Compositions and methods for treating glycogen storage disease type 1a

Abstract

The present invention provides compositions comprising novel adenosine base editors (e.g., ABE8) with improved efficiency and methods of using base editors comprising adenosine deaminase variants to alter mutations associated with glycogen storage disease type 1a (GSD1a).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is an International PCT (International PCT) application claiming priority to and the benefit of U.S. Provisional Application Nos. 62 / 805,271, filed on February 13, 2019; 62 / 852,228, filed on May 23, 2019; 62 / 852,224, filed on May 23, 2019; 62 / 876,354, filed on July 19, 2019; 62 / 912,992, filed on October 9, 2019; 62 / 931,722, filed on November 6, 2019; 62 / 941,569, filed on November 27, 2019; and 62 / 966,526, filed on January 27, 2020, the entire contents of which are incorporated herein by reference in their entirety.

[0003] Reference Addition

[0004] 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 was specifically and individually indicated to be incorporated by reference. Unless otherwise indicated, the publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure provides compositions comprising novel adenosine base editors (e.g., ABE8) with improved efficiency and methods of using base editors comprising adenosine deaminase variants to alter mutations associated with glycogen storage disease type 1a (GSD1a). Background Art

[0006] For most known genetic diseases, point mutations in the target locus need to be corrected, rather than randomly damaging genes, to study or address the root cause of the disease. Current genome editing technologies utilize the clustered regularly interspaced short palindromic repeat (CRISPR) system to introduce double-stranded DNA breaks at the target locus as the first step in gene correction. In response to double-stranded DNA breaks, the repair process of cellular DNA primarily involves joining non-homologous ends at the DNA cut site, resulting in random insertions and deletions (indels). Although most genetic diseases are caused by point mutations, current point mutation correction methods are inefficient and typically induce a large number of random insertions and deletions (indels) at the target locus, which are caused by the cell's response to dsDNA breaks. Therefore, there is a need for an improved form of genome editing that is more efficient and produces far fewer unexpected products, such as random insertions and deletions (indels) or translocations.

[0007] Glycogen storage disease type 1 (also known as GSD1 or Von Gierke disease) is an inherited disorder that results in deficient glycogenolysis and gluconeogenesis, leading to accumulation of glycogen and lipids in tissues, causing life-threatening hypoglycemia and lactic acidosis, and potentially leading to central nervous system damage and long-term liver and kidney complications such as steatosis, hepatic adenoma, and hepatocellular carcinoma.

[0008] There are two types of GSD1, type 1a (GSD1a) and type 1b (GSD1b), which are caused by different gene mutations. GSD1a is caused by mutations in the glucose-6-phosphatase (G6PC) gene and affects approximately 80% of GSD1 patients. In the United States, GSD1a affects approximately one in every 100,000 births, with approximately 22% of patients carrying the recessive mutation Q347* and 37% carrying the recessive mutation R83C.

[0009] There are no approved drug therapies for GSD1a. Although liver transplantation is curative, there are no approved therapies, and current treatment options involve almost exclusively continuous cornstarch feeding. If left untreated for a long time, patients develop severe lactic acidosis, which can progress to renal failure and death in infancy or childhood. GSD1a represents an area of ​​significant unmet medical need. Therefore, new compositions and methods for treating patients with GSD1a are needed.

[0010] Reference Addition

[0011] 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 was specifically and individually indicated to be incorporated by reference. Unless otherwise indicated, the publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety. Summary of the Invention

[0012] The present invention is characterized in that a composition and method for accurately correcting pathogenic amino acids using a programmable nucleobase editor. In particular, the compositions and methods of the present invention can be used to treat glycogen storage disease type 1a (GSD1a). Therefore, the present invention provides compositions and methods for treating GSD1a, using an adenosine (A) base editor (ABE) (e.g., ABE8) to accurately correct single nucleotide polymorphisms in the endogenous G6PC gene to correct harmful mutations (e.g., Q347X, R83C).

[0013] On the one hand, the present invention provides a method for editing a G6PC polynucleotide comprising a single nucleotide polymorphism (SNP) associated with glycogen storage disease type 1a (GSD1a), the method comprising contacting the G6PC polynucleotide with an adenosine deaminase base editor 8 (ABE8) in a complex with one or more guide polynucleotides, wherein the ABE8 comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein the one or more guide polynucleotides target the base editor to achieve an A·T to G·C change of the SNP associated with GSD1a. On the other hand, the present invention provides a cell comprising the adenosine deaminase base editor 8 (ABE8), or a polynucleotide encoding the base editor, which comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and one or more guide polynucleotides targeting the base editor to achieve an A·T to G·C change of the SNP associated with GSD1a. On the other hand, the present invention provides a method for treating GSD1a in an individual, comprising administering to the individual: an adenosine deaminase base editor 8 (ABE8) or a polynucleotide encoding the base editor, wherein the adenosine deaminase base editor 8 (ABE8) comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and one or more guide polynucleotides targeting the adenosine deaminase base editor 8 (ABE8) to achieve an A·T to G·C change of the SNP associated with GSD1a. On the other hand, the present invention provides a method for producing hepatocytes or progenitor cells thereof, the method comprising: a) introducing a polynucleotide comprising the SNP associated with GSD1a, adenosine deaminase base editor 8 (ABE8), or encoding the adenosine deaminase base editor 8 (ABE8) or induced pluripotent stem cells or hepatocyte progenitor cells, wherein the base editor comprises a polynucleotide programmable nucleotide binding domain and an adenosine deaminase domain, and one or more guide polynucleotides, wherein one or more of the guide polynucleotides target the base editor to achieve the A·T to G·C change of the SNP associated with GSD1a; b) differentiating the induced pluripotent stem cells or hepatocyte progenitor cells into hepatocytes.

[0014] In one aspect, the present invention provides a method for editing a glucose-6-phosphatase (G6PC) polynucleotide comprising a single nucleotide polymorphism (SNP) associated with glycogen storage disease type 1a (GSD1a), the method comprising contacting the G6PC polynucleotide with an adenosine deaminase base editor 8 (ABE) in a complex with one or more guide polynucleotides, wherein the adenosine deaminase base editor 8 (ABE8) comprises an adenosine deaminase variant domain inserted into a Cas9 or Cas12 polypeptide, wherein one or more of the guide polynucleotides targets the base editor to effect an A·T to G·C change of the SNP associated with GSD1a. On the other hand, the present invention provides a method for treating glycogen storage disease type 1a (GSD1a) in an individual, the method comprising administering to the individual: an adenosine deaminase base editor 8 (ABE8) or a polynucleotide encoding the base editor 8 (ABE8), wherein the adenosine deaminase base editor 8 (ABE8) comprises an adenosine deaminase variant inserted into a Cas9 or Cas12 polypeptide, and one or more guide polynucleotides targeting adenosine deaminase base editor 8 (ABE8) to achieve an A·T to G·C change of the SNP associated with GSD1a, thereby treating GSD1a in the individual. In another aspect, the present invention provides a method for treating glycogen storage disease type 1a (GSD1a) in an individual, the method comprising administering to the individual: a fusion protein comprising an adenosine deaminase variant inserted into a Cas9 or Cas12 polypeptide or a polynucleotide encoding the fusion protein thereof, and one or more guide polynucleotides targeting the fusion protein to achieve an A·T to G·C change of a single nucleotide polymorphism (SNP) associated with GSD1a, thereby treating GSD1a in the individual.

[0015] On the one hand, the present invention provides a pharmaceutical composition for treating glycogen storage disease type 1a (GSD1a), comprising an effective amount of adenosine deaminase base editor 8 (ABE8), wherein the adenosine deaminase base editor 8 (ABE8) comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase variant domain. In some embodiments, the pharmaceutical composition comprises one or more guide polynucleotides that can target the adenosine deaminase base editor 8 (ABE8) to achieve a change from A·T to G·C of the SNP associated with GSD1a. On the other hand, the present invention provides a pharmaceutical composition for treating glycogen storage disease type 1a (GSD1a), comprising an effective amount of any cell provided herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0016] On the other hand, the present invention provides a kit for treating glycogen storage disease type 1a (GSD1a), the kit comprising an adenosine deaminase base editor 8 (ABE8), wherein the adenosine deaminase base editor 8 (ABE8) comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and one or more guide polynucleotides capable of targeting the adenosine deaminase base editor 8 (ABE8) to achieve a change of A·T to G·C of the SNP associated with GSD1a. In another aspect, the present invention provides a kit for treating glycogen storage disease type 1a (GSD1a), the kit comprising any cell provided herein.

[0017] In some embodiments, the contacting is performed in cells, eukaryotic cells, mammalian cells, or human cells. In some embodiments, the cells are in vivo. In some embodiments, the cells are isolated. In some embodiments, the cells are hepatocytes, hepatocyte precursors, or iPSc-derived hepatocytes. In some embodiments, the cells express a G6PC polypeptide. In some embodiments, the cells or hepatocyte progenitor cells are from an individual with GSD1a. In some embodiments, the individual is a mammal or a human. In some embodiments, the hepatocytes or hepatocyte progenitor cells are mammalian cells or human cells. In some embodiments, the adenosine deaminase base editor 8 (ABE8) or a polynucleotide encoding the adenosine deaminase base editor 8 (ABE8), and one or more guide polynucleotides are delivered to the cells of the individual.

[0018] In various embodiments of the foregoing or any other aspect of the invention described herein, the SNP associated with GSD1a is located in the glucose-6-phosphatase (G6PC) gene. In one embodiment, the change of A·T to G·C at the SNP associated with glycogen storage disease type 1a (GSD1a) changes glutamine (Q) to a non-glutamine (X) amino acid. In one embodiment, the change of A·T to G·C of the SNP associated with glycogen storage disease type 1a (GSD1a) changes arginine (R) in the G6PC polypeptide to non-arginine (X). In one embodiment, the SNP associated with GSD1a results in the expression of a G6PC polypeptide having a non-glutamine amino acid (X) at position 347 or a non-arginine amino acid (X) at position 83. In one embodiment, the correction of the base editor replaces the non-glutamine amino acid (X) at position 347 with glutamine. In another embodiment, the correction of the base editor replaces the non-arginine amino acid (X) at position 83 with arginine. In one embodiment, an A·T to G·C change at a SNP associated with GSD1a results in expression of a G6PC polypeptide that prematurely stops at amino acid position 347 or encodes a cysteine ​​at position 83. In some embodiments, the change at the SNP is one or more of Q347X and / or R83C.

[0019] In various embodiments of the above aspects or any other aspects of the invention described herein, the adenosine deaminase variant is inserted into a flexible loop, an alpha helical region, an unstructured portion, or a solvent accessible portion of a Cas9 or Cas12 polypeptide. In some embodiments, the adenosine deaminase variant is flanked by the N-terminal fragment and the C-terminal fragment of the Cas9 or Cas12 polypeptide. In some embodiments, the fusion protein or adenosine deaminase base editor 8 (ABE8) comprises a structure of NH2-[N-terminal fragment of the Cas9 or Cas12 polypeptide]-[adenosine deaminase variant]-[C-terminal fragment of the Cas9 or Cas12 polypeptide]-COOH, wherein each "]-[" 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 a portion of a flexible loop of the Cas9 or Cas12 polypeptide. In one embodiment, the flexible loop comprises an amino acid close to a target nucleobase. In some embodiments, the one or more guide polynucleotides guide the fusion protein or adenosine deaminase base editor 8 (ABE8) to achieve deamination of the target nucleobase. In some embodiments, deamination of the SNP target nucleobase replaces the target nucleobase with a non-wild-type nucleobase, and wherein deamination of the target nucleobase improves symptoms of GSD1a. In one embodiment, the target nucleobase is 1-20 nucleobases away from a PAM sequence in a target polynucleotide sequence. In one embodiment, the target nucleobase is 2-12 nucleobases upstream of the PAM sequence.

[0020] In one embodiment, the N-terminal fragment or C-terminal fragment of the Cas9 or Cas12 polypeptide binds to the target polynucleotide sequence. In one embodiment, the N-terminal fragment or C-terminal fragment comprises a RuvC domain; the N-terminal fragment or 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 domains, and wherein the deaminase is inserted at the partial or complete deletion of the Cas9 or Cas12 polypeptide. In one embodiment, the deletion is located within the RuvC domain; the deletion is located within the HNH domain; or the deletion bridges the RuvC domain and the C-terminal domain, the LI domain and the HNH domain, or the RuvC domain and the LI domain.

[0021] In various embodiments, the polynucleotide programmable DNA binding domain is a Cas9 polypeptide. In some embodiments, the fusion protein or adenosine deaminase base editor 8 (ABE8) comprises an adenosine deaminase variant domain inserted into the Cas9 polypeptide. In some embodiments, the Cas9 polypeptide is Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9) or a variant thereof. In some embodiments, the Cas9 polypeptide has the following amino acid sequence (Cas9 reference sequence):

[0022]

[0023] (Single underline: HNH domain; double underline: RuvC domain; (Cas9 reference sequence)), or their corresponding regions.

[0024] In some embodiments, the Cas9 polypeptide comprises a deletion of amino acids numbered 1017-1069 or their corresponding amino acids in the Cas9 polypeptide reference sequence; the Cas9 polypeptide comprises a deletion of amino acids numbered 792-872 or their corresponding amino acids in the Cas9 polypeptide reference sequence; or the Cas9 polypeptide comprises a deletion of amino acids numbered 792-906 or their corresponding amino acids in the Cas9 polypeptide reference sequence. In some embodiments, the adenosine deaminase variant is inserted into a flexible loop of the Cas9 polypeptide. In some embodiments, the flexible loop comprises a region selected from the group consisting of amino acid residues numbered 530-537, 569-579, 686-691, 768-793, 943-947, 1002-1040, 1052-1077, 1232-1248, and 1298-1300 in the Cas9 reference sequence, or amino acid positions corresponding thereto. In some embodiments, the deaminase is inserted at an amino acid position numbered 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 in the Cas9 reference sequence, or an amino acid position corresponding thereto. In some embodiments, the deaminase is inserted at an amino acid position numbered 768-769, 792-793, 1022-1023, 1026-1027, 1040-1041, 1068-1069, or 1247-1248 in the Cas9 reference sequence, or an amino acid position corresponding thereto. In some embodiments, the deaminase is inserted at an amino acid position numbered 1016-1017, 1023-1024, 1029-1030, 1040-1041, 1069-1070, or 1247-1248 of the Cas9 reference sequence, or at amino acid positions corresponding thereto. In some embodiments, the adenosine deaminase variant is inserted into a Cas9 polypeptide at a locus identified in Table 10A. In some embodiments, 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 the Cas9 reference sequence, or at residues corresponding thereto.In some embodiments, 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 the Cas9 reference sequence, or residues corresponding thereto.

[0025] In some embodiments, the Cas9 polypeptide is a nickase, or wherein the Cas9 polypeptide is nuclease-inactive. In some embodiments, the Cas9 polypeptide is a modified SpCas9 and is specific for an altered PAM or is specific for a non-G PAM. In some embodiments, the modified SpCas9 polypeptide comprises amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and is specific for an altered PAM 5'-NGC-3'.

[0026] In various embodiments, the polynucleotide programmable DNA binding domain is a modified Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof. In various embodiments of the above aspects or any other aspects of the invention described herein, the polynucleotide programmable DNA binding domain comprises the modified SpCas9 having an altered protospacer adjacent motif (PAM) specificity or specificity for a non-G PAM. In one embodiment, the modified SpCas9 is specific for the nucleic acid sequence 5'-NGA-3'. In one embodiment, the modified SpCas9 is specific for the nucleic acid sequence 5'-AGA-3' or 5'-GGA-3'. In one embodiment, the modified SpCas9 is specific for an NGA PAM variant.

[0027] In various embodiments, the polynucleotide programmable DNA binding domain is Staphylococcus aureus Cas9 (SaCas9) or a variant thereof. In one embodiment, the SaCas9 is specific for the nucleic acid sequence 5'-NNGRRT-3'. In one embodiment, the SaCas9 is specific for the nucleic acid sequence 5'-GAGAAT-3'. In one embodiment, the SaCas9 is specific for the NNGRRT PAM variant.

[0028] In various embodiments, the polynucleotide programmable DNA binding domain is 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 the following amino acid positions: a) 153-154, 255-256, 306-307, 980-981, 1019-1020, 534-535, 604-605, or 344-345 of BhCas12b, or the corresponding amino acid residues of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, or Cas12i; b) 147 and 148, 248 and 256 of BvCas12b In one embodiment, the adenosine deaminase variant is inserted into the Cas12 polypeptide at the locus identified in Table 10B. 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.

[0029] In various embodiments, the polynucleotide programmable DNA binding domain is a nuclease inactive variant. In other embodiments, the polynucleotide programmable DNA binding domain is a nickase variant. In one embodiment, the nickase variant comprises a D10A amino acid replacement or its corresponding amino acid replacement. In certain embodiments, the adenosine deaminase domain is capable of deaminating adenosine in deoxyribonucleic acid (DNA). In certain embodiments, the adenosine deaminase domain is a monomer comprising an adenosine deaminase variant. In certain embodiments, the adenosine deaminase domain is a heterodimer comprising a wild-type adenosine deaminase domain and an adenosine deaminase variant.

[0030] In some embodiments, the adenosine deaminase variant comprises the following amino acid sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD; wherein the amino acid sequence comprises at least one alteration. In some embodiments, the adenosine deaminase variant comprises an alteration at amino acid position 82 and / or 166 relative to the above sequence. In some embodiments, the at least one alteration relative to the above sequence comprises V82S, Y147T, Y147R, Q154S, Y123H, and / or Q154R. In some embodiments, the at least one alteration comprises a combination of alterations 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 In some embodiments, the at least one change relative to the above sequence is Y147T+Q154S.

[0031] In some embodiments, the adenosine deaminase variant comprises a C-terminal deletion beginning at a residue selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 157. In some embodiments, the adenosine deaminase variant is an adenosine deaminase monomer comprising a TadA*8 adenosine deaminase variant domain. In some embodiments, 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 some embodiments, the adenosine deaminase variant is an adenosine deaminase heterodimer comprising a TadA domain and a TadA*8 adenosine deaminase variant domain.

[0032] In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of:

[0033] a)GACCUAGGCGAGGCAGUAGG;

[0034] b)CCAGUAUGGACACUGUCCAAA;

[0035] c)CAGUAUGGACACUGUCCAAA; and

[0036] d)AGUAUGGACACUGUCCAAAG.

[0037] In some embodiments, the one or more guide RNAs comprise a CRISPR RNA (crRNA) and a trans-encoded small RNA (tracrRNA), wherein the crRNA comprises a nucleic acid sequence complementary to a G6PC nucleic acid sequence comprising the SNP associated with GSD1a. In some embodiments, the adenosine deaminase base editor 8 (ABE8) is complexed with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to a G6PC nucleic acid sequence comprising the SNP associated with GSD1a.

[0038] In some embodiments, the adenosine deaminase is a TadA deaminase. In one embodiment, the TadA deaminase is a TadA*8 variant. In some embodiments, the TadA*8 variant is selected from the group consisting of: 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 and TadA*8.24. In some embodiments, the adenosine deaminase base editor 8 (ABE8) is selected from the group consisting of: 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. BE8.23-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.

[0039] In some embodiments, the adenosine deaminase base editor 8 (ABE8) comprises or consists essentially of the following sequence having adenosine deaminase activity or a fragment thereof:

[0040] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0041] In some embodiments, the gRNA comprises a scaffold having the following sequence:

[0042] GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUU.

[0043] In some embodiments, the gRNA comprises a scaffold having the following sequence:

[0044] GUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU.

[0045] In one aspect, provided herein is a base editor comprising an adenosine deaminase base editor 8 (ABE8) complexed with one or more guide polynucleotides, wherein the adenosine deaminase base editor 8 (ABE8) comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein one or more of the guide polynucleotides targets the base editor to achieve an A·T to G·C change of the SNP associated with GSD1a. In some embodiments, the adenosine deaminase variant comprises a change in V82S and / or T166R. 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, and the adenosine deaminase heterodimer comprises a wild-type adenosine deaminase domain and an adenosine deaminase variant. In some embodiments, the adenosine deaminase variant is a truncated TadA8, wherein the truncated TadA8 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 N-terminal amino acid residues deleted relative to the full-length TadA8. In some embodiments, the adenosine deaminase variant is a truncated TadA8, wherein the truncated TadA8 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 C-terminal amino acid residues deleted relative to the full-length TadA8. In some embodiments, the polynucleotide programmable DNA binding domain is a modified Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), a modified Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof. In some embodiments, the polynucleotide programmable DNA binding domain is a variant of SpCas9 with specificity for an altered protospacer adjacent motif (PAM) 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.

[0046] In one aspect, provided herein is 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:

[0047]

[0048] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, and wherein one or more of the guide polynucleotides targets the base editor to effect an A·T to G·C change of the SNP associated with GSD1a.

[0049] In one aspect, a cell comprising any of the base editor systems described above is provided. In some embodiments, the cell is a human cell or a mammalian cell. In some embodiments, the cell is isolated, in vivo, or in vitro.

[0050] The description and examples herein describe embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and therefore may vary. Those skilled in the art will recognize that there are many variations and modifications to the present disclosure, which are all within the scope thereof.

[0051] Unless otherwise indicated, the practice of some embodiments disclosed herein employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); Current Protocols in Molecular Biology series (FM Ausubel et al., ed.); Methods In Enzymology series (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames, and GR Taylor, ed. (1995)), Harlow and Lane, ed. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (RI Freshney, ed. (2010)).

