protein). In some embodiments, the disclosure provides a method for editing a nucleobase pair of a double-stranded DNA sequence. In some embodiments, the method comprises a) contacting a target region of the double-stranded DNA sequence with a complex comprising a base editor and a guide nucleic acid (e.g. gRNA), wherein the target region comprises a target nucleobase pair, b) inducing strand separation of the target region, c) converting a first nucleobase of the target nucleobase pair in a single strand of the target region into a second nucleobase, d) cleaving no more than one strand of the target region, wherein a third nucleobase is complementary to the first nucleobase base is replaced by a fourth nucleobase which is complementary to the second nucleobase, and the second nucleobase is replaced by a fifth nucleobase complementary to the fourth nucleobase, thereby generating an intended edited base pair, wherein the efficiency of generating the intended edited base pair is at least 5%. It is understood that in some embodiments step b is omitted. In some embodiments, at least 5% of the intended base pairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended base pairs are edited. In some embodiments, the method causes less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2% or less than 0.1% indel formation. In some embodiments, the ratio of intended product to unintended products on the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1 , 80:1, 90:1, 100:1 or 200:1 or more. In some embodiments, the ratio of intended mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1 or more. In some embodiments, the cleaved single strand is hybridized to the lead nucleic acid. In some embodiments, the nicked single strand is opposite the strand comprising the first nucleobase. In some embodiments, the intended edited base pair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited base pair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the targeting region includes a targeting window, where the targeting window includes the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended base pair processed occurs within the target window. In some embodiments, the target window includes the intended base pair being processed. In some embodiments, the nucleobase editor is any of the base editors provided herein.
Expression of fusion proteins in a host cell
Fusion proteins of the invention comprising an adenosine deaminase variant can be expressed in virtually any host cell of interest, including but not limited to bacteria, yeast, fungi, insect, plant and animal cells, using routine methods known to those skilled in the art are. For example, a DNA encoding an adenosine deaminase of the present invention can be cloned by designing appropriate primers for the upstream and downstream of CDS based on the cDNA sequence. The cloned DNA can be ligated directly or, if desired, after digestion with a restriction enzyme or addition of an appropriate linker and/or nuclear localization signal, to DNA encoding one or more additional components of a base editing system. The basic editing system is translated in a host cell to form a complex.
A DNA encoding a protein domain described herein can be obtained by chemically synthesizing the DNA or by joining synthesized partially overlapping oligoDNA short chains using the PCR method and the Gibson assembly method to construct a DNA, which encodes their full length. The advantage of constructing a full-length DNA by chemical synthesis or a combination of PCR methods or Gibson assembly methods is that the codon to be used in full-length CDS can be designed according to the host into which the DNA will be introduced. When a heterologous DNA is expressed, it is expected that the protein expression level increases by converting the DNA sequence thereof into a codon which is very frequently used in the host organism. As the host codon usage frequency data to be used, for example, Genetic Code Use Frequency Database (www.kazusa.or.jp/codon/index.html) available on the home page of Kazusa DNA Research Institute or reference may be made to documents showing codon usage frequency in each host. Referring to the data obtained and the DNA sequence to be introduced, codons showing a low frequency of use in the host can be converted from those used for the DNA sequence into a codon coding for the same amino acid and a high frequency of use shows.
An expression vector containing a DNA encoding a nucleic acid sequence-recognizing module and/or a nucleic acid base-converting enzyme can be prepared, for example, by linking the DNA downstream of a promoter in an appropriate expression vector.
As an expression vectorEscherichia coli- derived plasmids (e.g. pBR322, pBR325, pUC12, pUC13);Bacillus subtilis- derived plasmids (e.g. pUB110, pTP5, pC194); yeast-derived plasmids (e.g. pSH19, pSH15); insect cell expression plasmids (e.g. pFast-Bac); animal cell expression plasmids (e.g. pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo); bacteriophages such as λ phage and the like; insect virus vectors such as baculovirus and the like (e.g. BmNPV, AcNPV); animal virus vectors such as retrovirus, vaccinia virus, adenovirus and the like and the like can be used.
As the promoter, any promoter suitable for a host to be used for gene expression can be used. In a conventional method using DSB, since the survival rate of the host cell sometimes remarkably decreases due to toxicity, it is desirable to increase the number of cells to start induction by using an inductive promoter. However, since sufficient cell proliferation can also be achieved by expressing the nucleic acid-modifying enzyme complex of the present invention, a constitutional promoter can also be used without limitation.
For example, if the host is an animal cell, SR.alpha. Promoter, SV40 Promoter, LTR Promoter, CMV (Cytomegalovirus) Promoter, RSV (Rous Sarcoma Virus) Promoter, MoMuLV (Moloney Murine Leukemia Virus) LTR, HSV-TK (Herpesvirus Simple Thymidine Kinase)- promoter and the like used. Of these, the CMV promoter, SR&agr; Promoter and the like are preferred.
If the host isEscherichia coli, trp promoter, lac promoter, recA promoter, lamda.PL promoter, lpp promoter, T7 promoter and the like are preferred.
When the host is genusBacillusB. SPO1 promoter, SPO2 promoter, penP promoter and the like are preferred.
When the host is a yeast, Gall/10 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferred.
When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferred.
When the host is a plant cell, CaMV35S promoter, CaMV19S promoter, NOS promoter and the like are preferred.
As the expression vector, in addition to those mentioned above, a vector containing enhancer, splicing signal, terminator, polyA addition signal, selection marker such as drug resistance gene, auxotrophic complement gene and the like, origin of replication and the like can be used according to need.
An RNA encoding a protein domain described herein can be produced, for example, by transcription into mRNA in a per se known vitro transcription system using a vector encoding DNA encoding the above-mentioned nucleic acid sequence-recognizing module and/or a nucleic acid are base-converting enzyme as a template.
A fusion protein of the invention can be expressed intracellularly by introducing an expression vector containing a DNA encoding a nucleic acid sequence-recognizing module and/or a nucleic acid base-converting enzyme into a host cell and culturing the host cell.
As host, genusEscherichia, genusBacillusB. yeast, insect cell, insect cell, animal cell and the like can be used.
As a genusEscherichia, Escherichia coliK12.cndot.DH1 [Proc. national Academic Science. USA, 60 , 160 (1968)],Escherichia coliJM103 [Nucleic Acids Research, 9, 309 (1981)],Escherichia coliJA221 [Journal of Molecular Biology, 120, 517 (1978)],Escherichia coli HB101 [Journal of Molecular Biology, 41, 459 (1969)],Escherichia coliC600 [Genetics, 39 , 440 (1954)] and the like are used.
As a genusBacillus, Bacillus subtilisM1114 [Gene, 24, 255 (1983)],Bacillus subtilis207-21 [Journal of Biochemistry, 95 , 87 (1984)] and the like are used.
as yeast,Saccharomyces cerevisiaeAH22, AH22R-, NA87-11A, DKD-5D, 20B-12,Schizosaccharomyces pombeNCYC1913, NCYC2036,shepherd figsKM71 and the like can be used.