[0052] Although various features of the present disclosure may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0053] The features of the present disclosure are set forth with particularity in the appended claims.A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description of illustrative embodiments, in which the principles of the present disclosure are utilized, and in view of the accompanying drawings as described below.

[0054] definition

[0055] The following definitions supplement those in the art and are specific to the present application and are not intended to imply any related or unrelated case, such as any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used to test the practice of the present disclosure, the preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the invention belongs. The following references provide general definitions of many of the terms used in the present invention for those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0057] In this application, unless otherwise specifically stated, the use of the singular includes the plural. It must be noted that the singular forms "a," "an," and "the" used in this specification include plural references unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" and is understood to be inclusive, unless otherwise stated. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is not limiting.

[0058] As used in this specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or combination of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0059] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 standard deviation or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, such as within 5 times or 2 times the value. Where specific values ​​are described in the application and claims, the term "about" should be assumed to be within an acceptable error range for the specific value unless otherwise indicated.

[0060] 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0061] References in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some, but not necessarily all, embodiments of the present disclosure.

[0062] "Adenosine deaminase" refers to a polypeptide or fragment thereof that can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain catalyzes the hydrolytic deamination of adenosine to inosine, or the hydrolytic deamination of deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) can be from any organism, such as bacteria.

[0063] 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 TadA*8. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as human, chimpanzee, gorilla, monkey, cow, dog, 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 International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is herein incorporated by reference in its entirety.See also Komor, AC et al., “Programmable base editing of atarget base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, AC et al., "Improved base excision repairinhibition 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., "Base editing: precision chemistry on the transcript genome and omeof living cells." Nat Rev Genet 2018 Dec; 19(12): 770-788. doi: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0064] Wild-type TadA (wt) adenosine deaminase has the following sequence (also known as the TadA reference sequence):

[0065] MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD

[0066] In some embodiments, the adenosine deaminase comprises a change in the following sequence:

[0067] MSEVEFSHEY WMRHALTLAK RARDEREVPVGAVLVLNNRV IGEGWNRAIG LHDPTAHAEIMALRQGGLVM QNYRLIDATL YVTFEPCVMC AGAMIHSRIG RVVFGVRNAK TGAAGSLMDV LHYPGMNHRVEITEGILADE CAALLCYFFR MPRQVFNAQK KAQSSTD

[0068] (also known as TadA*7.10).

[0069] In some embodiments, TadA*7.10 comprises at least one alteration. In some embodiments, TadA*7.10 comprises alterations at amino acids 82 and / or 166. In specific embodiments, variants of the above sequences comprise one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. Alteration Y123H is also referred to herein as H123H (alteration of H123Y in TadA*7.10 reverts to Y123H (wt)). In other embodiments, variants of the TadA*7.10 sequence comprise a combination of alterations selected from the following groups: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123 H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166 R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.

[0070] In other embodiments, the present invention provides adenosine deaminase variants comprising deletions, such as TadA*8, relative to the corresponding mutations in TadA*7.10, a TadA reference sequence, or another TadA, comprising a C-terminal deletion starting at residue 149, 150, 151, 152, 153, 154, 155, 156, or 157. In other embodiments, the adenosine deaminase variant is a TadA (e.g., TadA*8) monomer comprising one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R, relative to the corresponding mutations in TadA*7.10, a TadA reference sequence, or another TadA. In other embodiments, the adenosine deaminase variant is a monomer comprising a combination of alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H relative to corresponding mutations in TadA*7.10, a TadA reference sequence, or another TadA. +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.

[0071] In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each having one or more of the following alterations relative to TadA*7.10, a TadA reference sequence, or a corresponding mutation in another TadA: 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 alterations selected from the following groups relative to corresponding mutations in TadA*7.10, a TadA reference sequence, or another TadA: 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.

[0072] In other embodiments, the adenosine deaminase variant is a heterologous dimer comprising a wild-type TadA adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8), which comprises one or more of the following changes relative to TadA*7.10, a TadA reference sequence, or the corresponding mutations in another TadA: 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 an adenosine deaminase variant domain (e.g., TadA*8), which comprises a combination of alterations selected from the following groups relative to corresponding mutations in TadA*7.10, a TadA reference sequence, or another TadA: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y12 3H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I7 6Y;Y147R+Q154R+T166R;

[0073] In other embodiments, the adenosine deaminase variant is a heterologous dimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8), which comprises one or more of the following changes Y147T, Y147R, Q154S, Y123H, V82S, T166R and / or Q154R relative to the corresponding mutations in TadA*7.10, a TadA reference sequence, or another TadA. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8), comprising a combination of the following changes relative to the corresponding mutations in TadA*7.10, a TadA reference sequence, or another TadA: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I 76Y+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.

[0074] In one embodiment, the adenosine deaminase is TadA*8, which comprises or consists essentially of the following sequence having adenosine deaminase activity or a fragment thereof:

[0075] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0076] In some embodiments, the TadA*8 is truncated. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 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, 16, 17, 18, 19, or 20 C-terminal amino acid residues relative to full-length TadA*8. In some embodiments, the adenosine deaminase variant is full-length TadA*8.

[0077] In certain embodiments, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from one of the following:

[0078] Staphylococcus aureus (S. aureus) TadA:

[0079] MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN

[0080] Bacillus subtilis TadA:

[0081] MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE

[0082] Salmonella typhimurium (S.typhimurium) TadA:

[0083] MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV

[0084] Shewanella putrefaciens TadA:

[0085] MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE

[0086] Haemophilus influenzae F3031 TadA:

[0087] MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTAΗAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILH

[0088] SRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK

[0089] Caulobacter crescentus TadA:

[0090] MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI

[0091] Geobacter sulfurreducens TadA:

[0092] MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP

[0093] TadA*7.10

[0094] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0095] "Adenosine deaminase base editor 8 (ABE8) polypeptide" or "ABE8" refers to a base editor, as defined herein, comprising a variant of an adenosine deaminase, wherein the adenosine deaminase variant comprises an alteration at amino acid position 82 and / or 166 of the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD. In some embodiments, ABE8 comprises further alterations relative to the reference sequence, as described herein.

[0096] "Adenosine deaminase base editor 8 (ABE8) polynucleotide" refers to a polynucleotide encoding ABE8.

[0097] "Administering" as used herein refers to providing one or more compositions described herein to a patient or individual. For example, but not limited to, administration of the composition, such as injection, can be by intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more of the described routes can be used. Parenteral administration can be, for example, by bolus injection or gradual infusion over time. Alternatively, or concurrently, administration can be by oral route.

[0098] "Agent" refers to any small molecule compound, antibody, nucleic acid molecule or polypeptide, or fragments thereof.

[0099] "Alteration" refers to a change (e.g., an increase or decrease) in the structure, expression level, or activity of a gene or polypeptide, as detected by standard art-known methods, such as those described herein. As used herein, an alteration includes a change in the sequence of a polynucleotide or polypeptide or a change in expression level, such as a 25% change, a 40% change, a 50% change, or greater.

[0100] "Ameliorate" means to reduce, inhibit, attenuate, reduce, arrest, or stabilize the development or progression of a disease.

[0101] "Analogs" are molecules that are not identical but have similar functional or structural characteristics. For example, polynucleotide or polypeptide analogs retain the biological activity of the corresponding naturally occurring polynucleotide or polypeptide while possessing certain modifications that enhance the analog's function relative to the naturally occurring polynucleotide or polypeptide. Such modifications can 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. Analogs can include non-natural nucleotides or amino acids.

[0102] "Base editor (BE)" or "nucleobase editor (NBE)" refers to a substance that binds to a polynucleotide and has nucleobase modification activity. In various embodiments, the base editor comprises a nucleobase modification polypeptide (e.g., a deaminase) and a nucleic acid programmable nucleotide binding domain bound to a guide polynucleotide (e.g., a guide RNA). In various embodiments, the substance is a biomolecular complex comprising a protein domain with base editing activity, i.e., capable of modifying bases (e.g., deoxyribonucleic acids) within a nucleic acid molecule (e.g., A, T, C, G, or U). In some embodiments, the polynucleotide programmable DNA binding domain is fused or connected to a deaminase domain. In one embodiment, the substance is a fusion protein comprising a domain with base editing activity. In another embodiment, the protein domain with base editing activity is connected to a guide RNA (e.g., through an RNA binding motif on the guide RNA and an RNA binding domain fused to a deaminase). In some embodiments, the domain with base editing activity is capable of deaminating bases 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 adenosine (A) in DNA. In some embodiments, the base editor is an adenosine base editor (ABE).

[0103] In some embodiments, the base editor is produced by cloning an adenosine deaminase variant (e.g., TadA*8) into a scaffold comprising a circular replacement Cas9 (e.g., spCAS9 or saCAS9) and a bipartite nuclear localization sequence (e.g., ABE8). Circular arrangement Cas9s are known in the art and are described in, for example, Oakes et al., Cell 176, 254-267, 2019. An exemplary circular arrangement is as follows, where the bold sequence represents a sequence derived from Cas9, the italic sequence represents a linker sequence, and the underlined sequence represents a bipartite nuclear localization sequence.

[0104] CP5 (with MSP "NGG = Pam variant with mutated conventional Cas9 such as NGG" PID = protein interaction domain and "D10A" nickase):

[0105]

[0106] In some embodiments, the ABE8 is selected from the base editors of Table 7 or 9 below. In some embodiments, the ABE8 contains an adenosine deaminase variant evolved from TadA. In some embodiments, the adenosine deaminase variant of ABE8 is a TadA*8 variant as described in Table 7 or 9 below. In some embodiments, the adenosine deaminase variant is a TadA*7.10 variant (e.g., TadA*8) comprising one or more changes selected from Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In various embodiments, the ABE8 comprises a TadA*7.10 variant (e.g., TadA*8), and a combination of alterations selected from the following groups: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V In some embodiments, ABE8 is a monomeric construct. In some embodiments, ABE8 is a heterodimeric construct. In some embodiments, the adenosine deaminase base editor 8 (ABE8) comprises the following sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0107] In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-related (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. The details of the base editor are described in International PCT 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, AC et al., "Programmable base editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, NM et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage" Nature 551, 464-471 (2017); Komor, AC et al., "Improved base excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A baseeditors with higher efficiency and product purity" Science Advances 3:eaao4774 (2017) and Rees, HA et al., "Base editing: precision chemistry on the genome and transcriptome of living cells." Nat Rev Genet. 2018 Dec; 19(12): 770-788. doi: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0108] For example, the adenine base editor (ABE) used in the base editing compositions, systems, and methods described herein has the following nucleic acid sequence (8877 base pairs): (Addgene, Watertown, MA.; Gaudelli NM et al., Nature. 2017 Nov 23; 551(7681): 464-471. doi: 10.1038 / nature24644; Koblan LW et al., Nat Biotechnol. 2018 Oct; 36(9): 843-846. doi: 8 / 10 4172). Also included are polynucleotide sequences having at least 95% or higher identity to the ABE nucleic acid sequence.

[0109]

[0110] "Base editing activity" refers to the activity used to chemically change a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, such as converting the target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, such as converting A·T to G·C. In another embodiment, the base editing activity is cytidine deaminase activity, such as converting the target C·G to T·A and adenosine or adenine deaminase activity, such as converting A·T to G·C. In some embodiments, base editing activity is assessed by editing efficiency. Base editing efficiency can be measured in any suitable manner, for example, by Sanger sequencing or next-generation sequencing. In some embodiments, base editing efficiency is measured by the percentage of total sequencing reads of nuclear base conversions affected by the base editor, for example, the percentage of total sequencing reads of target AT base pairs converted to GC base pairs. In some embodiments, when base editing is performed in a cell population, the base editing efficiency is measured by the total percentage of cells of nuclear base conversions affected by the base editor.

[0111] The term "base editor system" refers to a system for editing the nucleobases 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., adenosine deaminase) for deaminating the nucleobase; (3) one or more guide polynucleotides (e.g., 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.

[0112] In some embodiments, a base editor system can include more than one base editing component. For example, a base editor system can include more than one deaminase. In some embodiments, a base editor system can include one or more adenosine deaminases. In some embodiments, a single guide 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 target different deaminases to a target nucleic acid sequence.

[0113] The deaminase domain and the polynucleotide programmable nucleotide binding component of the base editor system can be covalently or non-covalently associated, or by any combination of their association and interaction. For example, in some embodiments, the deaminase domain can target the target nucleotide sequence through the polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or connected to the deaminase domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the deaminase domain to the target nucleotide sequence by non-covalent interaction or association with the deaminase domain. For example, in some embodiments, the deaminase domain may include an additional heterologous portion or domain that is capable of interacting, associating or forming a complex with an additional heterologous portion or domain that is part of the polynucleotide programmable nucleotide binding domain. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide, interacting, associating or forming a complex. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide, interacting, associating or forming a complex. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous moiety may be capable of binding to a polynucleotide linker. The additional heterologous moiety may be a protein domain. In some embodiments, the additional heterologous moiety may be a Kappa homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, the telomerase Ku binding motif and Ku protein, the telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0114] The base editor system may further include a guide polynucleotide component. It should be understood that the components of the base editor system can be covalently or non-covalently associated, or by a combination of their association and interaction. In some embodiments, the deaminase domain can target the target nucleotide sequence by a guide 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), which can interact, associate or form a complex with a portion or segment (e.g., a polynucleotide motif) of the guide 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 connected to the deaminase domain. In some embodiments, the additional heterologous portion may be able to bind to, interact with, associate with or form a complex with a polypeptide. In some embodiments, the additional heterologous portion may be able to bind to, interact with, associate with or form a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be able to bind to a guide polynucleotide. In some embodiments, the additional heterologous portion is able to bind to a polypeptide linker. In some embodiments, the additional heterologous portion is able to bind to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0115] In some embodiments, the base editor system may further include an inhibitor of a base excision repair (BER) component. It should be understood that the components of the base editor system can associate with each other by covalent, non-covalent interactions, or any combination of their association and interaction. The inhibitor of the BER component may include a BER inhibitor. In some embodiments, the BER inhibitor may be a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the BER inhibitor may be an inosine BER inhibitor. In some embodiments, the BER inhibitor can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or connected to a BER inhibitor. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or connected to a deaminase domain and a BER inhibitor. In some embodiments, the polynucleotide programmable nucleotide binding domain can target a BER inhibitor to a target nucleotide sequence by non-covalent interaction or association with a BER inhibitor. For example, in some embodiments, the inhibitor of the BER component may include an additional heterologous portion or domain that is capable of interacting, associating, or forming a complex with an additional heterologous portion or domain that is part of a polynucleotide programmable nucleotide binding domain.

[0116] In some embodiments, the BER inhibitor can target a target nucleotide sequence via a guide polynucleotide. For example, in some embodiments, the BER inhibitor can comprise 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, associating, or forming a complex with a portion or segment (e.g., a polynucleotide motif) of a guide polynucleotide. In some embodiments, the additional heterologous portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) of the guide polynucleotide can be fused or linked to the BER inhibitor. In some embodiments, the additional heterologous portion may be capable of binding, interacting, associating, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion is capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion is capable of binding to a polynucleotide linker. The additional heterologous portion can be a protein domain. In some embodiments, the additional heterologous portion can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0117] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease comprising a Cas9 protein or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain, and / or a gRNA binding domain of Cas9). Cas9 nucleases are sometimes also referred to as Casn1 nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)-associated nucleases. CRISPR is an adaptive immune system that provides protection against mobile genetic components (viruses, transposons, and conjugative plasmids). The CRISPR cluster comprises a spacer, a sequence complementary to the aforementioned mobile component, and a target invading nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, proper processing of pre-crRNA requires transcoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. tracrRNA acts as a guide for ribonuclease 3 to assist in processing pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleases the linear or circular dsDNA target complementary to the spacer. The target strand that is not complementary to the crRNA is first cut by endonucleolytic cutting and then trimmed 3′-5′ by exonucleolytic cutting. In nature, DNA binding and cutting usually require proteins and two kinds of RNA. However, single guide RNA ("sgRNA", or simply "gRNA") can be engineered to integrate various aspects of crRNA and tracrRNA into a single RNA species. See, for example, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes short motifs in CRISPR repeat sequences (PAM or protospacer adjacent motifs) to help distinguish self from non-self.The Cas9 nuclease sequence and structure are well known to those skilled in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., JJ, McShan W.M., Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., 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. Natl. Acad. Sci. USA 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski J., et al., “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease inadaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including but not limited to Streptococcus pyogenes and Streptococcus 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 organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunitysystems" (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0118] An example of a Cas9 is Streptococcus pyogenes Cas9 (spCas9), the amino acid sequence of which is provided below:

[0119]

[0120]

[0121] (Single underline: HNH domain; double underline: RuvC domain)

[0122] Nuclease-inactive Cas9 proteins are interchangeably referred to as "dCas9" proteins (for nuclease-"dead" Cas9) or catalytically inactive Cas9. Methods for generating Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains 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).

[0123] For example, it is known that the DNA cleavage domain of Cas9 includes two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cuts the chain complementary to the gRNA, while the RuvC1 subdomain cuts the non-complementary chain. Mutations in these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science.337:816-821 (2012); Qi et al., Cell.28;152(5):1173-83 (2013)). In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, that is, Cas9 is a nickase, referred to as "nCas9" protein (for "nickase" Cas9). In some embodiments, a protein comprising a Cas9 fragment is provided. For example, in some embodiments, the 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, a protein comprising Cas9 or a fragment thereof is referred to as a "Cas9 variant". 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, 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 variants may have 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, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., 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, 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% identical to the amino acid length of the corresponding wild-type Cas9.

[0124] 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, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0125] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, nucleotide and amino acid sequences are below).

[0126]

[0127]

[0128] (Single underline: HNH domain; double underline: RuvC domain)

[0129] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence:

[0130]

[0131]

[0132] (Single underline: HNH domain; double underline: RuvC domain)

[0133] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2 (nucleotide sequence below); and Uniprot Reference Sequence: Q99ZW2 (amino acid sequence below).

[0134]

[0135] (SEQ ID NO: 1. Single underline: HNH domain; double underline: RuvC domain)

[0136] In some embodiments, Cas9 is Cas9 from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense, China (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella balticola (NCBI Ref: NC_021284.1); baltica) (NCBI Ref: NC_018010.1); Psychroflexus Torquis I (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1), or Neisseria meningitidis (NCBI Ref.: YP_002342100.1), or Cas9 from any other organism.

[0137] In some embodiments, Cas9 is from Neisseria meningitidis (Nme). In some embodiments, Cas9 is Nme1, Nme2, or Nme3. In some embodiments, the PAM interaction domains of Nme1, Nme2, or Nme3 are N4GAT, N4CC, and N4CAAA, respectively (see, e.g., Edraki, A. et al., A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing, Molecular Cell (2018)). An example of a Neisseria meningitidis Cas9 protein, Nme1Cas9, (NCBI reference: WP_002235162.1; type II CRISPR RNA-guided endonuclease Cas9) has the following amino acid sequence:

[0138]

[0139]

[0140] Another example of a Neisseria meningitidis Cas9 protein, Nme2Cas9, (NCBI reference: WP_002230835; type II CRISPR RNA-guided endonuclease Cas9) has the following amino acid sequence:

[0141]

[0142] In some embodiments, dCas9 corresponds to, or partially or completely comprises, a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises D10A and H840A mutations or corresponding mutations in another Cas9. In some embodiments, dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A):

[0143]

[0144] (Single underline: HNH domain; double underline: RuvC domain).

[0145] In some embodiments, the Cas9 domain comprises a D10A mutation, while residue 840 remains a histidine in the amino acid sequences provided above, or at the corresponding position in any of the amino acid sequences provided herein.

[0146] In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which, for example, result in nuclease-inactive Cas9 (dCas9). For example, such mutations include other amino acid substitutions at D10 and H840, or other substitutions within the Cas9 nuclease domain (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs 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, at least 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 shorter or longer amino acid sequences of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or more.

[0147] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of the Cas9 protein, such as one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but only comprise one or more fragments thereof. Example amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and suitable sequences of additional Cas9 domains and fragments will be apparent to those skilled in the art.

[0148] It should be understood that additional Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including variants and homologs thereof, are within the scope of the present disclosure. Example Cas9 proteins include, but are not limited to, 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.

[0149] Exemplary catalytically inactive Cas9 (dCas9):

[0150]

[0151] Exemplary catalytic Cas9 nickase (nCas9):

[0152]

[0153] Exemplary catalytically active Cas9:

[0154]

[0155] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., nanoarchaea), which constitute the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, Cas9 refers to CasX or CasY, which has been described in, for example, "New CRISPR-Cas system from uncultivated microbes" Cell Res.2017Feb 21.doi:10.1038 / cr.2017.21 by Burstein et al., the entire contents of which are incorporated herein by reference. Using genome-analyzed metagenomics, many CRISPR-Cas systems have been identified, including Cas9, which was first reported in the archaeal domain. As part of an active CRISPR-Cas system, this diverse Cas9 protein was found in nanoarchaea, which have rarely been studied. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, were found, which are among the most compact systems discovered so far. In some embodiments, Cas9 refers to CasX, or a variant of CasX. In some embodiments, Cas9 refers to CasY or a variant of CasY. It will be understood that other RNA-guided DNA binding proteins can be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0156] In certain embodiments, napDNAbps useful in the methods of the present invention include circular arrangements known in the art and described, for example, by Oakes et al., Cell 176, 254–267, 2019. An exemplary circular arrangement is shown below, where bold sequences represent sequences derived from Cas9, italic sequences represent linker sequences, and underlined sequences represent bipartite nuclear localization sequences.