As an insect cell, when the virus is AcNPV, cabbage armyworm larvae-derived established lineage cells (Spodoptera frupperdaCell; Sf cell), MG1 cells are derived from the midgut ofis trichoplusia, High Five™ cells derived from an egg ofTrichoplusia ni, Mamestra brassicae-derived cells,stigma-derived cells and the like can be used. If the virus is BmNPV, cells fromBombyx mori-derived established line (Bombyx moriN cell; BmN cell) and the like are used as insect cells. As the Sf cell, for example, an Sf9 cell (ATCC CRL1711), an Sf21 cell [all above, In Vivo, 13, 213-217 (1977)] and the like are used.
B. die Insekt LarvaBombyx mori, Drosophila, cricket and the like [Nature, 315, 592 (1985)].
As the animal cell, cell lines such as monkey COS-7 cell, monkey Vero cell, Chinese hamster ovary (CHO) cell, dhfr gene-deficient CHO cell, mouse L cell, mouse AtT-20 cell B. cell, mouse myeloma cell, rat GH3 cell, human FL cell and the like, pluripotent stem cells such as iPS cells, ES cells and the like of humans and other mammals, and primary cultured cells prepared from various tissues are used. In addition, zebrafish embryo,XenopusOvum and the like can also be used.
As a plant cell, cultured cells, callus, protoplasts, leaf segments, root segments and the like produced from various plants (e.g. crops such as rice, wheat, corn and the like, product plants such as tomato, cucumber, eggplant and the like) are suspended from the like, garden plants such as Clove,Eustoma russelianumand the like, experimental plants such as tobacco,Arabidopsis thalianaand the like and the like) can be used.
All of the above host cells may be haploid (monoploid) or polyploid (e.g., diploid, triploid, tetraploid, and the like). In the conventional mutation introduction methods, the mutation is introduced only into a homologous chromosome in principle to produce a heterogeneous type. Therefore, the desired phenotype will not be expressed unless a dominant mutation occurs, and homozygosity disadvantageously requires labor and time. In contrast, according to the present invention, since a mutation can be introduced into any allele on the homologous chromosome in the genome, the desired phenotype can be expressed in a single generation even in the case of a recessive mutation, which is extremely useful because of the problem with the conventional method can be solved.
An expression vector can be introduced by a known method (e.g. lysozyme method, competent method, PEG method, CaCl2) Coprecipitation method, electroporation method, microinjection method, particle gun method, lipofection method,agrobacteriummethod and the like) according to the type of host.
Escherichia colican be transformed according to the methods described, for example, in Proc. national Academic Science. USA, 69 , 2110 (1972), Gene, 17 , 107 (1982) and the like.
The genusBacilluscan be introduced into a vector according to the methods described, for example, in Molecular & General Genetics, 168 , 111 (1979) and the like.
A yeast can be isolated according to the methods described, for example, in Methods in Enzymology, 194, 182-187 (1991), Proc. national Academic Science. USA, 75 , 1929 (1978) and the like.
An insect cell and an insect can be introduced into a vector according to the methods described in, for example, Bio/Technology, 6, 47-55 (1988) and the like.
An animal cell can be introduced into a vector according to the methods described in, for example, Cell Engineering Additional Volume 8, New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha) and Virology, 52, 456 (1973).
A cell introduced with a vector can be cultured according to a known method according to the kind of the host.
When for exampleEscherichia colior genusBacillusis cultured, a liquid medium is preferred as the medium used for the culture. The medium preferably contains a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of the transformant. examples of the carbon source include glucose, dextrin, soluble starch, sucrose and the like; Examples of the nitrogen source include inorganic or organic substances such as ammonium salts, nitrate salts, corn steep liquor, peptone, casein, meat extract, soybean cake, potato extract and the like; and examples of the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. The medium can contain yeast extract, vitamins, growth promoting factors and the like. The pH of the medium is preferably about 5 to about 8.
as a culture mediumEscherichia coliB. M9 medium containing glucose, casamino acid [Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972] is preferred. If necessary, agents such as 3β-indolylacrylic acid, for example, can be added to the medium in order to ensure efficient functioning of a promoter.Escherichia coliis cultivated at generally about 15-43°C. If necessary, aeration and stirring can be carried out.
The genusBacillusis cultivated at generally about 30-40°C. If necessary, aeration and stirring can be carried out.
Examples of the medium for culturing yeast include Burkholder's minimal medium [Proc. national Academic Science. USA, 77 , 4505 (1980)], SD medium with 0.5% casamino acid [Proc. national Academic Science. USA, 81 , 5330 (1984)] and the like. The pH of the medium is preferably about 5 - about 8. The cultivation is generally carried out at about 20°C - about 35°C. If necessary, aeration and stirring can be carried out.
As a medium for culturing an insect cell or an insect, for example, Grace's Insect is used
Medium [Nature, 195, 788 (1962)] containing an additive such as inactivated 10% bovine serum and the like as appropriate and the like is used. The pH of the medium is preferably about 6.2 to about 6.4. Culture is generally performed at about 27°C. If necessary, aeration and stirring can be carried out.
As a medium for culturing an animal cell, for example, Minimum Essential Medium (MEM) containing about 5-about 20% fetal bovine serum [Science, 122, 501 (1952)], Dulbecco's Modified Eagle's Medium (DMEM) [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association, 199 , 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73 , 1 (1950)] and the like are used. The pH of the medium is preferably about 6 to about 8. The cultivation is generally carried out at about 30°C to about 40°C. If necessary, aeration and stirring can be carried out.
As a medium for culturing a plant cell, for example, MS medium, LS medium, B5 medium and the like are used. The pH of the medium is preferably about 5 - about 8. The cultivation is generally carried out at about 20°C - about 30°C. If necessary, aeration and stirring can be carried out.
When a higher eukaryotic cell such as an animal cell, insect cell, plant cell and the like is used as a host cell, a DNA encoding a base editing system of the present invention (e.g. comprising an adenosine deaminase variant) is converted into a introduced host cell under the regulation of an inducible promoter (e.g., metallothionein promoter (induced by heavy metal ions), heat shock protein promoter (induced by heat shock), Tet-ON/Tet-OFF system promoter (induced by addition or removal of tetracycline ).or a derivative thereof), steroid-responsive promoter (induced by steroid hormone or a derivative thereof), etc.), the inducing substance is added to the medium (or removed from the medium) at an appropriate stage to induce the expression of the nucleic acid -modifying enzyme complex, culture is performed for a given period of time to perform base editing, and by introducing mutation into a target gene, transient expression of the base editing system can be realized.
Prokaryotic cells such asEscherichia coliand the like can use an inducible promoter. Examples of the inducible promoter include, but are not limited to, lac promoter (induced by IPTG), cspA promoter (induced by cold shock), araBAD promoter (induced by arabinose), and the like.
Alternatively, the above-mentioned inductive promoter can also be used as a vector removal mechanism when higher eukaryotic cells such as animal cells, insect cells, plant cells and the like are used as the host cell. That is, a vector is assembled with an origin of replication that functions in a host cell and a nucleic acid encoding a protein necessary for replication (eg, SV40 on and large T antigen, oriP, and EBNA-1 etc. for animal cells). expression of the nucleic acid encoding the protein is regulated by the inducible promoter mentioned above. Thus, while the vector is autonomously replicable in the presence of an inducer, autonomous replication is not available when the inducer is removed, and the vector naturally falls off along with cell division (autonomous replication is not possible by the addition). of tetracycline and doxycycline in the Tet-OFF system vector).
delivery system
Nucleic acid-based delivery of a nucleobase editor and gRNAs
Nucleic acids encoding base-editing systems according to the present disclosure can be administered to subjects or delivered into cells in vitro or in vivo by methods known in the art or as described herein. In one embodiment, nucleobase editors can e.g. B. be delivered by vectors (e.g. viral or non-viral vectors), non-vector based methods (e.g. using naked DNA, DNA complexes, lipid nanoparticles) or a combination thereof. In one embodiment, nucleobase editors are selectively delivered to cells (e.g., hepatocytes, embryonic stem cells, induced pluripotent stem cells (iPSCs), organoids). In other embodiments, nucleic acids encoding nucleobase editors are delivered to hepatocyte (liver) cells or their progenitors and/or induced pluripotent stem cells comprising mutations in the alpha1-antitrypsin (A1A7) gene. Such cells can be used to study the functional effects of alpha1 anti-trypsin gene editing. In one embodiment, the effect of an altered alpha1-antitrypsin gene in a hepatocyte is examined.
Nucleic acids encoding nucleobase editors may or may be delivered directly to cells (e.g., hematopoietic cells or their progenitors, hematopoietic stem cells, and/or induced pluripotent stem cells) as naked DNA or RNA, for example by transfection or electroporation conjugated to molecules (e.g. N-acetylgalactosamine) that promote uptake by target cells. Nucleic acid vectors, such as the vectors described herein, can also be used.
Nucleic acid vectors can comprise one or more sequences encoding a domain of a fusion protein described herein. A vector may also include a sequence encoding a signal peptide (eg, for nuclear localization, nucleolar localization, or mitochondrial localization) associated with (eg, inserted into or fused to) a sequence encoding a protein. As an example, a nucleic acid vector may include a Cas9 coding sequence that includes one or more nuclear localization sequences (e.g., an SV40 nuclear localization sequence) and an adenosine deaminase variant (e.g., ABE8).
The nucleic acid vector may also contain any suitable number of regulatory/controlling elements, e.g. B. promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences or internal ribosome entry sites (IRES). These elements are well known in the art. For hematopoietic cells, suitable promoters may include IFNbeta or CD45.
Nucleic acid vectors according to this disclosure include recombinant viral vectors. Exemplary viral vectors are set forth herein. Other viral vectors known in the art can also be used. In addition, viral particles can be used to deliver base editing system components in nucleic acid and/or peptide form. For example, "empty" virus particles can be assembled to contain any suitable cargo. Viral vectors and viral particles can also be engineered to incorporate targeting ligands to alter the specificity of the target tissue.
In addition to viral vectors, non-viral vectors can be used to deliver nucleic acids encoding genome editing systems according to the present disclosure. An important category of non-viral nucleic acid vectors are nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art. Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components. For example, organic (e.g., lipid and/or polymeric) nanoparticles may be suitable for use as delivery vehicles in certain embodiments of this disclosure. Exemplary lipids for use in nanoparticle formulations and/or gene transfer are shown in Table 10 (below).
Table 11 lists exemplary polymers for use in gene delivery and/or nanoparticle formulations.
Table 12 summarizes delivery methods for a polynucleotide encoding a fusion protein described herein.
In another aspect, delivery of genome editing system components or nucleic acids encoding such components, such as a nucleic acid-binding protein such as Cas9 or variants thereof, and a gRNA that targets a genomic nucleic acid sequence of interest, can be accomplished by delivery of a ribonucleoprotein (RNP) to cells. The RNP comprises the nucleic acid binding protein, e.g. B. Cas9, in complex with the target gRNA. RNPs can be delivered to cells using known methods, such as. B. electroporation, nucleofection or cationic lipid-mediated methods as reported by Zuris, J.A. et al., 2015, Nat. biotechnology,33(1):73-80. RNPs are advantageous for use in CRISPR baseline processing systems, particularly for cells that are difficult to transfect, such as. B. Primary cells. In addition, RNPs can also alleviate difficulties encountered in protein expression in cells, particularly when eukaryotic promoters, e.g. B. CMV or EF1A, which can be used in CRISPR plasmids, are not well expressed. Advantageously, the use of RNPs does not require delivery of foreign DNA into cells. In addition, since an RNP comprising a nucleic acid-binding protein and a gRNA complex degrades over time, the use of RNPs has the potential to limit off-target effects. In a manner similar to plasmid-based techniques, RNPs can be used to deliver binding protein (e.g. Cas9 variants) and direct homology-directed repair (HDR).
A promoter used to drive base editor-encoding nucleic acid molecule expression may include AAV ITR. This can be advantageous in order to dispense with an additional promoter element that can take up space in the vector. The extra space freed up can be used to drive the expression of additional elements, such as a guide nucleic acid or a selectable marker. The ITR activity is relatively weak, so it can be used to reduce potential toxicity due to over-expression of the chosen nuclease.
Any suitable promoter can be used to drive expression of the base editor and optionally the guide nucleic acid. For ubiquitous expression, promoters including CMV, CAG, CBh, PGK, SV40, ferritin heavy or light chains, etc. can be used. For brain or other CNS cell expression, suitable promoters may include: SynapsinI for all neurons, CaMKIIalpha for excitatory neurons B. GAD67 or GAD65 or VGAT for GABAergic neurons, etc. For liver cell expression, suitable promoters include the albumin promoter. For lung cell expression, suitable promoters may include SP-B. For endothelial cells, suitable promoters may include ICAM. For hematopoietic cells, suitable promoters may include IFNbeta or CD45. Promoters suitable for osteoblasts may include OG-2.
In some embodiments, a base editor of the present disclosure is small enough to allow separate promoters to drive expression of the base editor and a compatible guide nucleic acid within the same nucleic acid molecule. For example, a vector or viral vector may comprise a first promoter operably linked to a nucleic acid encoding the base editor and a second promoter operably linked to the guide nucleic acid.
The promoter used to drive expression of a target nucleic acid can include: Pol III promoters such as U6 or H1. Use of Pol II promoter and intron cassettes to express Adeno-Associated Virus (AAV) gRNA.