[0157] CP5 (with MSP "NGG = Pam variant with mutated conventional Cas9 like NGG" PID = protein interaction domain and "D10A" nickase).

[0158]

[0159]

[0160] Non-limiting examples of polynucleotide programmable nucleotide-binding domains that can be incorporated into base editors include CRISPR protein-derived domains, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs).

[0161] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can 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 amino acid sequence included in the napDNAbp 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 amino acid sequence included in the napDNAbp 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 will be appreciated that Cas12b / C2c1, CasX, and CasY from other bacterial species may also be used in accordance with the present disclosure.

[0162] Cas12b / C2c1(uniprot.org / uniprot / T0D7A2#2)

[0163] sp|T0D7A2|C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acido-terrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB13137 / GD3B) GN=c2c1 PE=1 SV=1

[0164]

[0165] CasX

[0166] (uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53)

[0167] >tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx proteins OS=Sulfolobus islandicus (strain HVE10 / 4) GN=SiH_0402 PE=4 SV=1

[0168] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG

[0169] SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0170] >tr|F0NH53|F0NH53_SULIR CRISPR-associated protein, Casx OS=Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4SV=1

[0171] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0172] Deltaproteobacteria CasX <0>

[0173] MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQK WYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0174] CasY(ncbi.nlm.nih.gov / protein / APG80656.1)

[0175] >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacterium]

[0176]

[0177] The term "Cas12" or "Cas12 domain" refers to an RNA-guided nuclease comprising a Cas12 protein or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas12, and / or a gRNA binding domain of Cas12). Cas12 belongs to Class 2, Type V CRISPR / Cas systems. Cas12 nucleases are sometimes also referred to as CRISPR (clustered regularly interspaced short palindromic repeats)-associated nucleases. The sequence of an exemplary Bacillus chondroitinib Cas 12b (BhCas12b) Cas 12 domain is provided below:

[0178]

[0179] Amino acid sequences having at least 85% or greater identity to the BhCas12b amino acid sequence can also be used in the methods of the invention.

[0180] The term "conservative amino acid substitution" or "conservative mutation" refers to the substitution of an amino acid by another amino acid with a common property. A functional method for defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Based on such analysis, groups of amino acids can be defined in which the amino acids within the group are preferentially interchangeable and are therefore most similar to each other in terms of their impact on overall protein structure (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include amino acid substitutions of amino acids, such as lysine for arginine and vice versa, so that a positive charge can be maintained; glutamic acid for aspartic acid and vice versa, to maintain a negative charge; serine for threonine, so that a free –OH can be maintained; and glutamine for asparagine, so that a free –NH2 can be maintained.

[0181] As used interchangeably herein, the terms "coding sequence" or "protein coding sequence" refer to a polynucleotide segment that encodes a protein. The region or sequence has a start codon near the 5' end and a stop codon near the 3' end. The coding sequence may also be referred to as an open reading frame.

[0182] As used herein, the term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In certain embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenine to hypoxanthine. In certain embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine or adenine (A) to inosine (I). In certain embodiments, the deaminase or deaminase domain catalyzes the hydrolytic deamination of adenosine or deoxyadenosine to an adenosine deaminase that is inosine or deoxyinosine. In certain embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) can be from any organism, such as bacteria. In some embodiments, the adenosine deaminase is from a bacterium, such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Caulobacter crescentus.

[0183] 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 TadA*8. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as human, chimpanzee, gorilla, monkey, cow, dog, 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 International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is herein incorporated by reference in its entirety.See also Komor, AC et al., "Programmable base editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, NM et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage" Nature 551, 464-471 (2017); Komor, AC et al., "Improved base excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A baseeditors with higher efficiency and product purity" Science Advances 3:eaao4774 (2017)), and Rees, HA, et al., "Base editing: precision chemistry on the genome and transcriptome of living cells." Nat Rev Genet. 2018 Dec; 19(12): 770-788. doi: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0184] "Detection" refers to identifying the presence, absence, or amount of an analyte to be detected. In one embodiment, sequence changes in a polynucleotide or polypeptide are detected. In another embodiment, the presence of an indel is detected.

[0185] "Detectable label" refers to a composition that, when attached to a molecule of interest, renders the latter detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., commonly used in ELISA), biotin, digoxigenin, or haptens.

[0186] "Disease" refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. An example of a disease includes glycogen storage disease type 1 (also known as GSD1 or von Gierke's disease). In some embodiments, GSD1 is type 1a (GSD1a).

[0187] An "effective amount" refers to the amount required to improve the symptoms of the disease relative to an untreated patient. The effective amount of the active compound used to implement the present invention to treat the disease depends on the mode of administration, the age, weight, and general health of the individual. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. This amount is referred to as an "effective" amount. In one embodiment, the effective amount is an amount of the base editor of the present invention sufficient to introduce a change in the gene of interest (e.g., G6PC) in a cell (e.g., in vitro or in vivo). In one embodiment, the effective amount is the amount of the base editor required to achieve a therapeutic effect (e.g., reducing or controlling GSD1a or its symptoms or conditions). This therapeutic effect does not need to be sufficient to change G6PC in all cells of an individual, tissue, or organ, but only needs to change the G6PC present in about 1%, 5%, 10%, 25%, 50%, 75% or more cells in the individual, tissue, or organ. In one embodiment, the effective amount is sufficient to improve one or more symptoms of GSD1a.

[0188] "Fragment" refers to a portion of a polypeptide or nucleic acid molecule. The portion comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of a reference nucleic acid molecule or polypeptide. A fragment can comprise 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.

[0189] "Glucose-6-phosphatase (G6PC) polypeptide" refers to a polypeptide or fragment thereof having at least about 95% amino acid sequence identity to NCBI accession number AAA16222.1. In a specific embodiment, the present invention provides a method for editing a G6PC polynucleotide comprising a single nucleotide polymorphism (SNP) associated with glycogen storage disease type 1a (GSD1a). In one embodiment, the change of A·T to G·C at the SNP associated with GSD1a changes the glutamine (Q) in the G6PC polypeptide to a non-glutamine (X) amino acid. In another embodiment, the change of A·T to G·C at the SNP associated with GSD1a changes the arginine (R) in the G6PC polypeptide to a non-arginine (X). In one embodiment, the SNP associated with GSD1a results in the expression of the G6PC polypeptide having a non-glutamine (X) amino acid at position 347 or a non-arginine (X) amino acid at position 83. In one embodiment, the base editor correction replaces the glutamine at position 347 with a non-glutamine amino acid (X). In another embodiment, the correction of the base editor replaces the arginine at position 83 with a non-arginine amino acid (X).

[0190] In certain embodiments, G6PC comprises one or more alterations relative to the following reference sequences. In certain embodiments, G6PC associated with GSD1a comprises one or more mutations selected from Q347X and R83C. An exemplary G6PC amino acid sequence from Homo sapiens is provided below:

[0191]

[0192] "Glucose-6-phosphatase polynucleotide" refers to a polynucleotide encoding a G6PC polypeptide. An exemplary G6PC nucleotide sequence from Homo sapiens is provided below (GenBank: U01120.1):

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200] "Guide RNA" or "gRNA" refers to a polynucleotide that is specific for a target sequence and that can form 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). The gRNA can exist as a complex of two or more RNAs or as a single RNA molecule. A gRNA that exists as a single RNA molecule may be referred to as a single guide RNA (sgRNA), but "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or a complex of two or more molecules. Typically, a gRNA that exists as a single RNA species contains two domains: (1) a domain that has homology to the target nucleic acid (e.g., that guides the binding of the Cas9 complex to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence called tracrRNA and comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical to or homologous to the tracrRNA 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 that include domain 2) can be found in U.S. Provisional Patent Application No. USSN 61 / 874,682, filed on September 6, 2013, entitled “Switchable Cas9 Nucleases and Uses Thereof,” and U.S. Provisional Patent Application No. USSN 61 / 874,746, filed on September 6, 2013, entitled “Delivery System For Functional Nucleases,” the entire contents of each of which are incorporated herein by reference in their entirety. In some embodiments, a gRNA comprises two or more of domains (1) and (2) and can be referred to as an “extended gRNA.” As described herein, an extended gRNA will bind to two or more Cas9 proteins and bind to a target nucleic acid at two or more different regions. The gRNA comprises a nucleotide sequence complementary to the target site that mediates binding of the nuclease / RNA complex to the target site, providing sequence specificity for the nuclease:RNA complex. As will be understood by those skilled in the art, RNA polynucleotide sequences, such as gRNA sequences, include the nucleobase uracil (U), a pyrimidine derivative, rather than the nucleobase thymine (T) contained in DNA polynucleotide sequences. In RNA, uracil base pairs with adenine and replaces thymine during DNA transcription.

[0201] "Heterodimer" refers to a fusion protein comprising two domains, such as a wild-type TadA domain and a variant TadA domain (such as TadA*8) or two variant TadA domains (such as TadA*7.10 and TadA*8 or two TadA*8 domains).

[0202] "Hybridization" refers to hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds.

[0203] The term "inhibitor of base repair" or "IBR" refers to a protein that can inhibit the activity of a nucleic acid repair enzyme, such as a base excision repair (BER) enzyme. In some embodiments, an IBR is an inhibitor of inosine base excision repair. Exemplary inhibitors of base repair include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGG1, hNEIL1, T7 Endol, T4PDG, UDG, hSMUG1, and hAAG. In some embodiments, an IBR is an inhibitor of Endo V or hAAG. In some embodiments, an IBR is a catalytically inactive EndoV or a catalytically inactive hAAG. In some embodiments, a base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, a base repair inhibitor is a catalytically inactive EndoV or a catalytically inactive hAAG.

[0204] In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI refers to a protein capable of inhibiting uracil-DNA glycosylase base excision repair enzymes. In some embodiments, the UGI domain comprises a fragment of wild-type UGI or wild-type UGI. In some embodiments, the UGI protein provided herein includes a fragment of UGI and a protein homologous to 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 a "dead inosine-specific nuclease." Without wishing to be bound by any particular theory, catalytically inactive inosine glycosylases (e.g., alkyladenine glycosylase (AAG)) can bind to inosine, but cannot produce abasic sites or remove inosine, thereby spatially blocking the newly formed inosine portion from DNA damage / repair mechanisms. In some embodiments, catalytically inactive inosine-specific nucleases can bind to inosine in nucleic acids but do not cut nucleic acids. Non-limiting, exemplary catalytically inactive inosine-specific nucleases include catalytically inactive alkyl adenine glycosylase (AAG nuclease), e.g., from humans, and catalytically inactive endonuclease V (EndoV nuclease), e.g., from Escherichia coli. In some embodiments, the catalytically inactive AAG nuclease comprises an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0205] By "increase" is meant a positive change of at least 10%, 25%, 50%, 75% or 100%.

[0206] An "intein" is a protein fragment that can self-excise and connect the remaining fragment (extein) with a peptide bond in a process called protein splicing. Inteins are also called "protein introns." The process in which an intein self-excises and connects to the remaining part of a protein is referred to herein as "protein splicing" or "intein-mediated protein splicing." In some embodiments, the intein of a precursor protein (a protein containing an intein before intein-mediated protein splicing) comes from two genes. Such inteins are referred to herein as split inteins (e.g., split intein-N and split intein-C). For example, in cyanobacteria, DnaE, ​​the catalytic subunit a of DNA polymerase III, is encoded by two independent 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."

[0207] Other intein systems may also be used. For example, synthetic inteins based on the dnaE intein, Cfa-N (e.g., split intein-N), and Cfa-C (e.g., split intein-C) intein pairs have 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 intein pairs that can be used in accordance with 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., described in U.S. Pat. No. 8,394,604, incorporated herein by reference).

[0208] Exemplary nucleotide and amino acid sequences of inteins are provided below.

[0209] DnaE intein-N DNA:

[0210] TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTTGCCAATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCGATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGGGGA GAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAGGGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTTCCTAAT

[0211] DnaE intein-N protein:

[0212] CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN

[0213] DnaE intein-C DNA:

[0214] ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGATATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAGCTTCTAAT

[0215] Intein-C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN

[0216] Cfa-N DNA:

[0217] TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCCTATTGGAAAGATTGTCGAAGAGAGAATTGAATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGGCACAATCGCGGCGAACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACGAGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAATAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTGCCA

[0218] Cfa-N protein:

[0219] CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP

[0220] Cfa-C DNA:

[0221] ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAGGAAAGTAAAGATAATATCTCGAAAAAGTCTTGGTACCCAAAATGTCTATGATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTAGCCAGCAAC

[0222] Cfa-C protein:

[0223] MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN

[0224] Intein-N and intein-C can be fused to the N-terminal portion of split-Cas9 and the C-terminal portion of split-Cas9, respectively, for connecting the N-terminal portion of split-Cas9 and the C-terminal portion of split-Cas9. For example, in some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of split-Cas9, i.e., forming a structure of N-[N-terminal portion of split-Cas9]-[intein-N]--C. In some embodiments, intein-C is fused to the N-terminus of the C-terminal portion of split-Cas9, i.e., forming N-[intein-C]--[C-terminal portion of split-Cas9]-C. Intein-mediated protein splicing mechanisms for connecting proteins to which inteins are fused (e.g., split-Cas9) are known in the art, for example, as described by Shah et al., ChemSci. 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 herein incorporated by reference in its entirety.

[0225] The terms "isolated," "purified," or "biologically pure" refer to materials that are free, to varying degrees, from components that normally accompany them in their native state. "Isolated" refers to a degree of separation from the original source or surrounding environment. "Purified" refers to a degree of separation that is greater than the degree of isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that a nucleic acid or protein produces essentially a single band in an electrophoretic gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications may result in different isolated proteins that can be purified separately.

[0226] An "isolated polynucleotide" refers to a nucleic acid (e.g., DNA) that is free of the genes that flank the genes in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. Thus, the term includes, for example, recombinant DNA that is integrated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryotic or eukaryotic organism; or that exists as an independent molecule (e.g., cDNA or genomic or cDNA fragments generated by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes RNA molecules transcribed from a DNA molecule, as well as recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0227] An "isolated polypeptide" refers to a polypeptide of the present invention that has been separated from naturally associated components. Generally, a polypeptide is isolated when it is at least 60% free by weight of proteins and naturally occurring organic molecules. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the polypeptide of the present invention. An isolated polypeptide of the present 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, such as column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0228] As used herein, the term "linker" may refer to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group, or a molecule or ribonucleosome that connects two molecules or parts (e.g., two components of a protein complex), or two domains of a fusion protein, such as a polynucleotide programmable DNA binding domain (e.g., dCas9) and a deaminase domain (e.g., adenosine deaminase). The linker can connect different components or different parts of a base editor system. For example, in some embodiments, the linker can connect the guide polynucleotide binding domain of a polynucleotide programmable nucleotide binding domain and the catalytic domain of a deaminase. In some embodiments, the linker can connect a CRISPR polypeptide and a deaminase. In some embodiments, the linker can connect Cas9 and a deaminase. In some embodiments, the linker can connect dCas9 and a deaminase. In some embodiments, the linker can connect nCas9 and a deaminase. In some embodiments, the linker can connect a guide polynucleotide and a deaminase. In some embodiments, the linker can connect the deamination component of the base editor system and the polynucleotide programmable nucleotide binding component. In some embodiments, the linker can connect the deamination component of the base editor system and the RNA binding portion of the polynucleotide programmable nucleotide binding component. In some embodiments, a linker can connect the RNA binding portion of the deamination component of the base editor system and the RNA binding portion of the polynucleotide programmable nucleotide binding component. The linker can be located between or on both sides of two groups, molecules or other parts and connected to each by a covalent bond or non-covalent interaction, thereby connecting the two. In some embodiments, the linker can be an organic molecule, group, polymer or chemical part. 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, the linker can comprise an aptamer capable of binding a ligand. In some embodiments, the ligand can be a carbohydrate, peptide, protein or nucleic acid. In some embodiments, the linker can comprise an aptamer that can be derived from a riboswitch. The riboswitch from which the aptamer is derived can be selected from the group consisting of a theophylline riboswitch, a thiamine pyrophosphate (TPP) riboswitch, an adenosylcobalamin (AdoCbl) riboswitch, an S-adenosylmethionine (SAM) riboswitch, a SAH riboswitch, a flavin mononucleotide (FMN) riboswitch, a tetrahydrofolate riboswitch, a lysine riboswitch, a glycine riboswitch, a purine riboswitch, a GlmS riboswitch, and a prequinine (PreQ1) riboswitch. In some embodiments, the linker can comprise an aptamer that binds to a polypeptide or protein domain, such as a polypeptide ligand.In some embodiments, the polypeptide ligand can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and 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.

[0229] In some embodiments, the linker can be one or more amino acids (e.g., peptides or proteins). In some embodiments, the length of the linker can be about 5-100 amino acids, such as 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 acid lengths. 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 acid lengths. Longer or shorter linkers are also contemplated.

[0230] In some embodiments, the linker connects the gRNA binding domain of the RNA programmable nuclease, including the Cas9 nuclease domain and the catalytic domain of the nucleic acid editing protein (e.g., adenosine deaminase). In some embodiments, the linker connects dCas9 and the nucleic acid editing protein. For example, the linker is located between or on both sides of two groups, molecules or other parts and is connected to each by a covalent bond, thereby connecting the two. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer or chemical part. In some embodiments, the length of the linker is 5-200 amino acids, e.g., 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. Longer or shorter linkers are also contemplated.

[0231] In some embodiments, the domain of the nucleobase editor is fused via a linker comprising the amino acid sequence: SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or

[0232] GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGG SGGS. In some embodiments, the domain of the base editor is fused via a linker comprising the amino acid sequence SGSETPGTSESATPES, which may also be referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker comprises (SGGS) n 、(GGGS) n 、(GGGGS) n 、(G) n 、(EAAAK) n 、(GGS) n , SGSETPGTSESATPES, or (XP) n motif, or any combination of these, wherein n is independently an integer between 1 and 30, and wherein 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.

[0233] In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS. In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence

[0234] PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0235] "Marker" refers to any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or condition.

[0236] As used herein, the term "mutation" refers to the replacement of a residue in a sequence (such as a nucleic acid or amino acid sequence) by another residue, or the deletion or insertion of one or more residues in the sequence. Mutation is generally described herein by identifying the original residue followed by the position of the residue in the sequence, and by the identity of the newly replaced residue. Various methods for performing amino acid replacement (mutation) provided herein are well known in the art and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). In some embodiments, the currently disclosed base editors can effectively produce "expected mutations" such as point mutations in nucleic acids (such as nucleic acids in individual genomes) without producing a large number of unexpected mutations, such as unexpected point mutations. In some embodiments, the expected mutation is a mutation produced by a specific base editor (such as an adenosine base editor) combined with a guide polynucleotide (such as a gRNA), which is specifically designed to produce an expected mutation.

[0237] Typically, mutations generated or identified in a sequence (e.g., an amino acid sequence described herein) are numbered relative to a reference (or wild-type) sequence, i.e., a sequence that does not contain the mutation. Those skilled in the art will readily understand how to determine the position of a mutation in an amino acid or nucleic acid sequence relative to a reference sequence.

[0238] The term "non-conservative mutation" refers to amino acid substitutions between different groups, for example, tryptophan for lysine or serine for phenylalanine. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, thereby increasing the biological activity of the functional variant compared to the wild-type protein.

[0239] The terms "nuclear localization sequence," "nuclear localization signal," or "NLS" refer to an amino acid sequence that facilitates the import of proteins into the nucleus. Nuclear localization sequences are known in the art and are described, for example, in International PCT Application No. PCT / EP2000 / 011690 by Plank et al., filed November 23, 2000, and published as WO / 2001 / 038547 on May 31, 2001, the contents of which are incorporated herein by reference for disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS, such as described by Koblan et al., Nature Biotech. 2018, doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises an amino acid sequence selected from the group consisting of KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0240] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to compounds comprising a core base and an acidic moiety, such as a polymer of nucleosides, nucleotides or nucleotides. Typically, polymeric nucleic acids, such as nucleic acid molecules comprising three or more nucleotides, are linear molecules in which adjacent nucleotides are interconnected by phosphodiester bonds. In certain embodiments, "nucleic acid" refers to a single nucleic acid residue (such as a nucleotide and / or nucleoside). In certain embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" are used interchangeably to refer to a polymer of nucleotides (such as a chain of at least three nucleotides). In certain embodiments, "nucleic acid" includes RNA and single-stranded and / or double-stranded DNA. Nucleic acid can be naturally occurring, such as in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid or other naturally occurring nucleic acid molecules. On the other hand, nucleic acid molecules can be non-naturally occurring molecules, such as recombinant DNA or RNA, artificial chromosomes, engineered genomes or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids or include non-naturally occurring nucleotides or nucleosides. In addition, the terms "nucleic acid", "DNA", "RNA" and / or similar terms include nucleic acid analogs, such as analogs having backbones other than phosphodiester backbones. Nucleic acids can be purified from natural sources, produced and optionally purified using a recombinant expression system, chemically synthesized, and the like. Where appropriate, for example, in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs, such as analogs having chemically modified bases or sugar and backbone modifications. Unless otherwise indicated, nucleic acid sequences are presented in a 5' to 3' direction. In some embodiments, nucleic acids are or comprise natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methyl cytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanosine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (2'-e.g., fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0241] 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 associated with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or a guide polynucleotide (e.g., gRNA), that guides 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. The Cas9 protein can be associated with a guide RNA that guides 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 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, 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, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Cs c2, 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 protein, type V Cas effector protein, type VI Cas effector protein, CARF, DinG, homologs thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, although they may not be specifically listed in the present disclosure. See, for example, Makarova et al., "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR J. 2018 Oct; 1: 325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., "Functionally diverse type V CRISPR-Cas systems" Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, the entire contents of each of which are incorporated herein by reference.