Viral Vectors
A base editor described herein can therefore be supplied with viral vectors. In some embodiments, a base editor disclosed herein may be encoded on a nucleic acid contained in a viral vector. In some embodiments, one or more components of the basic editor system may be encoded on one or more viral vectors. For example, a base editor and a guide nucleic acid can be encoded on a single viral vector. In other embodiments, the base editor and the guide nucleic acid are encoded on different viral vectors. In either case, the base editor and guide nucleic acids may each be operably linked to a promoter and terminator. The combination of components encoded on a viral vector may be dictated by the cargo size limitations of the chosen viral vector.
The use of viral RNA or DNA systems to deliver a base editor utilizes sophisticated processes for targeting a virus to specific cells in culture or in the host and transporting the viral payload to the nucleus or host cell genome. Viral vectors can be administered directly to cells in culture or patients (in vivo), or they can be used to treat cells in vitro and the modified cells optionally administered to patients (ex vivo). Conventional virus-based systems could include retrovirus, lentivirus, adenovirus, adeno-associated, and herpes simplex virus vectors for gene transfer. Integration into the host genome is possible using the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. In addition, high transduction efficiencies have been observed in many different cell types and target tissues.
Viral vectors can include lentivirus (e.g., HIV and FIV-based vectors), adenovirus (e.g., AD100), retrovirus (e.g., Maloney murine leukemia virus, MML-V), herpesvirus vectors (e.g., B. HSV-2) and adenovirus. associated viruses (AAVs) or other plasmid or viral vector types, particularly using formulations and doses from e.g. B. US Pat. No. 8,454,972 (formulations, doses for adenovirus), US Pat. No. 8,404,658 (formulations, doses for AAV) and US Pat. No. 5,846,946 (formulations, dosages for DNA plasmids) and from clinical studies and publications relating to the clinical studies with lentivirus, AAV and adenovirus. For example, for AAV, the route of administration, formulation and dose can be as described in US Pat. No. 8,454,972 and as in AAV clinical trials. For adenovirus, the route of administration, formulation and dose can be as described in US Pat. No. 8,404,658 and as in clinical trials with adenovirus. For plasmid delivery, the route of administration, formulation and dose can be as described in US Pat. No. 5,846,946 and as in clinical studies with plasmids. Doses may be based on or extrapolated from an average 70 kg individual (eg, adult male) and may be adjusted for patients, subjects, mammals of different weights and species. The frequency of administration is within the range of the medical practitioner or veterinarian (e.g., physician, veterinarian) depending on usual factors including age, gender, general health, other conditions of the patient or subject and the particular condition or symptoms being treated. The viral vectors can be injected into the tissue of interest. For cell-type specific base editing, expression of the base editor and optional guide nucleic acid can be driven by a cell-type specific promoter. In some aspects, the disclosure relates to viral delivery of a nucleobase editor targeting an alpha1-antitrypsin mutation using, for example, a viral vector such as a lentiviral vector or a recombinant adeno-associated virus vector.
A retrovirus' tropism can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically produce high titers of virus. The selection of a retroviral gene delivery system would therefore depend on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimal cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon simian leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g. Buchscher et al, J Virol 66: 2731-2739 (1992), Johann et al, J Virol 66: 1635-1640 (1992), Sommnerfelt et al, Virol 176: 58-59 (1990). ), Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700).
Retroviral vectors, particularly lentiviral vectors, may require polynucleotide sequences smaller than a given length for efficient integration into a target cell. For example, retroviral vectors greater than 9 kb in length may result in low viral titers compared to those of smaller size. In some aspects, a base editor of the present disclosure is of sufficient size to allow efficient packaging and delivery into a target cell via a retroviral vector. In some embodiments, a base editor is sized to allow for efficient packaging and delivery even when co-expressed with a guide nucleic acid and/or other components of a targeting nuclease system.
In applications where transient expression is preferred, adenovirus-based systems can be used. Adenovirus-based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. High titers and levels of expression have been obtained with such vectors. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors can also be used to transduce cells with target nucleic acids, e.g. B. in the in vitro production of nucleic acids and peptides and for in vivo and ex vivo gene therapy methods (see, e.g., West et al., Virology 160: 38-47 (1987); US Patent No. 4,797,368; WO 93/24641, Kotin, Human Gene Therapy 5:793-801 (1994), Muzyczka, J Clin Invest 94:1351 (1994). U.S. Patent No. 5,173,414, Tratschin et al Mol Cell Biol 5:3251-3260 (1985) Tratschin et al Mol Cell Biol 4:2072-2081 (1984) Hermonat & Muzyczka , PNAS 81 :6466-6470 (1984) and Samulski et al., J Virol 63 :03822-3828 (1989).
AAV is a small, single-stranded DNA-dependent virus that belongs to the parvovirus family. The 4.7 kb wild-type (wt) AAV genome consists of two genes encoding four replication proteins, respectively. encode three capsid proteins, and is flanked on either side by 145 bp inverted terminal repeats (ITRs). The virion consists of three capsid proteins, Vp1, Vp2, and Vp3, produced in a 1:1:10 ratio from the same open reading frame, but differential splicing (Vp1) and alternative translational start sites (Vp2 and Vp3, respectively). . Vp3 is the most abundant subunit in the virion and participates in cell surface receptor recognition that defines the tropism of the virus. A phospholipase domain that functions in viral infectivity has been identified in the unique N-terminus of Vp1.
Similar to wt AAV, recombinant AAV (rAAV) uses the cis-acting 145 bp ITRs to flank vector transgene cassettes, providing up to 4.5 kb for packaging of foreign DNA. After infection, rAAV can express a fusion protein of the invention and persist without integration into the host genome by existing episomally in head-to-tail circular concatemers. Although there are numerous examples of the success of rAAV using this system, in vitro and in vivo, the limited packaging capacity has limited the use of AAV-mediated gene delivery when the length of the gene's coding sequence is equal to or greater than that of wt AAV -genome.
Viral vectors can be chosen based on the application. For example, AAV may be advantageous for in vivo gene delivery over other viral vectors. In some embodiments, AAV allows for low toxicity, which may be due to the purification process not requiring ultracentrifugation of cellular particles capable of activating the immune response. In some embodiments, AAV allows for a low probability of causing insertional mutagenesis because it does not integrate into the host genome. Adenoviruses are commonly used as vaccines because of the powerful immunogenic response they induce. The packaging capacity of viral vectors can limit the size of the basic editor that can be packaged into the vector.
AAV has a packaging capacity of approximately 4.5 Kb or 4.75 Kb including two 145 base inverted terminal repeats (ITRs). This means that the disclosed base editor as well as a promoter and a transcription terminator can fit into a single viral vector. Constructs larger than 4.5 or 4.75 Kb can result in significantly reduced virus production. For example, SpCas9 is quite large, the gene itself is over 4.1 Kb, making it difficult to pack into AAV. Therefore, embodiments of the present disclosure include the use of a disclosed base editor that is shorter than conventional base editors. In some examples, the base editors are smaller than 4 KB. Exposed basic editors can be smaller than 4.5kb, 4.4kb, 4.3kb, 4.2kb, 4.1kb, 4kb, 3.9kb, 3.8kb, 3.7kb, 3, 6kb, 3.5kb, 3.4kb, 3.3kb, 3.2kb, 3.1kb, 3kb, 2.9kb, 2.8kb, 2.7kb, 2.6kb, 2.5KB, 2KB or 1.5KB. In some embodiments, the basic editors disclosed are 4.5 kb or less in length.
An AAV can be AAV1, AAV2, AAV5, or any combination thereof. One can choose the AAV type with regard to the cells to be targeted; e.g. eg one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 to target heart tissue. AAV8 is useful for delivery to the liver. A tabulation of specific AAV serotypes relative to these cells can be found in Grimm, D. et al., J. Virol. 82: 5887-5911 (2008)).
Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both mitotic and postmitotic cells. The best-known lentivirus is Human Immunodeficiency Virus (HIV), which uses the envelope glycoproteins of other viruses to attack a wide range of cell types.
Lentiviruses can be produced as follows. After cloning pCasES10 (which contains a lentiviral transfer plasmid backbone), HEK293FT was reduced to 50% at low passage (p=5) the day before transfection in DMEM with 10% fetal bovine serum and no antibiotics in a T-75 flask. seeded confluence. After 20 hours, the medium is changed to OptiMEM (serum-free) medium and transfection was performed 4 hours later. Cells are transfected with 10 µg lentiviral transfer plasmid (pCasES10) and the following packaging plasmids: 5 µg pMD2.G (VSV-g pseudotype) and 7.5 µg psPAX2 (gag/pol/rev/tat) . Transfection can be done in 4 mL OptiMEM with a cationic lipid delivery agent (50 μL Lipofectamine 2000 and 100 μL Plus Reagent). After 6 hours, the medium is changed to antibiotic-free DMEM with 10% fetal bovine serum. These methods use serum during cell culture, but serum-free methods are preferred.
Lentivirus can be purified as follows. Viral supernatants are harvested after 48 hours. The supernatants are first cleaned of debris and filtered through a 0.45 µm low protein binding (PVDF) filter. They are then spun in an ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets are resuspended in 50 µl DMEM overnight at 4°C. They are then aliquoted and immediately frozen at -80°C.
In another embodiment, non-primate lentiviral minimal vectors based on Equine Infectious Anemia Virus (EIAV) are also contemplated. In another embodiment, RetinoStat® is a equine infectious anemia virus-based lentiviral gene therapy vector expressing the angiostatic proteins endostatin and angiostatin to be administered via subretinal injection. In another embodiment, the use of self-inactivating lentiviral vectors is contemplated.
Each RNA of the systems, for example a guide RNA or a base editor-encoding mRNA, can be supplied in the form of RNA. Base editor-encoding mRNA can be generated using in vitro transcription. For example, nuclease mRNA can be synthesized using a PCR cassette containing the following elements: T7 promoter, optional Kozak sequence (GCCACC), nuclease sequence, and 3' UTR, such as a beta globin 3' UTR -PolyA tail. The cassette can be used for transcription by T7 polymerase. Leader polynucleotides (e.g. gRNA) can also be transcribed using in vitro transcription from a cassette containing a T7 promoter followed by the sequence "GG" and the leader polynucleotide sequence.
In order to enhance expression and reduce possible toxicity, the base editor coding sequence and/or the guide nucleic acid can be modified to contain one or more modified nucleosides, e.g. using pseudo-U or 5-methyl-C.
The low packaging capacity of AAV vectors makes delivery of a range of genes larger than this size and/or the use of large physiological regulatory elements challenging. These challenges can be addressed, for example, by splitting the protein(s) to be delivered into two or more fragments, fusing the N-terminal fragment to a split intein N and the C-terminal fragment to a Split is fused intotein-C. These fragments are then packaged into two or more AAV vectors. As used herein, "intein" refers to a self-splicing protein intron (e.g., peptide) that ligates flanking N-terminal and C-terminal exteins (e.g., fragments to be joined). The use of certain inteins to join heterologous protein fragments is described, for example, in Wood et al., J. Biol. Chem. 289(21); 14512-9 (2014). For example, when fused to separate protein fragments, the inteins IntN and IntC recognize each other, splice themselves out, and simultaneously ligate the flanking N- and C-terminal exteins of the protein fragments to which they have been fused, rendering them recover a full-length protein from the two protein fragments. Other suitable proteins will be apparent to those skilled in the art.
A fragment of a fusion protein of the invention can vary in length. In some embodiments, a protein fragment is from 2 amino acids to about 1000 amino acids in length. In some embodiments, a protein fragment ranges in length from about 5 amino acids to about 500 amino acids. In some embodiments, a protein fragment is from about 20 amino acids to about 200 amino acids in length. In some embodiments, a protein fragment ranges in length from about 10 amino acids to about 100 amino acids. Suitable protein fragments of other lengths will be apparent to those skilled in the art.
In one embodiment, dual AAV vectors are generated by splitting a large transgene expression cassette into two separate halves (5' and 3' ends or head and tail), with each half of the cassette inserted into a single AAV vector (from < 5kB). Reassembly of the full-length transgene expression cassette is then achieved after co-infection of the same cell by both dual AAV vectors, followed by: (1) homologous recombination (HR) between 5' and 3' genomes (dual AAV overlap vectors); (2) ITR-mediated tail-to-head concatemerization of 5' and 3' genomes (AAV dual trans-splicing vectors); or (3) a combination of these two mechanisms (double AAV hybrid vectors). The use of dual AAV vectors in vivo results in the expression of full-length proteins. Using the dual AAV vector platform represents an efficient and viable gene delivery strategy for transgenes >4.7 kb in size.
guts
In some embodiments, a portion or fragment of a nuclease (e.g., Cas9) is fused to an intein. The nuclease can be fused to the N-terminus or the C-terminus of the intein. In some embodiments, a portion or fragment of a fusion protein is fused to an intein and fused to an AAV capsid protein. The intein, nuclease, and capsid protein can be fused together in any configuration (e.g., nuclease-intein-capsid, intein-nuclease-capsid, capsid-intein-nuclease, etc.). In some embodiments, the N-terminus of an intein is fused to the C-terminus of a fusion protein, and the C-terminus of the intein is fused to the N-terminus of an AAV capsid protein.
Inteins (intervening protein) are self-processing domains found in a variety of different organisms that perform a process known as protein splicing. Protein splicing is a multistep biochemical reaction involving both cleavage and peptide bond formation. While the endogenous substrates of protein splicing are proteins found in intein-containing organisms, inteins can also be used to chemically manipulate virtually any polypeptide backbone.
During protein splicing, the intein excises itself from a precursor polypeptide by cleaving two peptide bonds, thereby ligating the flanking extein (external protein) sequences via the formation of a new peptide bond. This rearrangement occurs post-translationally (or possibly co-translationally). Intein-mediated protein splicing occurs spontaneously and requires only the folding of the intein domain.