[0242] The terms "nucleobase," "nitrogenous base," or "base" are used interchangeably herein and refer to nitrogenous biological compounds that form nucleosides, which in turn are components of nucleotides. The ability of nucleobases to form base pairs and stack on each other directly leads to long helical structures, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The five nucleobases—adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U)—are referred to as bases or archetypes. Adenine and guanine are derived from purines, while cytosine, uracil, and thymine are derived from pyrimidines. DNA and RNA may also contain other (non-primary) modified bases. 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 produced by the presence of mutagens, and both are produced by deamination (replacing an amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be produced by deamination of cytosine. "Nucleoside" consists of a nucleobase and a five-carbon sugar (ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides with modified nucleobases include inosine (I), xanthine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). "Nucleotide" consists of a nucleobase, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group.

[0243] As used herein, the term "nucleobase editing domain" or "nucleobase editing protein" refers to a protein or enzyme that catalyzes the modification of nucleobases in RNA or DNA, such as the deamination of cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine) and adenine (or adenosine) to hypoxanthine (or inosine), as well as non-templated nucleotide additions and insertions. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., adenine deaminase or adenosine deaminase). In some embodiments, the nucleobase editing domain can be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase editing domain can be an engineered or evolved nucleobase editing domain from a naturally occurring nucleobase editing domain. The nucleobase editing domain can be from any organism, such as bacteria, humans, chimpanzees, gorillas, monkeys, cows, dogs, rats, or mice.

[0244] As used herein, "obtaining" as in "obtaining a substance" includes synthesizing, purchasing, or otherwise obtaining the substance.

[0245] As used herein, "patient" or "individual" refers to a mammalian individual or individuals who have been diagnosed with, are at risk of having or developing, or are suspected of having or developing a disease or condition. In some embodiments, the term "patient" refers to a mammalian individual who has a higher than average likelihood of developing a disease or condition. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cows, 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 patients can be male and / or female.

[0246] A "patient in need thereof" or "individual in need thereof" herein refers to a patient diagnosed with or suspected of having a disease or disorder, such as, but not limited to, glycogen storage disease type 1 (GSD1 or von Gierke's disease).

[0247] The terms "pathogenic mutation," "disease-causing variant," "disease envelope mutation," "pathogenic variant," "deleterious mutation," or "susceptibility mutation" refer to a genetic alteration or mutation that increases an individual's susceptibility to a disease or condition. In some embodiments, the pathogenic mutation comprises a substitution of at least one wild-type amino acid with at least one pathogenic amino acid in the protein encoded by the gene.

[0248] The terms "protein," "peptide," "polypeptide," and their grammatical equivalents are used interchangeably herein to refer to polymers of amino acid residues linked together by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides 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 may refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide may be modified, for example, by adding chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modification, and the like. A protein, peptide, or polypeptide may also be a single molecule or may be a multimolecular complex. A protein, peptide, or polypeptide may be simply a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. A protein can be located at the amino terminal (N-terminal) portion or carboxyl terminal (C-terminal) protein of the fusion protein, thereby forming an amino terminal fusion protein or a carboxyl terminal fusion protein respectively. Protein can include different domains, for example, a nucleic acid binding domain (such as the gRNA binding domain of Cas9 guiding protein to bind to the target site) and a nucleic acid cleavage domain, or the catalytic domain of nucleic acid editing protein. In certain embodiments, the protein includes a protein portion, such as an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, such as a compound that can serve as a nucleic acid cleavage agent. In certain embodiments, the protein forms a complex or associates with nucleic acids such as RNA or DNA. Any protein provided herein can be produced by any method known in the art. For example, a protein provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising peptide linkers. Recombinant protein expression and purification methods are well known, including those described by Green and Sambrook in Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), the entire contents of which are incorporated herein by reference.

[0249] The polypeptides and proteins disclosed herein (including functional parts 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-acetylaminomethyl-cysteine, 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, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. Polypeptides and proteins can be associated with post-translational modifications of one or more amino acids of the polypeptide structure. 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, disulfide bond formation, sulfation, myristoylation, palmitoylation, prenylation, farnesylation, geranylation, glycosylation, fatty acylation, and iodination.

[0250] As used herein, the term "recombinant" in the context of a protein or nucleic acid refers to a protein or nucleic acid that does not occur in nature but is a 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.

[0251] "Decrease" refers to a negative change of at least 10%, 25%, 50%, 75% or 100%.

[0252] "Reference" refers to a standard or control condition. In one embodiment, the reference is a wild-type or healthy cell. In other embodiments and without limitation, the reference is an untreated cell that has not been subjected to the test condition, or has been subjected to a placebo or saline, culture medium, buffer, and / or a control vector that does not contain the polynucleotide of interest.

[0253] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a particular sequence; for example, a fragment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence is typically 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 typically 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 therebetween. In some embodiments, the reference sequence is the wild-type sequence of the protein of interest. In other embodiments, the reference sequence is a polynucleotide sequence encoding the wild-type protein.

[0254] The terms "RNA programmable nuclease" and "RNA-guided nuclease" are used with (e.g., bound to or associated with) one or more RNAs that are not targets for cleavage. In some embodiments, when a complex is formed with RNA, the RNA programmable nuclease may be referred to as a nuclease:RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs or as a single RNA molecule. A gRNA that exists as a single RNA molecule may be referred to as a single guide RNA (sgRNA), although "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or a complex of two or more molecules. Typically, a gRNA that exists as a single RNA species contains two domains: (1) a domain that has homology to the target nucleic acid (e.g., guides the binding of the Cas9 complex to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence called tracrRNA and contains a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA provided by Jinek et al. in Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in U.S. Provisional Patent Application No. USSN 61 / 874,682, filed on September 6, 2013, entitled "Switchable Cas9 Nucleases and Uses Thereof," and U.S. Provisional Patent Application No. USSN 61 / 874,746, filed on September 6, 2013, entitled "Delivery System For Functional Nucleases," the entire contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the gRNA comprises two or more of domains (1) and (2) and can be referred to as an "extended gRNA." For example, as described herein, an extended gRNA will, for example, bind to two or more Cas9 proteins and bind to a target nucleic acid at two or more different regions. The gRNA contains a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site and provides sequence specificity to the nuclease:RNA complex.

[0255] In some embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 from Streptococcus pyogenes (Casn1) (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C, Sezate S., Suvorov AN, Kenton S., Lai HS, Lin S.P., Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III."Deltcheva E.,Chylinski K.,Sharma CM.,Gonzales K.,Chao Y.,PirzadaZ.A.,Eckert MR,Vogel J.,Charpentier E.,Nature 471:602-607(2011).

[0256] Because RNA-programmable nucleases such as Cas9 use RNA:DNA hybrids to target DNA cleavage sites, these proteins can, in principle, target any sequence specified by a guide RNA. Methods for using RNA programmable nucleases (e.g., Cas9) for site-specific cleavage (e.g., genome modification) 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, WY et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013); the entire contents of which are incorporated herein by reference).

[0257] The term "single nucleotide polymorphism (SNP)" is a variation of a single nucleotide that occurs at a specific location in the genome, wherein each variation is present to a certain extent (e.g., >1%) in the population. For example, at a specific base position in the human genome, a C nucleotide may appear in most individuals, but in a minority of individuals, the position is occupied by an A. This means that a SNP exists at that specific location, and the two possible nucleotide variations, C or A, are called alleles at that location. SNPs are the basis for differences in disease susceptibility. The severity of the disease and the way our bodies respond to treatment are also manifestations of genetic variation. SNPs can fall into the coding region of a gene, the non-coding region of a gene, or the intergenic region (the region between genes). In some embodiments, due to the degeneracy of the genetic code, SNPs within the coding sequence do not necessarily change the amino acid sequence of the protein produced. There are two types of SNPs in the coding region: synonymous SNPs 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 aren't located in protein-coding regions can still affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNAs. Gene expression affected by these SNPs is called an eSNP (expression SNP) and can be located upstream or downstream of the gene. Single nucleotide variants (SNVs) are variations in a single nucleotide that occur in all cells without any frequency restrictions. Somatic SNVs are also referred to as single nucleotide alterations.

[0258] "Specific binding" refers to a nucleic acid molecule, polypeptide, or complex thereof (e.g., a nucleic acid programmable DNA binding domain and a guide nucleic acid), compound, or molecule that recognizes and binds to a polypeptide and / or nucleic acid molecule of the invention but does not substantially recognize and bind to other molecules in a sample, such as a biological sample.

[0259] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" refers to the pairing, under various stringency conditions, between complementary polynucleotide sequences (e.g., the genes described herein) or portions thereof to form a double-stranded molecule. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0260] For example, stringent salt concentrations are typically less than about 750 mM sodium chloride and 75 mM trisodium citrate, preferably less than about 500 mM sodium chloride and 50 mM trisodium citrate, and more preferably less than about 250 mM sodium chloride and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of an organic solvent such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions typically include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, the concentration of detergents such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of vector DNA, are well known to those skilled in the art. Different degrees of stringency can be achieved by combining these different conditions as needed. In one embodiment, hybridization will be performed at 30°C in 750 mM sodium chloride, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization will be performed in 500 mM sodium chloride, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C. In another embodiment, hybridization will be performed in 250 mM sodium chloride, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations on these conditions will be apparent to those skilled in the art.

[0261] For most applications, the post-hybridization wash step will also vary in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As described above, wash stringency can be increased by lowering the salt concentration or increasing the temperature. For example, the stringent salt concentration for the wash step is preferably less than about 30 mM sodium chloride and 3 mM trisodium citrate, and most preferably less than about 15 mM sodium chloride and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash step 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. In one embodiment, the wash step is performed at 25°C in 30 mM sodium chloride, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash step is performed at 42°C in 15 mM sodium chloride, 1.5 mM trisodium citrate, and 0.1% SDS. In an even more preferred embodiment, the wash step is performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Other variations on these conditions will be 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.

[0262] "Split" means to separate into two or more fragments.

[0263] "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 "reconstructed" Cas9 protein. In a particular embodiment, the Cas9 protein is divided into two fragments within the disordered region of the protein, for example, as described in Nishimasu et al. in Cell, Volume 156, Issue 5, pp. 935-949, 2014, or 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 between about amino acids A292-G364, F445-K483, or E565-T637 within the SpCas9 region, or 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 a protein into two fragments is referred to as "splitting" the protein.

[0264] In other embodiments, the N-terminal portion of the Cas9 protein comprises amino acids 1-573 or 1-637 of Streptococcus pyogenes Cas9 wild type (SpCas9) (NCBI reference sequence: NC_002737.2, Uniprot reference sequence: Q99ZW2), and the C-terminal portion of the Cas9 protein comprises amino acids 574-1368 or 638-1368 of SpCas9 wild type, or a portion of the corresponding positions thereof.

[0265] The C-terminal portion of the split Cas9 can be connected to the N-terminal portion of the split Cas9 to form a complete Cas9 protein. In some embodiments, the C-terminal portion of the Cas9 protein starts where the N-terminal portion of the Cas9 protein ends. Therefore, in some embodiments, the C-terminal portion of the split Cas9 comprises a portion of amino acids (551-651)-1368 of spCas9. "(551-651)-1368" refers to the amino acids starting from amino acids 551-651 (inclusive) and ending at amino acid 1368.For example, the C-terminal portion of the split-Cas9 can include a portion of any one of the following spCas9 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, 575-1368, 576-1368, 577-1368, 578-1368, 579-1368, 580-1368, 581-1368, 582-1368, 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, 605-1368, 606-1368, 607-1368, 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, 625-1368 68, 626-1368, 627-1368, 628-1368, 629-1368, 630-1368, 631-1368, 632-1368, 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.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.

[0266] "Subject" refers to a mammal, including but not limited to a human or non-human mammal, such as a cow, horse, dog, sheep, or cat. 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.

[0267] "Substantially identical" refers to a polypeptide or nucleic acid molecule that is at least 60%, 80%, 85%, 90%, 95%, or even 99% identical to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (any of the nucleic acid sequences described herein). In one embodiment, such a sequence is at least 60%, 80%, 85%, 90%, 95%, or even 99% identical to the sequence used for comparison at the amino acid or nucleic acid level.

[0268] Sequence identity is typically determined using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, COBALT, EMBOSS Needle, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by specifying various substitutions, deletions, and / or other modified degrees of homology. 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 method for determining the degree of identity, the BLAST program can be used, where e -3 and e -100 Probability scores between and indicate closely related sequences.

[0269] For example, COBALT is used with the following parameters:

[0270] a) Alignment parameters: Gap penalties -11, -1 and End-Gappenalties -5, -1,

[0271] b) CDD parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved columns and Recompute on, and

[0272] c) Query Clustering parameters: Use query clusters on; Word Size4; Max clusterdistance 0.8; Alphabet Regular.

[0273] For example, when using EMBOSS Needle, the following parameters are available:

[0274] a) Matrix: BLOSUM62;

[0275] b) GAP OPEN: 10;

[0276] c) GAP EXTEND: 0.5;

[0277] d) Output format (OUTPUT FORMAT): pair;

[0278] e)END GAP PENALTY: false;

[0279] f) END GAP OPEN: 10; and

[0280] g) END GAP EXTEND: 0.5.

[0281] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a base editor. In one embodiment, the target site is deaminated by a deaminase or a fusion protein comprising a deaminase (e.g., adenine deaminase).

[0282] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disorder and / or symptom associated therewith or obtaining a desired pharmacological and / or physiological effect. It should be understood that, although not excluded, treating a disorder or condition does not require complete elimination of the disorder, condition, or symptoms associated therewith. In some embodiments, the effect is therapeutic, that is, but not limited to, the effect partially or completely reduces, attenuates, eliminates, diminishes, alleviates, reduces the intensity of the disease, or cures the disease and / or adverse symptoms attributable to the disease. In some embodiments, the effect is preventative, that is, the effect protects or prevents the occurrence or recurrence of a disease or condition. To this end, the presently disclosed methods comprise administering a therapeutically effective amount of a composition as described herein.

[0283] "Uracil glycosylase inhibitor" or "UGI" refers to a substance that inhibits the uracil excision repair system. In one embodiment, the substance is a protein or fragment thereof that binds to the host uracil-DNA glycosylase and prevents the removal of uracil residues from DNA. In one embodiment, a UGI is a protein, fragment, or domain thereof that is capable of inhibiting the uracil-DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain comprises wild-type UGI or a modified version thereof. In some embodiments, the UGI domain comprises a fragment of the exemplary amino acid sequence set forth below. In some embodiments, the UGI fragment comprises an amino acid sequence comprising 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 exemplary UGI sequence provided below. In some embodiments, the UGI comprises an amino acid sequence homologous to the exemplary UGI amino acid sequence described below or a fragment thereof. 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%, at least 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 a portion thereof. An exemplary UGI comprises the following amino acid sequence:

[0284] >splP14739IUNGI_BPPB2 uracil-DNA glycosylase inhibitor

[0285] MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML.

[0286] The term "vector" refers to a means for introducing a nucleic acid sequence into a cell to produce a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, and episomes. An "expression vector" is a nucleic acid sequence that contains a nucleotide sequence to be expressed in a recipient cell. An expression vector may include additional nucleic acid sequences to facilitate and / or promote expression of the introduced sequence, such as initiation, termination, enhancer, promoter, and secretion sequences.

[0287] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0288] DNA editing has emerged as a viable means to alter disease states by correcting pathogenic mutations at the genetic level. Until recently, all DNA editing platforms functioned by inducing DNA double-strand breaks (DSBs) at specific genomic loci and relying on endogenous DNA repair pathways to determine product outcomes in a semi-stochastic manner, thereby generating complex populations of gene products. While precise, user-defined repair outcomes can be achieved through the homology-directed repair (HDR) pathway, numerous challenges have hindered the efficient use of HDR for repair in therapeutically relevant cell types. In practice, this pathway is inefficient relative to competing, error-prone non-homologous end-joining pathways. Furthermore, HDR is strictly restricted to the G1 and S phases of the cell cycle, preventing precise repair of DSBs in post-mitotic cells. Consequently, it has proven difficult or impossible to efficiently alter genomic sequence in these populations in a user-defined, programmable manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0289] The features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description of illustrative embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings:

[0290] Figure 1 The G6PC nucleotide target sequence and corresponding amino acid sequence are depicted, indicating the bystander and target A>G bases to correct the GSD1a Q347X mutation.

[0291] Figure 2 depicts precise base correction and bystander editing. Figure 2A The positions of target and bystander nucleobases are depicted. Figure 2B Depicted are the precise on-target and bystander correction percentages of the GSD1a G6PC Q347X mutation in HEK293T cells using ABE8 variants.

[0292] Figure 3A and 3B Depicted are editor optimizations for correction of the GSD1a G6PC Q347X mutation in HEK293T cells. Figure 3A The G6PC nucleotide target sequence and indicated bystanders and the corresponding amino acid sequences at the target A>G bases and the GGA PAM sequence for correction of the GSD1a Q347X mutation are depicted. Figure 3B is a graph depicting the percentage of correction of the GSD1a G6PC Q347X mutation using ABE8 monomeric and heterodimeric variants.

[0293] Figure 4Graph depicting the percentage of correction of the GSD1aG6PC Q347X mutation using the ABE8 double mutant variant in HEK293T cells, comparing bystander (A2) and on-target (A6) A>G bases.

[0294] Figure 5 is a graph depicting the percentage of GSD1a Q347X mutations accurately corrected using ABE8 variants in patient-derived B lymphocytes.

[0295] Figure 6A and 6B Depicts precise correction of the GSD1a G6PCQ347X mutation in compound heterozygous (Q347X, G222R) patient iPS-derived hepatocytes. Figure 6A Depicted are the G6PC nucleotide target sequence, the corresponding amino acid sequence, and the GGA PAM sequence, indicating bystander and on-target A>G bases for correction of the GSD1a Q347X mutation. Figure 6B is a graph depicting the comparison of A>G base editing efficiency of the GSD1a Q347X mutation using ABE8 variants for on-target versus bystander correction.

[0296] Figure 7A and 7B Depicted are editor optimization for correction of the GSD1a Q347X mutation in patient iPS-derived hepatocytes. Figure 7A The NGA PAM sequence of GSD1a and the corresponding target sequence are shown, indicating bystander and on-target A>G bases. Figure 7B is a graph depicting base editing efficiency of the GSD1a Q347X mutation using the ABE8 variant.

[0297] Figure 8A and 8B A timeline for in vitro transduction of the GSD1a Q347X mutation in a primary hepatocyte co-culture system is provided. Figure 8A A timeline showing the schedule of in vitro transduction in hepatocyte monolayers or hepatocyte co-cultures at representative time points is provided. Figure 8B Shown are images of transduced primary hepatocytes from donors in a co-culture system for the GSD1aQ347X mutation.

[0298] Figure 9 Images showing GFP expression (GFP, bright field, merged) on day 6 (D6) in primary hepatocyte co-culture cells transduced with a lentiviral vector containing the GSD1a Q347X mutation at a multiplicity of infection (MOI) of 30, 100, and 300 lentiviruses.

[0299] Figure 10A 、 10Band 10C depict correction of the GSD1a Q347X mutation in a lentiviral-transduced primary hepatocyte co-culture system. Figure 10A Images of GFP expression in primary hepatocyte co-culture cells (donor RSE) transduced with a lentiviral vector containing the GSD1aQ347X mutation at an MOI of 500 are shown. Figure 10B Figure 2 is a graph depicting the efficiency of A>G base editing for on-target correction of the GSD1a Q347X mutation and indels in transduced primary hepatocyte co-cultures. The dashed line represents the A>G base editing efficiency for therapeutic benefit. Figure 10C Graph depicting the efficiency of A>G base editing of the GSD1a Q347X mutation in co-cultures of primary hepatocytes transduced with culture medium containing or without polyethylene glycol 8000 (PEG8K) and treated with collagenase types III, IV, and hyaluronic acid or left untreated.

[0300] Figure 11 The G6PC nucleotide target sequence and corresponding amino acid sequence are depicted, indicating bystander and on-target A>G bases to correct the GSD1a R83C mutation.

[0301] Figure 12A and 12B Depicted is the precise correction of the GSD1a G6PC R83C mutation in HEK293T cells. Figure 12A Depicted are the G6PC nucleotide target sequence and corresponding amino acid sequence, indicating bystander, synonymous, and on-target A>G bases, for correction of the GSD1a R83C mutation. Figure 12B Graph depicting the A>G base editing efficiency of the GSD1a R83C mutation using the ABE8 variant, comparing on-target versus bystander correction.

[0302] Figure 13A and 13B Depicts base editing of the G6PC R83C mutation via plasmid transfection in HEK293T lentiviral model cells. Figure 13A The GAGAAT PAM sequence of GSD1a gRNA#820 and the corresponding target sequence and the AGA PAM sequence of GSD1a gRNA#1121 and the corresponding target sequence are shown, indicating the on-target A>G bases of the bystander and target sequences. Figure 13B is a graph depicting the percentage of on-target and bystander correction of the GSD1aR83C mutation using ABE base editors with gRNA1121 or gRNA820.

[0303] Figure 14 is a graph depicting the A>G base editing efficiency of the GSD1a R83C mutation using the saABE8 variant.

[0304] Figure 15 Graph depicting the A>G base editing efficiency of the GSD1a R83C mutation using the saABE8 double mutant variant.