About 5% of inteins are split inteins, which are transcribed and translated as two separate polypeptides, the N-intein and the C-intein, each fused to an extein. During translation, the intein fragments spontaneously and non-covalently assemble into the canonical intein structure to perform protein splicing in trans. The mechanism of protein splicing involves a series of acyl transfer reactions that result in the cleavage of two peptide bonds at the intein-extein junctions and the formation of a new peptide bond between the N- and C-exteins. This process is initiated by activation of the peptide bond that connects the N-extein and the N-terminus of the intein. Almost all inteins have a cysteine or serine at their N-terminus, which attacks the carbonyl carbon of the C-terminal N-extein residue. This N-to-O/S acyl shift is facilitated by a conserved threonine and histidine (termed the TXXH motif) along with an abundant aspartate, resulting in the formation of a linear (thio)ester intermediate. Next, this intermediate undergoes trans-(thio)-esterification by nucleophilic attack of the first C-extein residue (+1), which is a cysteine, serine, or threonine. The resulting branched (thio)ester intermediate is separated by a unique transformation: cyclization of the intein's highly conserved C-terminal asparagine. This process is facilitated by the histidine (found in a highly conserved HNF motif) and the penultimate histidine, and may also affect aspartate. This succinimidation reaction excises the intein from the reactive complex, leaving the exteins bound by a non-peptidic bond. This structure rapidly rearranges into a stable peptide bond in an intein-independent manner.
In some embodiments, an N-terminal fragment of a base editor (e.g., ABE, CBE) is fused to a split intein-N and a C-terminal fragment is fused to a split intein-C. These fragments are then packaged into two or more AAV vectors. The use of certain inteins to join heterologous protein fragments is described, for example, in Wood et al., J. Biol. Chem. 289(21); 14512-9 (2014). For example, when fused to separate protein fragments, the inteins IntN and IntC recognize each other, splice themselves out, and simultaneously ligate the flanking N- and C-terminal exteins of the protein fragments to which they have been fused, rendering them recover a full-length protein from the two protein fragments. Other suitable proteins will be apparent to those skilled in the art.
In some embodiments, an ABE has been cleaved into N- and C-terminal fragments at Ala, Ser, Thr, or Cys residues within selected regions of SpCas9. These regions correspond to loop regions identified by Cas9 crystal structure analysis. The N-terminus of each fragment is fused to an intein-N and the C-terminus of each fragment is fused to an intein-C at amino acid positions S303, T310, T313, S355, A456, S460, A463, T466, S469. T472, T474, C574, S577, A589 and S590 given in bold capital letters in the following order.
Using nucleobase editors to target mutations
The suitability of nucleobase editors targeting mutation is evaluated as described herein. In one embodiment, a single cell of interest is transduced with a base editing system along with a small amount of a vector encoding a reporter (e.g., GFP). These cells can be any cell line known in the art, including immortalized human cell lines such as 293T, K562 or U20S. Alternatively, primary cells (e.g., human) can be used. Such cells may be relevant to the ultimate cell goal.
Delivery can be performed using a viral vector. In one embodiment, transfection can be performed using lipid transfection (such as Lipofectamine or Fugene) or by electroporation. After transfection, the expression of GFP can be determined either by fluorescence microscopy or by flow cytometry to confirm consistent and high levels of transfection. These preliminary transfections can involve different nucleobase editors to determine which combinations of editors yield the greatest activity.
The activity of the nucleobase editor is assessed as described herein, i. H. by sequencing the cells' genome to detect changes in a target sequence. For Sanger sequencing, purified PCR amplicons are cloned into a plasmid backbone, transformed, miniprepared, and sequenced with a single primer. Sequencing can also be performed using next generation sequencing techniques. Using next-generation sequencing, amplicons can be 300-500 bp in length with the intended cleavage site placed asymmetrically. After PCR, next-generation sequencing adapters and barcodes (e.g., Illumina multiplex adapters and indexes) can be attached to the ends of the amplicon, e.g. B. for use in high-throughput sequencing (e.g. on an Illumina MiSeq).
The fusion proteins that induce the greatest levels of target-specific changes in initial tests can be selected for further evaluation.
In certain embodiments, the nucleobase editors are used to target polynucleotides of interest. In one embodiment, a nucleobase editor of the invention is delivered to cells (e.g., hematopoietic cells or their progenitors, hematopoietic stem cells, and/or induced pluripotent stem cells) in association with a guide RNA that is used to target a mutation of interest to be targeted within a cell's genome, thereby altering the mutation. In some embodiments, a base editor is targeted by a guide RNA to introduce one or more edits into the sequence of a gene of interest.
The system can include one or more different vectors. In one aspect, the base editor is codon-optimized for expression of the desired cell type, preferably a eukaryotic cell, preferably a mammalian cell or a human cell.
In general, codon optimization refers to a method of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15 , 20, 25, 50 or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell, while retaining the native amino acid sequence. Different species show a particular preference for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with translation efficiency of messenger RNA (mRNA), which in turn is thought to depend, among other things, on the properties of the codons to be translated and the availability of certain ones RNA (tRNA) molecules transferred. The prevalence of selected tRNAs in a cell generally reflects the codons most commonly used in peptide synthesis. Accordingly, genes can be tailored based on codon optimization for optimal gene expression in a given organism. For example, codon usage tables are readily available in the "Codon Usage Database" available at www.kazusa.orjp/codon/ (accessed July 9, 2002), and these tables can be customized in a number of ways. See Nakamura, Y., et al. "Codon Usage Tabulated from the International DNA Sequence Databases: Status for the Year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon-optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.) are also available. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more or all codons) in a sequence encoding an engineered nuclease conform to the am commonly used codon for a specific amino acid.
Packaging cells are typically used to form virus particles that can infect a host cell. Such cells include 293 cells, which package adenovirus, and psi.2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by making a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, replacing other viral sequences with an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans from the packaging cell line. For example, AAV vectors used in gene therapy typically have only ITR sequences from the AAV genome required for packaging and integration into the host genome. Viral DNA can be packaged into a cell line containing a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line can also be infected with adenovirus as a helper. The helper virus can promote replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts in some cases due to a lack of ITR sequences. Adenovirus contamination can e.g. by heat treatment, to which adenovirus is more sensitive than AAV.
pharmaceutical compositions
Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the base editors, fusion proteins or fusion protein-leader-polynucleotide complexes described herein. The term "pharmaceutical composition" as used herein refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition includes additional active ingredients (e.g., for specific delivery, half-life enhancement, or other therapeutic compounds).
The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle such as a pharmaceutically acceptable vehicle. or stearic acid) or solvent encapsulation material involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body to another site (e.g., organ, tissue, or part of the body). A pharmaceutically-acceptable carrier is "acceptable" in the sense that it is compatible with the other ingredients of the formulation and is not harmful to the subject's tissues (e.g., physiologically compatible, sterile, physiological pH, etc.).