[0305] Figure 16 is a graph depicting the A>G base editing efficiency of on-target, bystander, and synonymous bystander correction of the GSD1a R83C mutation in HEK293T cells using ABE8 variants.

[0306] Figure 17A and 17B Depicted is the correction of the GSD1aQ347X mutation in primary mouse hepatocytes isolated from a GSD1a transgenic mouse model. Figure 17A Shown are images of primary mouse hepatocytes isolated from the ASC transgenic mouse model huG6PC, R83C(V166L). Figure 17B Graph depicting base editing efficiency at positions A12G, A10G, A6G, and indels for correction of the GSD1a R83C mutation in primary mouse hepatocytes isolated from a GSD1a transgenic mouse model using the ABE8 variant.

[0307] Figure 18 Graph depicting the levels of A>G base editing at on-target (12A) and off-target (6A) sites using the TadA-SaCas9 ABE editor in combination with guide RNAs of varying lengths as shown. Data were generated in HEK293T cells. Target site and other editing details are also provided.

[0308] Figure 19 Graph depicting the levels of A>G base editing (percent edited) at on-target (12A) and off-target (6A) sites using ABE8s (TadA*8 variant - SaCas9) in combination with 20nt and 21nt guide RNAs. Data were acquired in HEK293T cells.

[0309] Figure 20 Graph depicting the levels of A>G base editing (percentage correction of R83C) at on-target (12A) and off-target (6A) sites using the ABE base editor (TadA variant - SaCas9) in combination with 20nt or 21nt guide RNAs. Data were acquired in HEK293T cells.

[0310] Figure 21 Graph depicting the levels of A>G base editing (%) at on-target (12A) and off-target (6A) sites using the ABE base editor (TadA variant - SaCas9) in combination with 20nt or 21nt guide RNAs. Data were obtained in a primary human hepatocyte lentiviral model of GSD1a R83C.

[0311] Figure 22 Graph depicting the levels of A>G base editing (percent correction of R83C) at on-target (12A) and off-target (6A) sites using the ABE base editor (TadA variant - SaCas9) in combination with 20nt or 21nt guide RNAs. Data were obtained in a primary human hepatocyte lentiviral model of GSD1a R83C.

[0312] Figure 23 Graph depicting the levels of A>G (%) precise base editing at on-target and off-target sites in heterozygous transgenic GSD1a R83C mice.

[0313] Figure 24 This is a table describing all possible PAM-accessing Cas9 variants for the NRNN PAM. Only Cas9 variants that are required to recognize three or fewer defined nucleotides in their PAM are listed. Non-G PAM variants include SpCas9-NRRH, SpCas9-NRTH, and SpCas9-NRCH. DETAILED DESCRIPTION

[0314] Provided herein are compositions comprising novel adenosine base editors (e.g., ABE8) with improved efficiency and methods of using base editors comprising adenosine deaminase variants to alter mutations associated with glycogen storage disease type 1a (GSD1a).

[0315] The present invention is based, at least in part, on the discovery that base editors characterized by an adenosine deaminase variant (i.e., ABE8) can precisely correct single nucleotide polymorphisms in the endogenous glucose 6-phosphatase (G6PC) gene (e.g., R83C, Q347X).

[0316] The GSD1a mutations R83C and Q347X are cytidine-to-thymidine (C→T) conversion mutations that result in C·G to T·A base pair substitutions. These substitutions can be restored to the wild-type, non-pathogenic genomic sequence with adenosine base editors (ABEs), which catalyze the A·T to G·C substitutions. By extension, mutations caused by GSD1a are potential targets for restoration to the wild-type sequence using ABEs without the risk of overexpression of the G6PC gene as would be the case with gene therapy. Thus, A·T to G·C DNA base editing precisely corrects one or more of the most prevalent GSD1a-induced mutations in the G6PC gene.

[0317] Nucleobase editors

[0318] Disclosed herein are base editors or nuclear base editors for editing, modifying or changing a target nucleotide sequence of a polynucleotide. A nuclear base editor or base editor comprising a polynucleotide programmable nucleotide binding domain and a nuclear base editing domain (e.g., adenosine deaminase) is described herein. The polynucleotide programmable nucleotide binding domain, when bound to a bound guide polynucleotide (e.g., gRNA), can specifically bind to a target polynucleotide sequence (i.e., by a complementary base pairing sequence between the bases of the bound guide nucleic acid and the bases of the target polynucleotide), thereby targeting the base editor to a target nucleic acid sequence that needs 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.

[0319] Polynucleotide programmable nucleotide binding domain

[0320] It should be understood that the polynucleotide programmable nucleotide binding domain 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 guides the polynucleotide programmable nucleotide binding domain to RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, although they are not specifically listed in the present disclosure.

[0321] The polynucleotide programmable nucleotide binding domain of the base editor itself may include one or more domains. For example, the polynucleotide programmable nucleotide binding domain may include one or more nuclease domains. In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain may include a nucleic acid endonuclease or a nucleic acid exonuclease. In this article, the term "exonuclease" refers to a protein or polypeptide that can decompose nucleic acids (such as RNA or DNA) from free ends, and the term "endonuclease" refers to a protein or polypeptide that can catalyze (e.g., cut) an internal region in a nucleic acid (e.g., DNA or RNA). In some embodiments, the endonuclease can cut a single strand of a double-stranded nucleic acid. In some embodiments, the endonuclease can cut two chains of a double-stranded nucleic acid molecule. In some embodiments, the polynucleotide programmable nucleotide binding domain may be a deoxyribonuclease. In some embodiments, the polynucleotide programmable nucleotide binding domain may be a ribonuclease.

[0322] In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain can cut zero, one or two chains of the target polynucleotide.In some embodiments, the polynucleotide programmable nucleotide binding domain may include a nickase domain.Herein, the term "nickase" refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain that can cut only one of the two chains in a double-stranded nucleic acid molecule (such as DNA).In some embodiments, nickase can be derived from a polynucleotide programmable nucleotide binding domain in a completely catalytically active (such as natural) form by introducing one or more mutations into an active polynucleotide programmable nucleotide binding domain.For example, in the case where the polynucleotide programmable nucleotide binding domain includes a nickase domain derived from Cas9, the nickase domain derived from Cas9 can include a D10A mutation and histidine at position 840.In such embodiments, the residue H840 retains catalytic activity and can thus cut a single strand of a nucleic acid duplex.In another embodiment, the nickase domain derived from Cas9 can include an H840A mutation, and the amino acid residue at position 10 is still D. In some embodiments, the nickase can be derived from a polynucleotide programmable nucleotide binding domain in a fully catalytically active (e.g., native) form by removing all or part of the nuclease domain that is not required for nickase activity. For example, in the case where the polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the nickase domain derived from Cas9 may comprise all or part of a deletion of a RuvC domain or a HNH domain.

[0323] The amino acid sequence of an exemplary catalytically active Cas9 is as follows:

[0324]

[0325] A base editor comprising a polynucleotide programmable nucleotide binding domain comprising a nickase domain is therefore capable of producing single-stranded DNA breaks (nicks) at a specific polynucleotide target sequence (e.g., determined by the complementary sequence of the bound guide nucleic acid). In some embodiments, the chain of the nucleic acid duplex target polynucleotide sequence cut by a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain) is a chain that is not edited by a base editor (i.e., the chain cut by the base editor is opposite to the chain comprising the base to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain) can cut the chain of a DNA molecule targeted for editing. In such embodiments, the non-target chain is not cut.

[0326] Also provided herein is a base editor comprising a polynucleotide programmable nucleotide binding domain that is catalytically dead (i.e., cannot cut the target polynucleotide sequence).Herein, the terms "catalytically dead" and "nuclease dead" are used interchangeably to refer to a polynucleotide programmable nucleotide binding domain that has one or more mutations and / or deletions that cause it to be unable to cut a nucleic acid chain. In some embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain base editor may lack nuclease activity due to specific point mutations in one or more nuclease domains. For example, in the case where the base editor comprises a Cas9 domain, Cas9 may comprise a D10A mutation and an H840A mutation. Such mutations inactivate the two nuclease domains, resulting in loss of nuclease activity. In other embodiments, the 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 domain). In a further embodiment, the 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.

[0327] Also contemplated herein are mutations of polynucleotide programmable nucleotide binding domains that are capable of producing catalytically dead polynucleotide programmable nucleotide binding domains from previously functional versions of the polynucleotide programmable nucleotide binding domains. For example, in the case of catalytically dead Cas9 ("dCas9"), variants with mutations other than D10A and H840A are provided, which result in nuclease-inactivated Cas9. For example, such mutations include other amino acid replacements at D10 and H840, or other replacements within the Cas9 nuclease domain (e.g., replacements in the HNH nuclease subdomain and / or the RuvC1 subdomain). Based on the present disclosure and knowledge in the art, other suitable nuclease-inactive dCas9 domains will be apparent to those skilled in the art and are within the scope of the present 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 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9):833-838, the entire contents of which are incorporated herein by reference).

[0328] Non-limiting examples of polynucleotide programmable nucleotide binding domains that can be incorporated into base editors include CRISPR protein-derived domains, restriction nucleases, large-range nucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some embodiments, the base editor comprises a polynucleotide programmable nucleotide binding domain comprising a natural or modified protein or portion thereof, which is capable of binding to a nucleic acid sequence during CRISPR (i.e., clustered regularly interspaced short palindromic repeats)-mediated nucleic acid modification through a bound guide nucleic acid. Such proteins are referred to herein as “CRISPR proteins.” Therefore, disclosed herein are base editors comprising polynucleotide programmable nucleotide binding domains, wherein the polynucleotide programmable nucleotide binding domains comprise all or part of a CRISPR protein (i.e., a base editor comprising all or part of a CRISPR protein as a domain, also referred to as a “CRISPR protein-derived domain” of the base editor). The CRISPR protein-derived domain incorporated into the base editor can be modified compared to the wild-type or natural version of the CRISPR protein. For example, as described below, a CRISPR protein-derived domain can comprise one or more mutations, insertions, deletions, rearrangements, and / or recombinations relative to a wild-type or native form of a CRISPR protein.

[0329] CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). The CRISPR cluster consists of a spacer, a sequence complementary to the preceding mobile element, and the target invading nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, proper processing of pre-crRNA requires transcoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. tracrRNA serves as a guide for RNase 3-assisted processing of pre-crRNA. Cas9 / crRNA / tracrRNA then endonucleases the linear or circular dsDNA target complementary to the spacer. Target strands that are non-complementary to the crRNA are first endonucleolytically cleaved, followed by 3′-5′ exonucleolytic trimming. In nature, DNA binding and cleavage typically require a protein and two RNAs. However, single guide RNAs ("sgRNAs" or simply "gRNAs") can be engineered to integrate aspects of both crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), incorporated herein by reference in its entirety. Cas9 recognizes a short motif within the CRISPR repeat sequence (PAM or protospacer adjacent motif) to help distinguish self from non-self.

[0330] In some embodiments, the methods described herein can utilize engineered Cas proteins. A guide RNA (gRNA) is a short synthetic RNA consisting of a scaffold sequence required for Cas binding and a user-defined ~20 nucleotide spacer that defines the genomic target to be modified. Thus, technicians can alter the genomic target specificity of the Cas protein, depending in part on the specificity of the gRNA targeting sequence for the genomic target compared to the rest of the genome.

[0331] In some embodiments, the gRNA scaffold sequence is as follows: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAUAAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU.

[0332] In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is an endonuclease (e.g., a deoxyribonuclease or ribonuclease) that can bind to a target polynucleotide when bound to a bound guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is a nickase that can bind to a target polynucleotide when bound to a bound guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is a catalytic death domain that can bind to a target polynucleotide when bound to a bound guide nucleic acid. In some embodiments, the target polynucleotide bound by the CRISPR protein-derived domain of the base editor is DNA. In some embodiments, the target polynucleotide bound by the CRISPR protein-derived domain of the base editor is RNA.

[0333] Cas proteins useful herein include Class 1 and Class 2. Non-limiting examples of Cas proteins include: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, 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, homologs thereof, or modified versions thereof. Unmodified CRISPR enzymes can have DNA cleavage activity, such as Cas9, which has two functional nuclease domains: RuvC and HNH. The CRISPR enzyme can direct cleavage of one or both strands at a target sequence, for example, within the target sequence and / or within the complement of the target sequence. For example, the CRISPR enzyme can direct cleavage 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 the target sequence.

[0334] Vectors encoding CRISPR enzymes that are mutated relative to the corresponding wild-type enzyme such that the mutated 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 having 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 a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from Streptococcus pyogenes). Cas9 can refer to a polypeptide having at most or about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., from Streptococcus pyogenes). Cas9 may refer to a wild-type or modified form of the Cas9 protein, which may comprise amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0335] In some embodiments, the CRISPR protein-derived domain of the base editor may include all or part of Cas9 from the following: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheriae (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma aphididae (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanensis (NCBI Ref: NC_021846.1); Streptococcus (NCBI Ref: NC_021314.1); Bellella balticola (NCBI Ref: NC_018010.1); Campylobacter spirochetes (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: NC_021314.1); Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningitidis (NCBI Ref: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus.

[0336] Cas9 domains of nucleobase editors

[0337] The Cas9 nuclease sequence and structure are well known to those skilled in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus 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. Natl. Acad. Sci. USA 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales J., et al., “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471:602-607 (2011); and “A programmable dual-RNA-guided DNA endonuclease inadaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, Streptococcus pyogenes and Streptococcus 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 organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0338] In some embodiments, nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting exemplary Cas9 domains are provided herein. 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 cuts two chains of a double-stranded nucleic acid (e.g., two chains of a double-stranded DNA molecule). In some embodiments, the Cas9 domain comprises any amino acid sequence as described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence 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 amino acid sequence described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 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, 50 or more mutations compared to any one of the amino acid sequences listed herein. In some embodiments, the Cas9 domain comprises 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 described herein.

[0339] In some embodiments, proteins comprising Cas9 fragments are provided. For example, in some embodiments, the 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 a fragment thereof are referred to as "Cas9 variants". Cas9 variants have homology to Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70% identical to wild-type Cas9, 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, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the Cas9 variants may have 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, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., 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, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the corresponding wild-type Cas9 amino acid length. In some embodiments, the fragment is at least 100 amino acids long. 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.

[0340] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of the Cas9 protein, such as one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but only comprise one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and other suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0341] The Cas9 protein can be associated with a guide RNA that guides 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, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. In some embodiments, wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI reference sequence: NC_017053.1, nucleotide and amino acid sequences are as follows).

[0342]

[0343]

[0344]

[0345] (Single underline: HNH domain; double underline: RuvC domain)

[0346] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence:

[0347]

[0348]

[0349] (Single underline: HNH domain; double underline: RuvC domain).

[0350] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI reference sequence: NC_002737.2 (nucleotide sequence below); and Uniprot reference sequence: Q99ZW2 (amino acid sequence below):

[0351]

[0352]

[0353] (Single underline: HNH domain; double underline: RuvC domain)

[0354] In some embodiments, Cas9 refers to Cas9 from the following: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheriae (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma aphididae (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanensis (NCBI Ref: NC_021846.1); Streptococcus (NCBI Ref: NC_021314.1); Bellella balticola (NCBI Ref: NC_018010.1); Psychrocampus spirochetes I (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: NC_021314.1); Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1), or Neisseria meningitidis (NCBI Ref.: YP_002342100.1), or Cas9 from any other organism.

[0355] It should be understood that additional Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including variants and homologs thereof, are within the scope of the present disclosure. Exemplary Cas9 proteins include, but are not limited to, 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.

[0356] 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 (e.g., through a gRNA molecule) without cutting any chain of the double-stranded nucleic acid molecule. In some embodiments, the nuclease-inactivated dCas9 domain comprises a D10X mutation and an H840X mutation of an amino acid sequence listed herein, or a corresponding mutation in any amino acid sequence provided herein, wherein X is any amino acid change. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10A mutation and an H840A mutation of an amino acid sequence described herein, or a corresponding mutation in any amino acid sequence provided herein. As an example, the nuclease-inactive Cas9 domain is included in the amino acid sequence listed in the cloning vector pPlatTET-gRNA2 (accession number BAV54124).

[0357] The amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows:

[0358]

[0359] Based on the present disclosure and the knowledge in the art, other suitable nuclease-inactivating dCas9 domains will be apparent to those skilled in the art and are within the scope of the present 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 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9):833-838, the entire contents of which are incorporated herein by reference).

[0360] In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase, referred to as a "nCas9" protein (for "nickase" Cas9). Nuclease-inactive Cas9 proteins are interchangeably referred to as "dCas9" proteins (for nuclease-"dead" Cas9) or catalytically inactive Cas9. Methods for producing Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains 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 DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cuts the strand complementary to the gRNA, while the RuvC1 subdomain cuts the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337: 816-821 (2012); Qi et al., Cell. 28; 152(5): 1173-83 (2013)).

[0361] In some embodiments, the dCas9 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%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the dCas9 domains provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 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, 50, or more mutations compared to any one of the amino acid sequences listed herein. In some embodiments, the Cas9 domain comprises 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 described herein.

[0362] In some embodiments, the dCas9 corresponds to, or partially or completely comprises, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises D10A and H840A mutations or corresponding mutations in another Cas9.

[0363] In some embodiments, the dCas9 comprises the amino acid sequence of dCas9(D10A and H840A):

[0364] (Single underline: HNH domain; double underline: RuvC domain).

[0365] In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequences provided above, or at the corresponding position in any of the amino acid sequences provided herein.

[0366] In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which, for example, result in nuclease-inactive Cas9 (dCas9). For example, such mutations include other amino acid substitutions at D10 and H840, or other substitutions within the Cas9 nuclease domain (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs 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, at least 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 shorter or longer amino acid sequences of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or more.

[0367] In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase can be a Cas9 protein that can only cut one chain of a double-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, the Cas9 nickase cuts the target chain of the double-stranded nucleic acid molecule, meaning that the Cas9 nickase cuts the chain that is base-paired (complementary) with the gRNA (e.g., sgRNA) bound to Cas9. In some embodiments, the Cas9 nickase comprises a D10A mutation and has histidine at position 840. In some embodiments, the Cas9 nickase cuts the non-target, non-base editing chain of the double-stranded nucleic acid molecule, meaning that the Cas9 nickase cuts the chain that is not base-paired with the gRNA (e.g., sgRNA) bound to Cas9. In some embodiments, the 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 herein. Based on this disclosure and knowledge in the art, other suitable Cas9 nickases will be apparent to those skilled in the art and are within the scope of this disclosure.

[0368] The amino acid sequence of an exemplary catalytic Cas9 nickase (nCas9) is as follows:

[0369]

[0370] In some embodiments, the Cas9 refers to Cas9 from archaea (e.g., nanoarchaea), which constitute the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, the nucleic acid programmable DNA binding protein refers to CasX or CasY, which has been described in, for example, "New CRISPR-Cas systems from uncultivated microbes." by Burstein et al., Cell Res.2017Feb 21.doi:10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genome-analyzed metagenomics, many CRISPR-Cas systems have been identified, including Cas9, which was first reported in the field of archaea. This different Cas9 protein was found in the little-known nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, were found, which are one of the most compact systems discovered so far. In some embodiments, in the base editor system described herein, Cas9 is replaced by a variant of CasX or CasX. In some embodiments, in the base editor systems described herein, Cas9 is replaced by CasY or a variant of CasY. It should be understood that other RNA-guided DNA binding proteins can be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0371] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can 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 amino acid sequence included in the napDNAbp 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 will be appreciated that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.

[0372] An exemplary CasX ((uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53)tr|F0NN87|F0NN87_SULIHCRISPR-associated Casx protein OS=Sulfolobus icelandica (strain HVE10 / 4) GN=SiH_0402 PE=4SV=1) amino acid sequence is as follows:

[0373] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAAGWVLTRLGKAKV SEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0374] An exemplary CasX (>tr|F0NH53|F0NH53_SULIR CRISPR-associated protein, Casx OS=Sulfolobus icelandica (strain REY15A) GN=SiRe_0771 PE=4SV=1) amino acid sequence is as follows:

[0375] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0376] Proteus δ CasX

[0377] MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQ KWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0378] An exemplary CasY ((ncbi.nlm.nih.gov / protein / APG80656.1)>APG80656.1 CRISPR-associated protein CasY [uncultured Parabacterium bacteria]) amino acid sequence is as follows:

[0379]

[0380] The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Upon binding to its target, Cas9 undergoes a conformational change that positions the nuclease domains to cleave the opposite strand of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (approximately 3-4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired via one of two general repair pathways: (1) the efficient but error-prone nonhomologous end joining (NHEJ) pathway; or (2) the less efficient but high-fidelity homology-directed repair (HDR) pathway.

[0381] The "efficiency" of non-homologous end joining (NHEJ) and / or homology-directed repair (HDR) can be calculated by any convenient method. For example, in some embodiments, efficiency can be expressed as a percentage of successful HDR. For example, a surveyor nuclease assay can be used to produce a cleavage product, and the ratio of product to substrate can be used to calculate a percentage. For example, a surveyor nuclease can be used to directly cut the DNA containing the newly integrated restriction sequence as a result of successful HDR. More cracked substrates indicate a higher HDR percentage (higher HDR efficiency). As an illustrative example, the following equation [(cleavage product) / (substrate plus cleavage product)] can be used to calculate the score (percentage) of HDR (e.g., (b+c) / (a+b+c), where "a" is the band intensity of the DNA substrate, and "b" and "c" are cleavage products).

[0382] In some embodiments, 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 product to substrate can be used to calculate the percentage of NHEJ. T7 endonuclease I cuts the mismatched heteroduplex DNA produced by hybridization of wild-type and mutant DNA chains (NHEJ produces small random insertions and deletions (indels) at the original break site). More cutting indicates a higher NHEJ percentage (higher NHEJ efficiency). As an illustrative example, the score (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 Nov.; 8(11): 2281–2308).

[0383] The NHEJ repair pathway is the most active repair mechanism, which often results in small nucleotide insertions and deletions (indels) at the DSB site. The stochastic nature of NHEJ-mediated DSB repair has important practical significance because cell populations expressing Cas9 and gRNA or guide polynucleotides can lead to a variety of mutations. In some embodiments, the NHEJ generates small insertions and deletions in the target DNA, resulting in amino acid deletions, insertions, or frameshift mutations, leading to premature stop codons within the open reading frame (ORF) of the target gene. The ideal end result is a loss-of-function mutation within the target gene.

[0384] While NHEJ-mediated DSB repair typically disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to generate specific nucleotide changes, ranging from single nucleotide changes to large insertions, such as the addition of a fluorophore or tag. To utilize HDR for gene editing, a DNA repair template containing the desired sequence can be delivered to the cell type of interest using gRNA and Cas9 or Cas9 nickase. The repair template can contain the desired edit as well as additional homologous sequences immediately upstream and downstream of the target (called left and right homology arms). The length of each homology arm depends on the size of the introduced change, 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. Even in cells expressing Cas9, gRNA, and an exogenous repair template, the efficiency of HDR is typically low (<10% modified alleles). The efficiency of HDR can be improved by synchronizing cells, as HDR occurs during the S and G2 phases of the cell cycle. Chemical or genetic suppression of genes involved in NHEJ can also increase the frequency of HDR.

[0385] In some embodiments, Cas9 is a modified Cas9. A given gRNA targeting sequence can have additional sites throughout the genome where there is partial homology. These sites are called off-target sites and need to be considered when designing gRNA. In addition to optimizing gRNA design, the specificity of CRISPR can also be improved by modifying Cas9. Cas9 produces double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase is a D10A mutant of SpCas9 that retains one nuclease domain and produces DNA nicks instead of DSBs. The nickase system can also be combined with HDR-mediated gene editing for specific gene editing.

[0386] In some embodiments, Cas9 is a variant Cas9 protein. The variant Cas9 polypeptide has an amino acid sequence that differs by one amino acid compared to the amino acid sequence of the wild-type Cas9 protein (e.g., having a deletion, insertion, substitution, fusion). In some cases, the variant Cas9 polypeptide has an amino acid change (e.g., a deletion, insertion, or substitution) that reduces 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 compared to the corresponding wild-type Cas9 protein. In some embodiments, the variant Cas9 protein has no substantial nuclease activity. When the subject Cas9 protein is a variant Cas9 protein without substantial nuclease activity, it may be referred to as "dCas9".

[0387] In some embodiments, the variant Cas9 protein has reduced nuclease activity. For example, the variant Cas9 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% endonuclease activity compared to the wild-type Cas9 protein (e.g., wild-type Cas9 protein).

[0388] In some embodiments, variant Cas9 protein can cut the complementary chain of the guide target sequence, but the ability of cutting the non-complementary chain of the double-stranded guide target sequence is reduced. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, the variant Cas9 protein has D10A (aspartic acid to alanine at amino acid position 10) and can therefore cut the complementary chain of the double-stranded guide target sequence but cut the complementary chain of the non-double-stranded guide target sequence (therefore when the variant Cas9 protein cuts the double-stranded target nucleic acid, single-strand breaks (SSBs) are caused instead of double-strand breaks (DSBs)) (see, for example, Jinek et al., Science.2012Aug.17; 337(6096): 816-21).

[0389] In some embodiments, the variant Cas9 protein can cut the non-complementary chain of the double-stranded guide target sequence, but the ability to cut the complementary chain of the guide target sequence is reduced. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif). As a non-limiting example, in some embodiments, the variant Cas9 protein has an H840A (histidine to alanine at amino acid position 840) mutation, so the non-complementary chain of the guide target sequence can be cut, but the ability to cut the complementary chain of the guide target sequence is reduced (therefore, when the variant Cas9 protein cuts the double-stranded guide target sequence, SSB instead of DSB will be produced). The ability of such Cas9 proteins to cut guide target sequences (such as single-stranded guide target sequences) is reduced, but the ability to bind to guide target sequences (such as single guide target sequences) is retained.

[0390] In some embodiments, the variant Cas9 protein has a reduced ability to cut the complementary and non-complementary strands of a double-stranded target DNA. As a non-limiting example, in some embodiments, the variant Cas9 protein comprises both D10A and H840A mutations, such that the ability of the polypeptide to cut the complementary and non-complementary strands of a double-stranded target DNA is reduced. Such Cas9 proteins have a reduced ability to cut target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0391] As another non-limiting example, in some embodiments, the variant Cas9 protein contains W476A and W1126A mutations, which reduce the ability of the polypeptide to cut the target DNA. Such Cas9 proteins have a reduced ability to cut target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0392] As another non-limiting example, in some embodiments, the variant Cas9 protein contains P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, such that the ability of the polypeptide to cleave target DNA is reduced. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0393] As another non-limiting example, in some embodiments, the variant Cas9 protein includes H840A, W476A and W1126A mutations, so that the ability of polypeptide to cut target DNA is reduced. The ability of such Cas9 protein to cut target DNA (such as single-stranded target DNA) is reduced, but the ability to bind target DNA (such as single-stranded target DNA) is retained. As another non-limiting example, in some embodiments, the variant Cas9 protein includes H840A, D10A, W476A and W1126A mutations, so that the ability of polypeptide to cut target DNA is reduced. The ability of such Cas9 protein to cut target DNA (such as single-stranded target DNA) is reduced, but the ability to bind target DNA (such as single-stranded target DNA) is retained. In some embodiments, the variant Cas9 restores catalytic His residues at position 840 in the Cas9 HNH domain (A840H).

[0394] As another non-limiting example, in some embodiments, the variant Cas9 protein contains H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, so that the ability of the polypeptide to cut the target DNA is reduced. The ability of such Cas9 proteins to cut target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to target DNA (e.g., single-stranded target DNA) is retained. As another non-limiting example, in some embodiments, the variant Cas9 protein contains D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, so that the ability of the polypeptide to cut the target DNA is reduced. The ability of such Cas9 proteins to cut target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to target DNA (e.g., single-stranded target DNA) is retained. In some embodiments, when the variant Cas9 protein comprises W476A and W1126A mutations or when the variant Cas9 protein comprises P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, the variant Cas9 protein does not effectively bind to the PAM sequence. Therefore, in some such embodiments, when such variant Cas9 proteins are used in a binding method, the method does not require a PAM sequence. In other words, in some embodiments, when such variant Cas9 proteins are used in a binding method, the method can include a guide RNA, but the method can be performed in the absence of a PAM sequence (and the specificity of the binding is therefore provided by the targeting fragment of the guide RNA). Other residues can be mutated to achieve the above effects (even if one or other nuclease is partially inactivated). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986 and / or A987 can be changed (i.e., substituted). Furthermore, mutations other than alanine substitutions are also suitable.

[0395] In some embodiments, the variant Cas9 protein has reduced catalytic activity (for example, when the Cas9 protein has D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986 and / or A987 mutations, such as D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A and / or D986A), the variant Cas9 protein can still bind to the target DNA in a site-specific manner (because it is still guided to the target DNA sequence by the guide RNA) as long as it retains the ability to interact with the guide RNA.

[0396] In some embodiments, the variant Cas protein can be spCas9, spCas9-VRQR, spCas9-VRER, xCas9(sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.

[0397] In some embodiments, a modified SpCas9 comprising amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and having specificity for the altered PAM 5'-NGC-3' is used.

[0398] Alternatives to Streptococcus pyogenes Cas9 include RNA-guided endonucleases from the Cpf1 family, which have demonstrated cleavage activity in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of the class II CRISPR / Cas system. This acquired immunity mechanism is present in Prevotella and Francisella. The Cpf1 gene, associated with the CRISPR locus, encodes an endonuclease that uses a guide RNA to seek out and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the limitations of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, Cpf1-mediated DNA cleavage results in double-strand breaks with short 3' overhangs. The staggered cleavage pattern of Cpf1 could open up the possibility of targeted gene transfer, similar to traditional restriction enzyme cloning, and could increase the efficiency of gene editing. Like the aforementioned Cas9 variants and orthologs, Cpf1 also expands the number of CRISPR-targetable sites to AT-rich regions or AT-rich regions of the genome that lack the NGG PAM sites favored by SpCas9. The Cpf1 locus contains a mixed α / β domain, a RuvC-I followed by a helical region, a RuvC-II, and a zinc finger-like domain. The Cpf1 protein possesses a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, Cpf1 lacks the HNH endonuclease domain, and its N-terminus lacks the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain architecture indicates that Cpf1 is functionally unique and is classified as a Class 2, Type V CRISPR system. The Cas1, Cas2, and Cas4 proteins encoded by the Cpf1 locus are more similar to Type I and Type III systems than to Type II systems. Functional Cpf1 does not require transactivating CRISPR RNA (tracrRNA); therefore, only CRISPR (crRNA) is required. This facilitates genome editing because not only is Cpf1 smaller than Cas9, but its sgRNA molecule is also smaller (approximately half the nucleotides of Cas9). Compared to the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex cuts the target DNA or RNA by recognizing the adjacent motif 5'-YTN-3' or 5'-TTN-3'. After identifying the PAM, Cpf1 introduces a sticky-end-like DNA double-strand break with a 4- or 5-nucleotide overhang.

[0399] In some embodiments, the Cas9 is a Cas9 variant that is specific for a changed PAM sequence. In some embodiments, additional Cas9 variants and PAM sequences are described in Miller, SM et al., Continuous evolution of SpCas9 variants compatible with non-G PAMs, Nat. Biotechnol. (2020), the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 variants do not have specific PAM requirements. In some embodiments, Cas9 variants, such as SpCas9 variants, are specific for NRNH PAMs, wherein R is A or G and H is A, C or T. In some embodiments, the SpCas9 variants are specific for the PAM sequences AAA, TAA, CAA, GAA, TAT, GAT or CAC. In some embodiments, the SpCas9 variant comprises an amino acid replacement at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1218, 1219, 1221, 1249, 1256, 1264, 1290, 1318, 1317, 1320, 1321, 1323, 1332, 1333, 1335, 1337, or 1339, or their corresponding positions, as shown in SEQ ID NO: 1. In some embodiments, the SpCas9 variant comprises an amino acid replacement at position 1114, 1135, 1218, 1219, 1221, 1249, 1320, 1321, 1323, 1332, 1333, 1335, or 1337, or their corresponding positions, as shown in SEQ ID NO: 1. In some embodiments, the SpCas9 variant comprises an amino acid replacement at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1219, 1221, 1256, 1264, 1290, 1318, 1317, 1320, 1323, 1333, or their corresponding positions in SEQ ID NO: 1. In some embodiments, the SpCas9 variant comprises an amino acid replacement at position 1114, 1131, 1135, 1150, 1156, 1180, 1191, 1218, 1219, 1221, 1227, 1249, 1253, 1286, 1293, 1320, 1321, 1332, 1335, 1339, or their corresponding positions in SEQ ID NO: 1.In some embodiments, the SpCas9 variant comprises an amino acid substitution at position number 1114, 1127, 1135, 1180, 1207, 1219, 1234, 1286, 1301, 1332, 1335, 1337, 1338, 1349, or a corresponding position thereof in SEQ ID NO: 1. Exemplary amino acid substitutions and PAM specificities of SpCas9 variants are shown in Tables 1A-1D.

[0400] Table 1A.

[0401]

[0402]

[0403] Table 1B.

[0404]

[0405] Table 1C.

[0406]

[0407]

[0408] Table 1D.

[0409]

[0410] In some embodiments, Cas9 is Neisseria meningitidis Cas9 (NmeCas9) or a variant thereof. In some embodiments, NmeCas9 is specific for NNNNGAYW PAM, wherein Y is C or T and W is A or T. In some embodiments, NmeCas9 is specific for NNNNGYTT PAM, wherein Y is C or T. In some embodiments, NmeCas9 is specific for NNNNGTCTPAM. In some embodiments, NmeCas9 is Nme1 Cas9. In some embodiments, NmeCas9 has the following specificity: aNNNNGATT PAM, NNNNCCTA PAM, NNNNCCTC PAM, NNNNCCTT PAM, NNNNCCTG PAM, NNNNCCGT PAM, NNNNCCGGPAM, NNNNCCCA PAM, NNNNCCCT PAM, NNNNCCCC PAM, NNNNCCAT PAM, NNNNCCAG PAM, NNNNCCAT PAM, or NNNGATT PAM. In some embodiments, Nme1Cas9 is specific for NNNNGATTPAM, NNNNCCTA PAM, NNNNCCTC PAM, NNNNCCTT PAM, or NNNNCCTG PAM. In some embodiments, NmeCas9 is specific for CAA PAM, CAAA PAM, or CCA PAM. In some embodiments, NmeCas9 is Nme2 Cas9. In some embodiments, NmeCas9 is specific for NNNNCC (N4CC) PAM, where N is any one of A, G, C, or T. In some embodiments, NmeCas9 has the following specificity: NNNNCCGT PAM, NNNNCCGGPAM, NNNNCCCA PAM, NNNNCCCT PAM, NNNNCCCC PAM, NNNNCCAT PAM, NNNNCCAG PAM, NNNNCCATPAM, or NNNGATT PAM. In some embodiments, NmeCas9 is Nme3Cas9. In some embodiments, NmeCas9 is specific for NNNNCAAA PAM, NNNNCC PAM, or NNNNCNNN PAM. Additional NmeCas9 features and PAM sequences are described in Edraki et al., Mol. Cell. (2019) 73(4):714-726, which is incorporated herein by reference in its entirety.

[0411] An exemplary amino acid sequence of Nme1Cas9 is provided below:

[0412] Type II CRISPR RNA-guided endonuclease Cas9 [Neisseria meningitidis] WP_002235162.1

[0413]

[0414] An exemplary amino acid sequence of Nme2Cas9 is provided below:

[0415] Type II CRISPR RNA-guided endonuclease Cas9 [Neisseria meningitidis] WP_002230835.1

[0416]

[0417] Cas12 domain of nucleobase editors

[0418] Generally, 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 single protein effectors. For example, Cas9 and Cpf1 are Class 2 effectors, although of different types (Type II and Type V, respectively). In addition to Cpf1, Class 2 Type V CRISPR-Cas systems also include Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i). See, for example, Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems,” Mol. Cell, 2015 Nov. 5; 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, 2019 Jan. 4; 363: 88-91; the entire contents of each of which are incorporated herein by reference. Type V Cas proteins contain a RuvC (or RuvC-like) nuclease domain. Although the production of mature CRISPR RNA (crRNA) is generally independent of tracrRNA, for example, Cas12b / C2c1 requires tracrRNA to produce crRNA. Cas12b / C2c1 relies on crRNA and tracrRNA for DNA cleavage.

[0419] The nucleic acid programmable DNA binding proteins considered in the present invention include Cas proteins classified as the 2nd type V (Cas12 protein). Non-limiting examples of Cas 2 class V-type proteins include Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h and Cas12i, their homologues or modified forms thereof. As used herein, the Cas12 protein may also be referred to as Cas12 nuclease, Cas12 domain or Cas12 protein domain. In certain embodiments, the Cas12 protein of the present invention includes an amino acid sequence interrupted by an internal fusion protein domain such as a deaminase domain.

[0420] 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 forms a nick on one chain of a double-stranded nucleic acid (e.g., a double-stranded DNA molecule). In some embodiments, the Cas12 domain comprises any amino acid sequence as described herein. In some embodiments, the Cas12 domain comprises an amino acid sequence 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 amino acid sequence described herein. In some embodiments, the Cas12 domain comprises an amino acid sequence having 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 described herein. In some embodiments, the Cas12 domain comprises an amino acid sequence having 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 consecutive amino acid residues compared to any one of the amino acid sequences listed herein.

[0421] In some embodiments, proteins comprising Cas12 fragments are provided. For example, in some embodiments, the 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 a fragment thereof are referred to as "Cas12 variants". Cas12 variants have homology to Cas12 or a fragment thereof. For example, a Cas12 variant is at least about 70% identical to wild-type Cas12, 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, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. 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 (e.g., 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, 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 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 length of the corresponding wild-type Cas12 amino acids. In some embodiments, the fragment is at least 100 amino acids long. 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.

[0422] In some embodiments, Cas12 corresponds to, or partially or entirely comprises, a Cas12 amino acid sequence having one or more mutations that alter the Cas12 nuclease activity. For example, such mutations include amino acid replacements within the RuvC nuclease domain of Cas12. In some embodiments, variants or homologs 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, 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, Cas12 variants are provided that have the following shorter or longer amino acid sequences: about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids or more.

[0423] In some embodiments, the Cas12 fusion proteins provided herein include the full-length amino acid sequence of the Cas12 protein, such as one of the Cas12 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not include the full-length Cas12 sequence, but only include one or more fragments thereof. Exemplary amino acid sequences of suitable Cas12 domains are provided herein, and other suitable sequences of Cas12 domains and fragments will be apparent to those skilled in the art.

[0424] Typically, 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, the variant Cas12 polypeptide has an amino acid sequence that differs by 1, 2, 3, 4, 5 or more amino acids compared to the amino acid sequence of the wild-type Cas12 protein (e.g., having deletions, insertions, substitutions, fusions). In some cases, the variant Cas12 polypeptide has an amino acid change (e.g., a deletion, insertion, or substitution) that reduces the activity of the Cas12 polypeptide. For example, in some cases, the variant Cas12 is a Cas12b polypeptide having less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nickase activity of the corresponding wild-type Cas12b protein. In some cases, the variant Cas12b protein has no substantial nickase activity.

[0425] In some cases, the variant Cas12b protein has reduced nickase activity. For example, the variant 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 the wild-type Cas12b protein.

[0426] In some embodiments, the Cas12 protein includes an RNA-guided nuclease from the Cas12a / Cpf1 family that exhibits activity in mammalian cells. CRISPR of Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided nuclease of the class II CRISPR / Cas system. This acquired immunity mechanism is present in Prevotella and Francisella. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses guide RNA to find and cut viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the limitations of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, the result of Cpf1-mediated DNA cleavage is a double-strand break with a short 3' overhang. The staggered cleavage pattern of Cpf1 can open up the possibility of directional gene transfer, similar to traditional restriction enzyme cloning, which can improve the efficiency of gene editing. Like the aforementioned Cas9 variants and orthologs, Cpf1 can also expand the number of CRISPR-targetable sites to AT-rich regions or AT-rich regions of the genome that lack the NGG PAM sites favored by SpCas9. The Cpf1 locus contains a mixed α / β domain, a RuvC-I followed by a helical region, a RuvC-II, and a zinc finger-like domain. The Cpf1 protein possesses a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, unlike Cas9, Cpf1 lacks the HNH endonuclease domain, and its N-terminus lacks the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain architecture indicates that Cpf1 is functionally unique, classifying it as a Class 2, Type V CRISPR system. The Cas1, Cas2, and Cas4 proteins encoded by the Cpf1 locus are more similar to Type I and Type III systems than to Type II systems. Functional Cpf1 does not require transactivating CRISPR RNA (tracrRNA); therefore, only CRISPR (crRNA) is required. This facilitates genome editing because not only is Cpf1 smaller than Cas9, but its sgRNA molecule is also smaller (approximately half the nucleotides of Cas9). Compared to the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex cuts the target DNA or RNA by recognizing the adjacent motif 5'-YTN-3' or 5'-TTTN-3'. After recognizing the PAM, Cpf1 introduces a sticky-end-like DNA double-strand break with a 4- or 5-nucleotide overhang.

[0427] In some aspects of the invention, a vector encoding a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme such that the mutated 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 having 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 a wild-type exemplary Cas12 polypeptide (e.g., Cas12 from Bacillus jockii). Cas12 can refer to a polypeptide having at most or at most about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas12 polypeptide, such as from Bacillus hjöneri (BhCas12b), Bacillus V3-13 (BvCas12b), and Alicyclobacillus acidophilus (AaCas12b). Cas12 can refer to a wild-type or modified form of a Cas12 protein, which can include amino acid changes, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0428] Nucleic acid programmable DNA binding protein

[0429] Some aspects of the present disclosure provide a fusion protein comprising a domain acting as a nucleic acid programmable DNA binding protein, which can be used to guide a protein, such as a base editor, to a specific nucleic acid (e.g., DNA or RNA) sequence. In a particular embodiment, the 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 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, 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, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, 2. 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 protein, type V Cas effector protein, type VI Cas effector protein, CARF, DinG, homologs thereof, or modified or engineered versions thereof. Other nucleic acid-programmable DNA-binding proteins are also within the scope of the present disclosure, even though they may not be specifically listed in this disclosure.See, e.g., Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct; 1: 325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, the entire contents of each of which are incorporated herein by reference.

[0430] An example of a nucleic acid-programmable DNA-binding protein with a different PAM specificity than Cas9 is the clustered regularly interspaced short palindromic repeats from Proteobacteria and Francisella 1 (Cpf1). Similar to Cas9, Cpf1 is also a class 2 CRISPR effector. It has been shown that Cpf1 mediates robust DNA interference with characteristics distinct from those of Cas9. Cpf1 is a single RNA-guided endonuclease that lacks a tracrRNA and utilizes a T-rich protospacer-adjacent motif (TTN, TTTN, or YTN). Furthermore, Cpf1 cleaves DNA through staggered double-strand breaks. Of the 16 Cpf1 family proteins, two enzymes from the genera Acidinococcus and Lachnospiraceae have been shown to have potent genome editing activity in human cells. The Cpf1 protein is known in the art and has been described previously, for example, in 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 incorporated herein by reference.