Some non-limiting examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents such as polypeptides and amino acids (23) serum alcohols such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, colors, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. The terms such as "excipient," "carrier," "pharmaceutically acceptable carrier," "vehicle," or the like are used interchangeably herein.
Pharmaceutical compositions may include one or more pH buffering compounds to maintain the pH of the formulation at a predetermined level that reflects physiological pH, such as in the range of about 5.0 to about 8.0. The pH buffering compound used in the aqueous liquid formulation can be an amino acid or a mixture of amino acids, such as histidine, or a mixture of amino acids, such as histidine and glycine. Alternatively, the pH buffering compound is preferably an agent which maintains the pH of the formulation at a predetermined level, for example in the range from about 5.0 to about 8.0, and which does not chelate calcium ions. Illustrative examples of such pH buffering compounds include, but are not limited to, imidazole and acetate ion. The pH buffering compound can be present in any amount effective to maintain the pH of the formulation at a predetermined level.
Pharmaceutical compositions may also contain one or more osmotic modulators, i. H. a compound that modulates the osmotic properties (e.g., tonicity, osmolality, and/or osmotic pressure) of the formulation to a level that is acceptable to the bloodstream and blood cells of recipient subjects. The osmotically modulating agent can be an agent that does not chelate calcium ions. The osmotic modulating agent can be any compound known or available to those skilled in the art that modulates the osmotic properties of the formulation. One skilled in the art can empirically determine the suitability of a given osmotic modulating agent for use in the formulation of the present invention. Illustrative examples of suitable types of osmotic modulators include, but are not limited to: salts, such as sodium chloride and sodium acetate; sugars such as sucrose, dextrose and mannitol; amino acids such as glycine; and mixtures of one or more of these agents and/or types of agents. The osmotic modulating agent(s) can be present in any concentration sufficient to modulate the osmotic properties of the formulation.
In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, e.g. B. for gene editing. Suitable routes of administration of the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorganic, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous , periocular, intratumoral, intracerebral and intracerebroventricular administration.
In some embodiments, the pharmaceutical composition described herein is administered locally to a diseased site (e.g., a tumor site). In some embodiments, the pharmaceutical composition described herein is administered to a subject by injection, via a catheter, via a suppository, or via an implant, wherein the implant is made of a porous, non-porous, or gelatinous material such as a membrane such as a membrane. B. a sialastic membrane, or a fiber.
In other embodiments, the pharmaceutical composition described herein is delivered in a controlled release system. In one embodiment, a pump may be used (see, e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N Engl J Med 321:574). In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York , 1984), Ranger and Peppas, 1983, Macromol Sci Rev Macromol Chem 23:61 See also Levy et al 1985 Science 228:190 While et al 1989 Ann Neurol 25 :351 Howard et al., 1989, J Neurosurg 71:105.) Other controlled release systems are discussed, for example, in Langer, supra.
In some embodiments, the pharmaceutical composition is formulated according to routine procedures as a composition suitable for intravenous or subcutaneous administration to a subject, e.g. B. a human being adapted. In some embodiments, pharmaceutical compositions for administration by injection are solutions in sterile isotonic use as a solubilizing agent and a local anesthetic such as lignocaine to relieve pain at the injection site. In general, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate, in a hermetically sealed container, such as an ampoule or sachet, which indicates the quantity of the active ingredient. If the drug is to be administered by infusion, it can be delivered using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline solution may be provided so that the ingredients can be mixed prior to administration.
A pharmaceutical composition for systemic administration may be a liquid, e.g. B. sterile saline, Ringer's lactate or Hank's solution. In addition, the pharmaceutical composition may be in a solid form and reconstituted or suspended immediately before use. Lyophilized forms are also contemplated. The pharmaceutical composition may be contained in a lipid particle or vesicle, such as a liposome or microcrystal, also suitable for parenteral administration. The particles can have any suitable structure, e.g. B. unilamellar or plurilamellar, as long as compositions are contained therein. Compounds can be entrapped in "stabilized plasmid lipid particles" (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), small amounts (5-10 mol%) of cationic lipid and stabilized by a polyethylene glycol (PEG) coating (Zhang Y.P. et al., Gene Ther. 1999, 6:1438-47). Positively charged lipids such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate or "DOTAP" are particularly preferred for such particles and vesicles. The manufacture of such lipid particles is well known. See e.g. B. US Pat. No. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference.
For example, the pharmaceutical composition described herein may be administered or packaged as a unit dose. The term "unit dose" when used in relation to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as a unit dose for the subject, each unit containing a predetermined amount of active material which is calculated , in order to produce the desired therapeutic effect in conjunction with the necessary diluent; i.e. carrier or vehicle.
Further, the pharmaceutical composition may be provided as a pharmaceutical kit comprising (a) one container containing a compound of the invention in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g. sterile for reconstitution or dilution of the lyophilized compound of the invention Optionally, such containers may have associated therewith a notice in the form prescribed by any governmental agency which regulates the manufacture, use or sale of pharmaceutical or biological products, the notice permitting approval by the manufacturer - and use authority reflects or sale for human administration.
In another aspect, an article of manufacture containing materials useful for treating the diseases described above is included. In some embodiments, the article of manufacture includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic. In some embodiments, the container contains a composition effective to treat a disease as described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle. The active ingredient in the composition is a compound of the invention. In some embodiments, the label on the or associated with the container indicates that the composition is used to treat the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution or dextrose solution. It may also contain other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
In some embodiments, any of the fusion proteins, gRNAs, and/or complexes described herein are provided as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any of the fusion proteins provided herein. In some embodiments, the pharmaceutical composition comprises any of the complexes provided herein. In some embodiments, the pharmaceutical composition includes a ribonucleoprotein complex that includes an RNA-directed nuclease (e.g., Cas9) that forms a complex with a gRNA and a cationic lipid. In some embodiments, the pharmaceutical composition comprises a gRNA, a nucleic acid-programmable DNA-binding protein, a cationic lipid, and a pharmaceutically acceptable excipient. Pharmaceutical compositions can optionally comprise one or more additional therapeutically active substances.
In some embodiments, compositions provided herein are administered to a subject, such as a human subject, to effect targeted genomic modification within the subject. In some embodiments, cells are obtained from the subject and contacted with any of the pharmaceutical compositions provided herein. In some embodiments, cells removed from a subject and contacted ex vivo with a pharmaceutical composition are reintroduced into the subject, optionally after the desired genomic modification has been effected or detected in the cells. Methods for delivering pharmaceutical compositions comprising nucleases are known and are described, for example, in US Pat. No. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of which are incorporated herein by reference in their entireties. Although the descriptions of pharmaceutical compositions provided herein are primarily directed to pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to animals or organisms of any kind, e.g. veterinary use.
The modification of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals is well known and the veterinary pharmacologist of ordinary skill can design and/or such modification with little if any experimentation carry out. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, domestic animals, domestic animals and commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese and/or turkeys.