[0431] Useful in the present compositions and methods are nuclease-inactive Cpf1 (dCpf1) variants, which can be used as guide nucleotide sequence-programmable DNA binding protein domains. The Cpf1 protein has a RuvC-like nuclease domain similar to the RuvC domain of Cas9 but without an HNH nuclease domain, and the N-terminus of Cpf1 does not have the α-helical recognition lobe of Cas9. It is shown in Zetsche et al., Cell, 163, 759-771, 2015 (incorporated herein by reference), that the RuvC-like domain of the Cpf1 is responsible for cutting two DNA chains and the inactivation of the RuvC-like domain inactivates the Cpf1 nuclease activity. For example, mutations corresponding to D917A, E1006A or D1255A in Francisella novicida Cpf1 can inactivate the Cpf1 nuclease activity. In some embodiments, the dCpf1 of the present disclosure comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It will be appreciated that any mutation that inactivates the RuvC domain of Cpf1, such as a substitution mutation, deletion, or insertion, can be used in accordance with the present disclosure.

[0432] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In some embodiments, the Cpf1 protein is a nuclease-inactivated Cpf1 (dCpf1). In some embodiments, 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 comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It will be appreciated that Cpf1 from other bacterial species may also be used in accordance with the present disclosure.

[0433] Wild-type Francisella novicida Cpf1 (D917, E1006, and D1255 are in bold and underlined)

[0434]

[0435] Francisella newjersey Cpf1 D917A (A917, E1006, and D1255 are bold and underlined)

[0436]

[0437] Francisella newjersey Cpf1 E1006A (D917, A1006, and D1255 are bold and underlined)

[0438]

[0439]

[0440] Francisella newjersey Cpf1 D1255A (D917, E1006, and A1255 are bold and underlined)

[0441]

[0442]

[0443] Francisella newjersey Cpf1 D917A / E1006A (A917, A1006, and D1255 are in bold and underlined)

[0444]

[0445]

[0446] Francisella newjersey Cpf1 D917A / D1255A (A917, E1006, and A1255 are in bold and underlined)

[0447]

[0448] Francisella newjersey Cpf1 E1006A / D1255A (D917, A1006, and A1255 are in bold and underlined)

[0449]

[0450]

[0451] Francisella newjersey Cpf1 D917A / E1006A / D1255A (A917, A1006, and A1255 are bold and underlined)

[0452]

[0453]

[0454] In some embodiments, one of the Cas9 domains present in the fusion protein can be replaced with a guide nucleotide sequence-programmable DNA-binding protein domain that does not require a PAM sequence.

[0455] In some embodiments, the Cas9 domain is a Cas9 domain from Staphylococcus 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.

[0456] In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence having a NNGRRT or NNGRRT PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of E781X, N967X, and R1014X mutations, or corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of E781K, N967K, and R1014H mutations, or one or more corresponding mutations in any amino acid sequence provided herein. In some embodiments, the SaCas9 domain comprises E781K, N967K, or R1014H mutations, or corresponding mutations in any amino acid sequence provided herein.

[0457] Exemplary SaCas9 sequences

[0458]

[0459] Residue N579, underlined and bolded above, can be mutated (e.g., to A579) to generate a SaCas9 nickase.

[0460] Exemplary SaCas9n sequences

[0461]

[0462]

[0463] Residue A579 above, which can be mutated from N579 to generate the SaCas9 nickase, is underlined and in bold.

[0464] Exemplary SaKKH Cas9

[0465]

[0466] Residue A579 above can be mutated from N579 to generate the SaCas9 nickase, which is underlined and in bold. Residues K781, K967, and H1014 above can be mutated from E781, N967, and R1014 to generate the SaKKH Cas9, which are underlined and in italics.

[0467] In some embodiments, the napDNAbp is a circular displacement. In the following sequences, plain text represents the adenosine deaminase sequence, bold sequences represent sequences derived from Cas9, italic sequences represent linker sequences, and underlined sequences represent bipartite nuclear localization sequences.

[0468] CP5 (with MSP "NGC" PID and "D10A" nickase):

[0469]

[0470] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, but are not limited to, 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 single protein effectors. For example, Cas9 and Cpf1 are Class 2 effectors. In addition to Cas9 and Cpf1, Shmakov et al. described three different Class 2 CRISPR Cas systems (Cas12b / C2c1 and Cas12c / C2c3) in “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov. 5; 60(3): 385-397, the entire contents of which are incorporated herein by reference. The effectors of two of these systems, Cas12b / C2c1 and Cas12c / C2c3, contain a RuvC-like endonuclease domain associated with Cpf1. The third system contains an effector with two predicted HEPN RNase domains. Unlike the CRISPR RNA produced by Cas12b / C2c1, which relies on both CRISPR RNA and tracrRNA for DNA cleavage, the production of mature CRISPR RNA is independent of tracrRNA.

[0471] The crystal structure of Alicyclobaccillus acidoterrastris Cas12b / C2c1 (AacC2c1) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See, for example, Liu et al., “C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism”, Mol. Cell, 2017 Jan. 19; 65(2): 310-322, the entire contents of which are incorporated herein by reference. The crystal structure has also been reported in Alicyclobaccillus C2c1 bound to target DNA in a ternary complex. See, for example, Yang et al., “PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease”, Cell, 2016 Dec. 15; 167(7): 1814-1828, the entire contents of which are incorporated herein by reference. A catalytically competent conformation of AacC2c1, encompassing both the target and non-target DNA strands, has been independently trapped within a single RuvC catalytic pocket, with Cas12b / C2c1-mediated cleavage resulting in a seven-nucleotide staggered break in the target DNA. Structural comparisons between the Cas12b / C2c1 ternary complex and previously identified Cas9 and Cpf1 counterparts demonstrate the diversity of mechanisms employed by the CRISPR-Cas9 system.

[0472] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can be Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp is Cas12b / C2c1 protein. In some embodiments, the napDNAbp is Cas12c / C2c3 protein. In some embodiments, the amino acid sequence included in the napDNAbp 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 naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp is naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the amino acid sequence comprised by the napDNAbp 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 should be understood that Cas12b / C2c1 or Cas12c / C2c3 from other bacterial species can also be used according to the present disclosure.

[0473] The amino acid sequence of Cas12b / C2c1 ((uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2|C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS=Alicyclobacillus (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) GN=c2c1 PE=1SV=1) is as follows:

[0474]

[0475] AacCas12b (Alicyclobacillus) - WP_067623834

[0476]

[0477] BhCas12b (Bacillus jockii) NCBI reference sequence: WP_095142515

[0478]

[0479] Including a variant called BvCas12b V4 (S893R / K846R / E837G changes relative to the above). BhCas12b (V4) is represented as follows: 5'mRNA Cap---5'UTR---bhCas12b---STOP sequence---3'UTR---120polyA tail5'UTR:GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC

[0480] 3'UTR (TriLink standard UTR)

[0481] GCTGGAGCCTCGGTGGCCATGCTTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGA

[0482] Nucleic acid sequence of bhCas12b(V4)

[0483]

[0484] In some embodiments, the Cas12b is BvCas12B. In some embodiments, the Cas12b comprises amino acid substitutions S893R, K846R, and E837G, as numbered in the BvCas12B exemplary sequence provided below.

[0485] BvCas12b (Bacillus sp. V3-13) NCBI reference sequence: WP_101661451.1

[0486]

[0487] In some embodiments, Cas12b is BTCas12b. BTCas12b (Bacillus amylothermophilus) NCBI reference sequence: WP_041902512

[0488]

[0489] In some embodiments, napDNAbp refers to Cas12c. In some embodiments, the Cas12c protein is a variant of Cas12c1 or Cas12c1. In some embodiments, the Cas12 protein is a variant of Cas12c2 or Cas12c2. In some embodiments, the Cas12 protein is a variant of HI0009 (i.e., OspCas12c) Cas12c protein or OspCas12c from the genus Oleophilus. These Cas12c molecules have been described in Yan et al., "Functionally Diverse Type V CRISPR-Cas Systems," Science, 2019 Jan. 4; 363: 88-91;, the entire contents of which are incorporated herein by reference. In some embodiments, the amino acid sequence comprised by the napDNAbp 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 naturally occurring Cas12c1, Cas12c2 or OspCas12c protein. In some embodiments, the napDNAbp is a naturally occurring Cas12c1, Cas12c2 or OspCas12c protein. In some embodiments, the amino acid sequence comprised by the napDNAbp 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 Cas12c1, Cas12c2 or OspCas12c protein described herein. It will be appreciated that Cas12c1, Cas12c2, or OspCas12c from other bacterial species may also be used in accordance with the present disclosure.

[0490] Cas12c1

[0491]

[0492] Cas12c2

[0493]

[0494] OspCas12c

[0495]

[0496] In some embodiments, the napDNAbp refers to Cas12g, Cas12h or Cas12i, which are described in, for example, Yan et al., "Functionally Diverse Type VCRISPR-Cas Systems," Science, 2019 Jan. 4; 363: 88-91, each of which is incorporated herein by reference in its entirety. By aggregating more than 10TB of sequence data, a new classification of V-type Cas proteins was determined, which showed a slight similarity to previously characterized class V proteins (including Cas12g, Cas12h and Cas12i). In some embodiments, the Cas12 protein is a variant of Cas12g or Cas12g. In some embodiments, the Cas12 protein is a variant of Cas12h or Cas12h. In some embodiments, the Cas12 protein is a variant of Cas12i or Cas12i. It should be understood that other RNA-guided DNA binding proteins can be used as napDNAbp and are within the scope of the present disclosure. In some embodiments, the amino acid sequence included in the napDNAbp 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 naturally occurring Cas12g, Cas12h or Cas12i protein. In some embodiments, the napDNAbp is a naturally occurring Cas12g, Cas12h or Cas12i protein. In some embodiments, the amino acid sequence included in the napDNAbp 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 Cas12g, Cas12h or Cas12i protein described herein. It should be understood that Cas12g, Cas12h or Cas12i from other bacterial species can also be used according to the present disclosure. In some embodiments, Cas12i is Cas12i1 or Cas12i2.

[0497] Cas12g1

[0498] MAQASSTPAVSPRPRPRYREERTLVRKLLPRPGQSKQEFRENVKKLRKAFLQFNADVSGVCQWAIQFRPRYGKPAEPTETFWKFFLEPETSLPPNDSRSPEFRRLQAFEAAAGINGAAALDDPAFTNELRDSILAVASRPKTKEAQRLFSRLKDYQPAHRMILAKVAAEWIESRYRRAHQNWERNYEEWKKEKQEWEQNHPELTPEIREAFNQIFQQLEVKEKRVRICPAARLLQNKDNCQYAGKNKHSVLCNQFNEFKKNHLQGKAIKFFYKDAEKYLRCGLQSLKPNVQGPFREDWNKYLRYMNLKEETLRGKNGGRLPHCKNLGQECEFNPHTALCKQYQQQLSSRPDLVQHDELYRKWRREYWREPRKPVFRYPSVKRHSIAKIFGENYFQADFKNSVVGLRLDSMPAGQYLEFAFAPWPRNYRPQPGETEISSVHLHFVGTRPRIGFRFRVPHKRSRFDCTQEELDELRSRTFPRKAQDQKFLEAARKRLLETFPGNAEQELRLLAVDLGTDSARAAFFIGKTFQQAFPLKIVKIEKLYEQWPNQKQAGDRRDASSKQPRPGLSRDHVGRHLQKMRAQASEIAQKRQELTGTPAPETTTDQAAKKATLQPFDLRGLTVHTARMIRDWARLNARQIIQLAEENQVDLIVLESLRGFRPPGYENLDQEKKRRVAFFAHGRIRRKVTEKAVERGMRVVTVPYLASSKVCAECRKKQKDNKQWEKNKKRGLFKCEGCGSQAQVDENAARVLGRVFWGEIELPTAIP

[0499] Cas12h1

[0500] MKVHEIPRSQLLKIKQYEGSFVEWYRDLQEDRKKFASLLFRWAAFGYAAREDDGATYSPSQALLERRLLLGDAEDVAIKFLDVLFKGGAPSSSCYSLFYEDFALRDKAKYSGAKREFIEGLATMPLDKIIERIRQDEQLSKIPAEEWLILGAEYSPEEIWEQVAPRIVNVDRSLGKQLRERLGIKCRRPHDAGYCKILMEVVARQLRSHNETYHEY LNQTHEMKTKVANNLTNEFDLVCEFAEVLEEKNYGLGWYVLWQGVKQALKEQKKPTKIQIAVDQLRQPKFAGLLTAKWRALKGAYDTWKLKKRLEKRKAFPYMPNWDNDYQIPVGLTGGLGVFTLEVKRTEVVVDLKEHGKLFCSHSHYFGDLTAEKHPSRYHLKFRHKLKLRKRDSRVEPTIGPWIEAALREITIQKKPNWGLFYLGLPYALSHGIDNF QIAKRFFSAAKPDKEVINGLPSEMVVGAADLNLSNIVAPVKARIGKGLEGPLHALDYGYGELIDGPKILTPDGPRCGELISLKRDIVEIKSAIKEFKACQREGLTMSEETTTWLSEVESPDSPRCMIQSRIADTSRRLNSFKYQMNKEGYQDLAEALRLLDAMDSYNSLLESYQRMHLSPGEQSPKEAKFDTKRASFRDLRRRRVAHTIVEYFDDCDIVFFEDLDGPSDSDSRNNALVKLLSPRTLLLYIRQALEKRGIGMVEVAKDGTSQNNPISGHVGWRNKQNKSEIYFYEDKELLVMDADEVGAMNILCRGLNHSVCPYSFVTKAPEKKNDEKKEGDYGKRVKRFLKDRYGSSNVRFLVASMGFVTVTTKRPKDALVGKRLYYHGGELVTHDLHNRMKDEIKYLVEKEVLARRVSLSDSTIKSYKSFAHV

[0501] Cas12i1

[0502]

[0503] Cas12i2

[0504]

[0505] Representative nucleic acid and protein sequences of base editors are as follows:

[0506] BhCas12b GGSGGS-ABE8-Xten20 in P153

[0507]

[0508]

[0509]

[0510]

[0511] BhCas12b GGSGGS-ABE8-Xten20 in K255

[0512]

[0513]

[0514]

[0515]

[0516] BhCas12b GGSGGS-ABE8-Xten20 in D306

[0517]

[0518]

[0519]

[0520]

[0521] BhCas12b GGSGGS-ABE8-Xten20 in D980

[0522]

[0523]

[0524]

[0525]

[0526] BhCas12b GGSGGS-ABE8-Xten20 in K1019

[0527]

[0528]

[0529]

[0530]

[0531] For the above sequences, the Kozak sequence is indicated in bold and underlined; the N-terminal nuclear localization signal (NLS) is marked; lowercase letters indicate the GGGSGGS linker;

[0532] The sequence encoding ABE8 is marked, and the unmodified sequence encodes BhCas12b; the double underline indicates the Xten20 linker; the single underline indicates the C-terminal NLS; represents a GS linker; italic characters represent the coding sequence of a 3x hemagglutinin (HA) tag.

[0533] Guide polynucleotides

[0534] In one embodiment, the guide polynucleotide is a guide RNA. The RNA / Cas complex can help "guide" the Cas protein to the target DNA. Cas9 / crRNA / tracrRNA endonucleolytically cuts a linear or circular dsDNA target that is complementary to the spacer. The target chain that is not complementary to the crRNA is first endonucleolytically cut and then exonucleolytically cut at 3'-5'. In nature, DNA binding and cutting generally require proteins and two RNAs. However, single guide RNA ("sgRNA" or simply "gRNA") can be engineered to integrate various aspects of crRNA and tracrRNA into a single RNA species. See, for example, Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes short motifs in CRISPR repeats (PAM or protospacer adjacent motifs) to help distinguish self from non-self. Cas9 nuclease sequence and structure are well known to those skilled in the art (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti, JJ et al., Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded 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 orthologs have been described in various species, including but not limited to Streptococcus pyogenes and Streptococcus 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 organisms and loci disclosed in Chylinski, Rhun, and Charpentier "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference.In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase.

[0535] In some embodiments, the guide polynucleotide is at least one single guide RNA ("sgRNA" or "gRNA"). In some embodiments, the guide polynucleotide is at least one tracrRNA. In some embodiments, the guide polynucleotide does not require a PAM sequence to guide a polynucleotide-programmable DNA binding domain (e.g., Cas9 or Cpf1) to a target nucleotide sequence.

[0536] The polynucleotide programmable nucleotide binding domains (e.g., CRISPR-derived domains) of base editors disclosed herein can identify target polynucleotide sequences by associating with guide polynucleotides. Guide polynucleotides (e.g., gRNA) are typically single-stranded and can be programmed to site-specifically bind to (i.e., by complementary base pairing) a target sequence of a polynucleotide, thereby directing the base editor bound to the guide nucleic acid to the target sequence. The guide polynucleotide can be DNA. The guide polynucleotide can be RNA. In some embodiments, the guide polynucleotide comprises natural nucleotides (e.g., adenosine). In some embodiments, the guide polynucleotide comprises non-natural (or non-natural) nucleotides (e.g., peptide nucleic acids or nucleotide analogs). In some embodiments, the targeting region of the guide nucleic acid sequence can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The length of the targeting region of the guide nucleic acid can be between 10-30 nucleotides, or between 15-25 nucleotides in length, or between 15-20 nucleotides in length.

[0537] In some embodiments, the guide polynucleotide comprises two or more separate polynucleotides that can interact with each other through, for example, complementary base pairing (e.g., a dual guide polynucleotide). For example, a guide polynucleotide can comprise a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA). For example, a guide polynucleotide can comprise one or more transactivating CRISPR RNAs (tracrRNAs).

[0538] In type II CRISPR systems, CRISPR protein (e.g., Cas9) targeting nucleic acids typically requires complementary base pairing between a first RNA molecule (crRNA) containing a sequence that recognizes the target sequence and a second RNA molecule (trRNA) containing a repeat sequence that forms a scaffold region that stabilizes the guide RNA-CRISPR protein complex. Such dual-guide RNA systems can be used as guide polynucleotides to direct the base editors disclosed herein to the target polynucleotide sequence.

[0539] In some embodiments, the base editors provided herein utilize single guide polynucleotides (e.g., gRNA). In some embodiments, the base editors provided herein utilize dual guide polynucleotides (e.g., dual gRNA). In some embodiments, the base editors provided herein utilize one or more guide polynucleotides (e.g., multiple gRNAs). In some embodiments, single guide polynucleotides are used for different base editors described herein. For example, a single guide polynucleotide can be used for an adenosine base editor.

[0540] In other embodiments, the guide polynucleotide may comprise a polynucleotide targeting portion of a nucleic acid and a scaffold portion of a nucleic acid in a single molecule (i.e., a single-molecule guide nucleic acid). For example, a single-molecule guide polynucleotide may 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 a base editor and guiding the base editor to a target polynucleotide sequence.

[0541] Typically, a guide polynucleotide (e.g., a crRNA / trRNA complex or gRNA) comprises a "polynucleotide targeting fragment" that includes a sequence capable of recognizing and binding to a target polynucleotide sequence, and a "protein binding fragment" that stabilizes the guide polynucleotide within the polynucleotide programmable nucleotide binding domain component of the base editor. In some embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to a DNA polynucleotide, thereby facilitating the editing of bases in the DNA. In other embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to an RNA polynucleotide, thereby facilitating the editing of bases in the RNA. As used herein, a "segment" refers to a segment or region of a molecule, for example, a stretch of contiguous nucleotides in a guide polynucleotide. A segment may also refer to a region / segment of a complex, such that a segment may comprise a region of more than one molecule. For example, when a guide polynucleotide comprises a plurality of nucleic acid molecules, the protein binding segment may include, for example, all or a portion of a plurality of separate molecules that hybridize along a complementary region. In some embodiments, a protein-binding segment of a DNA-targeting RNA comprising two separate molecules can comprise (i) 40-75 base pairs of a first RNA molecule that is 100 base pairs in length; and (ii) 10-25 base pairs of a second RNA molecule that is 50 base pairs in length. Unless expressly defined otherwise in a particular context, the definition of "segment" is not limited to a specific total number of base pairs, is not limited to any specific number of base pairs from a given RNA molecule, is not limited to a specific number of separate molecules in a complex, and can include regions of RNA molecules of any total length and can include regions complementary to other molecules.

[0542] The guide RNA or guide polynucleotide may comprise two or more RNAs, such as CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). In some embodiments, the guide RNA or guide polynucleotide comprises single-stranded RNA or a single guide RNA (sgRNA) formed by fusing a portion (e.g., a functional portion) of crRNA and tracrRNA. The guide RNA or guide polynucleotide may also be a double RNA comprising crRNA and tracrRNA. In addition, crRNA can hybridize with target DNA.

[0543] As described above, the guide RNA or guide polynucleotide can be an expression product. For example, the DNA encoding the guide RNA can be a vector comprising a sequence encoding the guide RNA. The guide RNA or guide polynucleotide can be transferred into cells by transfecting the cells with an isolated guide RNA or plasmid DNA comprising a sequence encoding the guide RNA and a promoter. The guide RNA or guide polynucleotide can also be transferred into cells in other ways, for example, using viral-mediated gene delivery.