Formulations of the pharmaceutical compositions described herein may be prepared by any method known or later developed in the art of pharmacology. In general, such manufacturing processes involve the step of bringing the active ingredient(s) into association with an excipient and/or one or more other auxiliary ingredients and then, if necessary and/or desirable, forming and/or packaging the product therein in a desired individual or multi-dose unit. Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient which, as used herein, includes any and all solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, e.g. solid binders , lubricants, and the like, as appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety) discloses various excipients used in the formulation of pharmaceutical compositions, and known techniques for their manufacture. See also PCT application PCT/US2010/055131 (publication number WO2011/053982 A8, filed Nov. 2, 2010), which is incorporated herein by reference in its entirety, for additional suitable methods, reagents, excipients and solvents for preparing pharmaceutical compositions , comprising a nuclease .
Except to the extent that a conventional carrier medium is incompatible with a substance or its derivatives, such as by causing undesirable biological effects or otherwise deleterious interactions with other components of the pharmaceutical composition, their use is considered within the scope of this disclosure.
The compositions described above can be administered in effective amounts. The effective amount depends on the mode of administration, the particular condition being treated, and the result desired. It may also depend on the stage of the disease, the person's age and physical condition, the type of concomitant therapy, if any, and similar factors well known to the physician. For therapeutic uses, the amount is sufficient to produce a medically desirable result.
In some embodiments, compositions according to the present disclosure can be used to treat a variety of diseases, disorders, and/or conditions.
kits
Various aspects of this disclosure provide kits that include a basic editor system. In one embodiment, the kit comprises a nucleic acid construct comprising a nucleotide sequence encoding a nucleobase editor fusion protein. The fusion protein comprises a deaminase (e.g. adenosine deaminase) and a nucleic acid-programmable DNA binding protein (napDNAbp). In some embodiments, the kit includes at least one guide RNA capable of targeting a nucleic acid molecule of interest. In some embodiments, the kit comprises a nucleic acid construct comprising a nucleotide sequence encoding at least one guide RNA. In one embodiment, the kit comprises a nucleic acid construct comprising a nucleotide sequence encoding a nucleobase editor fusion protein comprising a deaminase and a guide RNA capable of targeting the alpha-1-antitrypsin polynucleotide.
In some embodiments, the kit provides instructions for using the kit to edit one or more mutations. The instructions generally provide information about using the kit to edit nucleic acid molecules. In other embodiments, the instructions include at least one of the following: precautions; warnings; clinical studies; and/or references. The instructions can be printed directly on the container (if available), or as a label on the container, or as a separate sheet, booklet, card or binder supplied in or with the container. In another embodiment, a kit may include instructions in the form of a label or a separate insert (package insert) for appropriate operating parameters. In yet another embodiment, the kit may include one or more containers with appropriate positive and negative controls or control samples to be used as standard(s) for detection, calibration or normalization. The kit may further comprise a second container containing a pharmaceutically acceptable buffer such as (sterile) phosphate buffered saline, Ringer's solution or dextrose solution. It may also contain other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
Fusion proteins with internal insertions
Provided herein are fusion proteins comprising a heterologous polypeptide fused to a nucleic acid-programmable nucleic acid binding protein, such as a napDNAbp. A heterologous polypeptide can be a polypeptide not found in the native or wild-type napDNAbp polypeptide sequence. The heterologous polypeptide may be fused to the napDNAbp at a C-terminal end of the napDNAbp, an N-terminal end of the napDNAbp, or inserted at an internal site of the napDNAbp. In some embodiments, the heterologous polypeptide is inserted at an internal site of the napDNAbp.
In some embodiments, the heterologous polypeptide is a deaminase or a functional fragment thereof. For example, a fusion protein may comprise a deaminase flanked by an N-terminal fragment and a C-terminal fragment of a Cas9 or Cas12 (e.g. Cas12b/C2c1) polypeptide. The deaminase in a fusion protein can be an adenosine deaminase. In some embodiments, the adenosine deaminase is a TadA (eg, TadA7.10 or TadA*8). In some embodiments, the TadA is a TadA*8. TadA sequences (e.g. TadA7.10 or TadA*8) as described herein are suitable deaminases for the fusion proteins described above.
The deaminase can be a circular permutant deaminase. For example, the deaminase can be a circularly permutant adenosine deaminase. In some embodiments, the deaminase is a circularly permutant TadA circularly permuted at amino acid residue 116 as numbered in the TadA reference sequence. In some embodiments, the deaminase is a circularly permutant TadA circularly permuted at amino acid residue 136 as numbered in the TadA reference sequence. In some embodiments, the deaminase is a circularly permutant TadA circularly permuted at amino acid residue 65 as numbered in the TadA reference sequence.
The fusion protein may include more than one deaminase. For example, the fusion protein may comprise 1, 2, 3, 4, 5 or more deaminases. In some embodiments, the fusion protein includes a deaminase. In some embodiments, the fusion protein includes two deaminases. The two or more deaminases in a fusion protein can be an adenosine deaminase. cytidine deaminase or a combination thereof, e.g. as described in PCT/US19/44935. The two or more deaminases can be homodimers. The two or more deaminases can be heterodimers. The two or more deaminases can be inserted into the napDNAbp in tandem. In some embodiments, the two or more deaminases may not be present in tandem in the napDNAbp.
In some embodiments, the napDNAbp in the fusion protein is a Cas9 polypeptide or fragment thereof. The Cas9 polypeptide may be a variant of the Cas9 polypeptide. In some embodiments, the Cas9 polypeptide is a Cas9 nickase (nCas9) polypeptide or fragment thereof. In some embodiments, the Cas9 polypeptide is a nuclease-dead Cas9 (dCas9) polypeptide or fragment thereof. The Cas9 polypeptide in a fusion protein may be a full-length Cas9 polypeptide. In some cases, the Cas9 polypeptide in a fusion protein may not be a full-length Cas9 polypeptide. For example, the Cas9 polypeptide may be truncated at an N-terminal or C-terminal end relative to a naturally occurring Cas9 protein. The Cas9 polypeptide can be a circularly permuted Cas9 protein. The Cas9 polypeptide can be a fragment, portion, or domain of a Cas9 polypeptide that is still capable of binding the target polynucleotide and a guide nucleic acid sequence.
In some embodiments, the Cas9 polypeptide is aStreptococcus pyogenesCas9 (SpCas9),Staphylococcus aureusCas9 (SaCas9),Streptococcus thermophilus1 Cas9 (St1Cas9) or fragments or variants thereof
The Cas9 polypeptide of a fusion protein may comprise an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% %, at least 99%, or at least 99.5% identical to a naturally occurring Cas9 polypeptide.
The Cas9 polypeptide of a fusion protein may comprise an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% %, at least 99% or at least 99.5% identical to the Cas9 amino acid sequence listed below (hereinafter referred to as "Cas9 reference sequence"):