[0544] The guide RNA or guide polynucleotide can be isolated. For example, the guide RNA can be transfected into a cell or organism in the form of isolated RNA. The guide RNA can be prepared by in vitro transcription using any in vitro transcription system known in the art. The guide RNA can be transferred into the cell in the form of isolated RNA rather than as a plasmid containing the guide RNA coding sequence.

[0545] A guide RNA or guide polynucleotide can contain three regions: a first region at the 5' end that can be complementary to a target site in a chromosomal sequence, a second internal region that can form a stem-loop structure, and a third 3' region that can be single-stranded. The first region of each guide RNA can also be different, allowing each guide RNA to guide the fusion protein to a specific target site. In addition, the second and third regions of each guide RNA can be identical across all guide RNAs.

[0546] The first region of the guide RNA or guide polynucleotide can be complementary to the sequence of the target site in the chromosome sequence, so that the first region of the guide RNA can be base paired with the target site. In some embodiments, the first region of the guide RNA may include or be from about 10 nucleotides to 25 nucleotides (i.e., from 10 nucleotides to about 25 nucleotides; or from about 10 nucleotides to about 25 nucleotides; or from 10 nucleotides to about 25 nucleotides; or about 10 nucleotides to 25 nucleotides) or more. For example, the base pairing region between the first region of the guide RNA and the target site in the chromosome sequence can be or can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length. In some embodiments, the length of the first region of the guide RNA can be or can be about 19, 20 or 21 nucleotides.

[0547] The guide RNA or guide polynucleotide may also include a second region that forms a secondary structure. For example, the secondary structure formed by the guide RNA may include a stem (or hairpin) and a loop. The length of the loop and the stem may vary. For example, the loop may have a length range of about 3 to 10 nucleotides, while the stem may have a length range of about 6 to 20 base pairs. The stem may include one or more protrusions of 1 to 10 or about 10 nucleotides. The total length of the second region may be in the range of about 16 to 60 nucleotides. For example, the length of the loop may be or may be about 4 nucleotides, and the stem may be or may be about 12 base pairs.

[0548] The guide RNA or guide polynucleotide can also include a third region at its 3' end that can be substantially single-stranded. For example, the third region can sometimes be non-complementary to any chromosomal sequence in the target cell and sometimes be non-complementary to the rest of the guide RNA. Furthermore, the length of the third region can vary. The length of the third region can be more than or equal to about 4 nucleotides. For example, the length of the third region can range from about 5 to 60 nucleotides.

[0549] A guide RNA or guide polynucleotide can target any exon or intron of a gene target. In some embodiments, a guide can target exon 1 or 2 of a gene; in other embodiments, a guide can 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 target different exons. Both exons and introns of a gene can be targeted.

[0550] The guide RNA or guide polynucleotide can target a nucleic acid sequence of about 20 nucleotides. The target nucleic acid can be less than about 20 nucleotides. The target nucleic acid can be at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or anywhere between 1-100 nucleotides in length. The target nucleic acid can be 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. The target nucleic acid sequence can be or can be about 20 bases 5' of the first nucleotide immediately adjacent to the PAM. The guide RNA can target a nucleic acid sequence. The 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.

[0551] A guide polynucleotide, such as a guide RNA, can refer to a nucleic acid that can hybridize to another nucleic acid, such as a target nucleic acid or a protospacer in the genome of a cell. A guide polynucleotide can be RNA. A guide polynucleotide can be DNA. A guide polynucleotide can be programmed or designed to site-specifically bind to a nucleic acid sequence. A guide polynucleotide can comprise a polynucleotide chain and can be referred to as a single guide polynucleotide. A guide polynucleotide can comprise two polynucleotide chains and can be referred to as a dual guide polynucleotide. A guide RNA can be introduced into a cell as an RNA molecule. For example, the RNA molecule can be transcribed in vitro and / or can be chemically synthesized. The RNA can be transcribed from a synthetic DNA molecule, for example Gene fragment. The guide RNA can then be introduced into the cell as an RNA molecule. The guide RNA can also be introduced into the cell in the form of a non-RNA nucleic acid molecule, such as a DNA molecule. For example, the DNA encoding the guide RNA can be operably linked to a promoter control sequence to express the guide RNA in the cell of interest. The RNA coding sequence can be operably linked to a promoter sequence recognized by RNA polymerase III (Pol III). Plasmid vectors that can be used to express the guide RNA include, but are not limited to, px330 vectors and px333 vectors. In some embodiments, the plasmid vector (e.g., px333 vector) may contain at least two DNA sequences encoding the guide RNA.

[0552] Methods for selecting, designing and validating guide polynucleotides such as guide RNAs and targeting sequences are described herein and are known to those skilled in the art. For example, in order to minimize the effects of potential substrate promiscuity of the deaminase domain in the nucleobase editor system (e.g., AID domain), the number of residues that may inadvertently become deamination targets (e.g., off-target C residues that may be present on ssDNA within the target nucleic acid locus) can be minimized. In addition, software tools can be used to optimize gRNAs corresponding to target nucleic acid sequences, for example, to minimize the total off-target activity of the entire genome. For example, for each possible targeting domain selection using Streptococcus pyogenes Cas9, all off-target sequences (before the selected PAM, such as NAG or NGG) can be identified in the genome, containing up to a specific number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base pairs. The first region of the gRNA that is complementary to the target site can be identified, and all first regions (e.g., crRNAs) can be ranked according to their total predicted off-target scores; the top-ranked targeting domains represent those that are likely to have the greatest on-target and least off-target activity. Candidate targeting gRNAs can be functionally assessed using methods known in the art and / or as described herein.

[0553] As a non-limiting example, a DNA sequence search algorithm can be used to identify the target DNA hybridization sequence in the crRNA of the guide RNA used with Cas9. GRNA design can be performed using the custom gRNA design software based on the public tool cas-offinder, as described in Bae S., Park J. and Kim J.-S. in Cas-OFFinder:A fast and versatile algorithm that searches for potential off-target sites of Cas9RNA-guided dendonucleases.Bioinformatics 30,1473-1475 (2014). The software scores the guide after calculating the whole genome off-target tendency. For guides with lengths ranging from 17 to 24, matches from perfect matches to 7 mismatches are generally considered. Once the off-target site is determined by calculation, a total score is calculated for each guide and is summarized in a table output using a web interface. In addition to identifying the potential target site adjacent to the PAM sequence, the software also identifies all PAM adjacent sequences that differ from the selected target site by 1, 2, 3 or more nucleotides. The genomic DNA sequence of the target nucleic acid sequence, such as the target gene, can be obtained, and publicly available tools such as the RepeatMasker program can be used to screen for repeat components. RepeatMasker searches for repeat elements and low complexity regions in the input DNA sequence. The output is a detailed annotation of the repeats present in the given query sequence.

[0554] After identification, the first region of the guide RNA, such as crRNA, can be graded based on their distance from the target site, their orthogonality, and the presence of 5' nucleotides to identify close matches to relevant PAM sequences (e.g., 5'G based on the identification of close matches containing relevant PAMs in the human genome, such as NGG PAM of Streptococcus pyogenes, NNGRRT or NNGRRV PAM of Staphylococcus aureus). As used herein, orthogonality refers to the number of sequences in the human genome that contain the least number of mismatches with the target sequence. For example, "high level of orthogonality" or "good orthogonality" can refer to a 20-mer targeting domain that does not have the same sequence in the human genome except for the intended target, nor does it contain one or two mismatches in the target sequence. Targeting domains with good orthogonality can be selected to minimize off-target DNA cleavage.

[0555] In some embodiments, a reporter system can be used to detect base editing activity and test candidate guide polynucleotides. In some embodiments, the reporter system can include a reporter gene-based assay, wherein the base editing activity results in the expression of the reporter gene. For example, the reporter system can include a reporter gene comprising an inactivation start codon, such as a mutation from 3'-TAC-5' to 3'-CAC-5' on the template chain. After the target C is successfully deaminated, the corresponding mRNA will be transcribed as 5'-AUG-3' instead of 5'-GUG-3', thereby achieving translation of the reporter gene. Suitable reporter genes are obvious to those skilled in the art. Non-limiting examples of reporter genes include encoding green fluorescent protein (GFP), red fluorescent protein (RFP), luciferase, secretory alkaline phosphatase (SEAP), or any other gene whose expression is detectable and obvious to those skilled in the art. The reporter system can be used to test many different gRNAs, for example, to determine which residues in the target DNA sequence will be targeted by the corresponding deaminase. sgRNAs targeting non-template chain nucleotide residues can also be tested to assess the off-target effects of specific base editing proteins (e.g., Cas9 deaminase fusion proteins). In some embodiments, such gRNAs can be designed so that the mutated start codon does not hybridize with the gRNA. The guide polynucleotide may comprise standard nucleotides, modified nucleotides (e.g., pseudouridine), nucleotide isomers, and / or nucleotide analogs. In some embodiments, the guide polynucleotide may comprise 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 any other suitable fluorescent dye), a detection label (e.g., biotin, digoxigenin, etc.), quantum dots, or gold particles.

[0556] The guide polynucleotide can be chemically and / or enzymatically synthesized. For example, a standard solid phase synthesis method based on phosphoramidite can be used to synthesize the guide RNA. Alternatively, the guide RNA can be synthesized in vitro by operably linking the 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 variants thereof. In embodiments where the guide RNA comprises two separate molecules (e.g., crRNA and tracrRNA), the crRNA can be chemically synthesized and the tracrRNA can be enzymatically synthesized.

[0557] In some embodiments, the base editor system can include multiple guide polynucleotides, such as gRNAs, that target the base editor to one or more loci (e.g., at least 1 gRNA, at least 2 gRNAs, at least 5 gRNAs, at least 10 gRNAs, at least 20 gRNAs, at least 30 g RNAs, or at least 50 gRNAs). In some embodiments, multiple gRNA sequences can be arranged in series in a single polynucleotide. In some embodiments, the gRNA sequences arranged in series are separated by directional repeats.

[0558] The DNA sequence encoding the guide RNA or guide polynucleotide can also be part of the vector. In addition, the vector can contain additional expression control sequences (such as enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selection marker sequences (such as GFP or antibiotic resistance genes, such as puromycin), replication origins, etc. The DNA molecule encoding the guide RNA or guide polynucleotide can be linear or circular.

[0559] In some embodiments, one or more components of the base editor system may be encoded by a DNA sequence. Such DNA sequences can be introduced into an expression system, such as a cell, together or separately. For example, a DNA sequence encoding a polynucleotide programmable nucleotide binding domain and a guide RNA can be introduced into a cell, and each DNA sequence can be part of a separate molecule (e.g., a vector comprising a polynucleotide programmable nucleotide binding domain encoding sequence and a second vector containing a guide RNA encoding sequence) or both can be part of the same molecule (e.g., a vector containing both a polynucleotide programmable nucleotide binding domain and a guide RNA encoding (and regulatory) sequence).

[0560] A guide polynucleotide can comprise one or more modifications to provide a nucleic acid with new or enhanced characteristics. A guide polynucleotide can comprise a nucleic acid affinity tag. A guide polynucleotide can comprise synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides.

[0561] In some embodiments, the gRNA or guide polynucleotide may comprise modifications. Modifications may be made at any position in the gRNA or guide polynucleotide. More than one modification may be made to a single gRNA or guide polynucleotide. The gRNA or guide polynucleotide may be quality controlled after modification. In some embodiments, quality control may include PAGE, HPLC, MS, or any combination thereof.

[0562] Modifications of the gRNA or guide polynucleotide can be substitutions, insertions, deletions, chemical modifications, physical modifications, stabilization, purification, or any combination thereof.

[0563] The gRNA or guide polynucleotide can also be modified with 5' adenylate, 5' guanosine triphosphate cap, 5' N7-methylguanosine triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' phosphorothioate, 5' phosphate, 5' phosphorothioate, Cis-Syn thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, d spacer, PC spacer, r spacer, Spacer 18, Spacer 9, 3'-3' modification, 5'-5' modification, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesterol TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, bis-biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3'DABCYL, black hole quencher 1, black hole quencher 2, DABCYLSE, 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 analog, wobble / universal base, fluorescent dye label, 2'-fluoro RNA, 2'-O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphorothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate, 5'-methylcytidine-5'-triphosphate, or any combination thereof.

[0564] In some embodiments, the modification is permanent. In other embodiments, the modification is temporary. In some embodiments, the gRNA or guide polynucleotide is modified multiple times. The gRNA or guide polynucleotide modifications can alter the physicochemical properties of the nucleotides, such as their conformation, polarity, hydrophobicity, chemical reactivity, base pairing interactions, or any combination thereof.

[0565] 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, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNNGATT, NNAGAAW, or NAAAC. Y is a pyrimidine; N is any nucleotide base; and W is A or T.

[0566] The modification can also be a phosphorothioate substitute. In some embodiments, natural phosphodiester bonds can be easily and rapidly degraded by cellular nucleases; modification of internucleotide bonds using phosphorothioate (PS) bond substitutes can be more stably hydrolyzed by cellular degradation. The modification can increase the stability of the gRNA or guide polynucleotide. The modification can also enhance biological activity. In some embodiments, the phosphorothioate-enhanced RNA gRNA can inhibit RNase A, RNase T1, calf serum nuclease, or any combination thereof. These properties can enable PS-RNA gRNA to be used in applications where there is a high likelihood of exposure to nucleases in vivo or in vitro. For example, a phosphorothioate (PS) bond can be introduced between the last 3-5 nucleotides at the 5'- or 3'-end of the gRNA, which can inhibit exonuclease degradation. In some embodiments, phosphorothioate bonds can be added throughout the gRNA to reduce endonuclease attack.

[0567] protospacer-adjacent motif

[0568] The term "protospacer adjacent motif (PAM)" or PAM-like motif refers to a 2-6 base pair DNA sequence that immediately follows the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. In some embodiments, the PAM can be a 5' PAM (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM (i.e., located downstream of the 5' end of the protospacer).

[0569] The PAM sequence is crucial for target binding, but the exact sequence depends on the type of Cas protein.

[0570] The base editors provided herein may include a CRISPR protein-derived domain capable of binding to a nucleotide sequence comprising a canonical or non-canonical protospacer adjacent motif (PAM) sequence. A PAM site is a nucleotide sequence close to a target polynucleotide sequence. Some aspects of the present disclosure provide base editors comprising all or part of a CRISPR protein with different PAM specificities.

[0571] For example, a typical Cas9 protein, such as Cas9 from Streptococcus pyogenes (spCas9), requires a typical NGGPAM sequence to bind to a specific nucleic acid region, where the "N" in "NGG" is adenine (A), thymine (T), guanine (G) or cytosine (C), and G is guanine. The PAM can be specific to the CRISPR protein and can be different between different base editors containing different CRISPR protein-derived domains. The PAM can be 5' or 3' of the target sequence. The PAM can be located upstream or downstream of the target sequence. The length of the PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. Typically, the length of the PAM is between 2-6 nucleotides. Several PAM variants are described in Table 2 below.

[0572] Table 2. Cas9 proteins and corresponding PAM sequences

[0573]

[0574]

[0575] 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, "MQKFRAER").

[0576] 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. In some embodiments, the NGT PAM variant is selected from a group of targeted mutations provided in Tables 3A and 3B below.

[0577] Table 3A: NGT PAM variant mutations at residues 1219, 1335, 1337, 1218

[0578]

[0579]

[0580] Table 3B: NGT PAM variant mutations at residues 1135, 1136, 1218, 1219, and 1335

[0581]

[0582]

[0583] In some embodiments, the NGT PAM variant is selected from variants 5, 7, 28, 31, or 36 in Tables 2 and 3. In some embodiments, the variant has improved NGT PAM recognition.

[0584] In some embodiments, the NGT PAM variants have mutations at residues 1219, 1335, 1337, and / or 1218. In some embodiments, NGT PAM variants having mutations to improve recognition are selected from the variants provided in Table 4 below.

[0585] Table 4: NGT PAM variant mutations at residues 1219, 1335, 1337 and 1218

[0586] Variants E1219V R1335Q T1337 G1218 1 F V T 2 F V R 3 F V Q 4 F V L 5 F V T R 6 F V R R 7 F V Q R 8 F V L R

[0587] In some embodiments, base editors specific for NGT PAMs can be generated as provided in Table 5 below.

[0588] Table 5. NGT PAM variants

[0589]

[0590]

[0591] 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 is variant 5. In some embodiments, the NGTN variant is variant 6.

[0592] In some embodiments, the Cas9 domain is a Cas9 domain from Streptococcus 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 D9X mutation, or a corresponding mutation in any amino acid sequence provided herein, wherein X is any amino acid other than D. In some embodiments, the SpCas9 comprises a D9A mutation, or a corresponding mutation in any amino acid sequence provided herein. In some embodiments, the SpCas9 domain, SpCas9d domain or SpCas9n domain can bind to a nucleic acid sequence with a non-canonical PAM. In some embodiments, the SpCas9 domain, SpCas9d domain or SpCas9n domain can bind to a nucleic acid sequence with an NGG, NGA or NGCG PAM sequence. In some embodiments, the SpCas9 domain comprises one or more of D1134X, R1335X and T1336X mutations, or a corresponding mutation in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1134E, R1335Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises D1134E, R1335Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises one or more of D1134X, R1335X, and T1336X mutations, or corresponding mutations in any of the amino acid sequences provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1134V, R1335Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises D1134V, R1335Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises one or more of D1134X, G1217X, R1335X, and T1336X mutations, or corresponding mutations in any of the amino acid sequences provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1134V, G1217R, R1335Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein.In some embodiments, the SpCas9 domain comprises D1134V, G1217R, R1335Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein.

[0593] In some embodiments, the amino acid sequence included in the Cas9 domain of any fusion protein provided herein 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 the Cas9 polypeptide described herein. In some embodiments, the Cas9 domain of any fusion protein provided herein comprises the amino acid sequence of any Cas9 polypeptide described herein. In some embodiments, the Cas9 domain of any fusion protein provided herein consists of the amino acid sequence of any Cas9 polypeptide described herein.

[0594] In some embodiments, a PAM recognized by a CRISPR protein-derived domain of a base editor disclosed herein can be presented to the cell on an oligonucleotide distinct from the insert encoding the base editor (e.g., an AAV insert). In such embodiments, providing the PAM on a separate oligonucleotide can allow cleavage of a target sequence that would otherwise not be cleaved because an adjacent PAM is not present on the same polynucleotide as the target sequence.

[0595] In one embodiment, Streptococcus pyogenes Cas9 (SpCas9) can be used as the CRISPR endonuclease for genome engineering. However, others can also be used. In some embodiments, different endonucleases can be used to target certain genomic targets. In some embodiments, synthetic SpCas9 derivatives with non-NGG PAM sequences can be used. In addition, other Cas9 orthologs from different species have been identified, and these "non-SpCas9" can be combined with a variety of PAM sequences that can also be used in the present disclosure. For example, the relatively large size of SpCas9 (approximately 4 kb coding sequence) can result in plasmids carrying SpCas9 cDNA being unable to be efficiently expressed in cells. In contrast, the coding sequence of Staphylococcus aureus Cas9 (SaCas9) is approximately 1 kilobase shorter than SpCas9, which may enable its efficient expression in cells. Similar to SpCas9, the SaCas9 endonuclease is capable of modifying target genes in mammalian cells in vitro and in vivo in mice. In some embodiments, Cas proteins can target different PAM sequences. In some embodiments, the target gene can be adjacent to the Cas9 PAM, for example, 5'-NGG. In other embodiments, other Cas9 orthologs may have different PAM requirements. For example, other PAMs, such as those from Streptococcus thermophilus (5'-NNAGAA for CRISPR1 and 5'-NGGNG for CRISPR3) and Neisseria meningitidis (5'-NNNNGATT), can also be adjacent to the target gene.

[0596] In some embodiments, for the Streptococcus pyogenes system, the target gene sequence can be before (i.e., 5' to) 5'-NGGPAM, and the 20-nt guide RNA sequence can be base-paired with the opposite strand to mediate Cas9 cleavage adjacent to the PAM. In some embodiments, the adjacent cut can be or can be about 3 base pairs upstream of the PAM. In some embodiments, the adjacent cut can be or can be about 10 base pairs upstream of the PAM. In some embodiments, the adjacent cut can be or can be about 0-20 base pairs upstream of the PAM. For example, the adjacent cut can be 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 the PAM. The adjacent cut can also be 1 to 30 base pairs downstream of the PAM. The sequence of an exemplary SpCas9 protein capable of binding to a PAM sequence is as follows:

[0597] The amino acid sequence of an exemplary PAM-bound SpCas9 is as follows:

[0598]

[0599] The amino acid sequence of an exemplary PAM-bound SpCas9n is as follows:

[0600]

[0601] The amino acid sequence of an exemplary PAM-bound SpEQR Cas9 is as follows:

[0602]

[0603] In the above sequence, residues E1134, Q1334, and R1336 can be mutated from D1134, R1335, and T1336 to generate SpEQR Cas9, which are underlined and bolded.

[0604] The amino acid sequence of an exemplary PAM-bound SpVQR Cas9 is as follows:

[0605]

[0606]

[0607] In the above sequence, residues V1134, Q1334, and R1336 can be mutated from D1134, R1335, and T1336 to generate SpVQR Cas9, which are underlined and in bold.

[0608] The amino acid sequence of an exemplary PAM-bound SpVRER Cas9 is as follows:

[0609]

[0610]

[0611] In the above sequence, residues V1134, R1217, Q1334, and R1336 can be mutated from D1134, G1217, R1335, and T1336 to generate SpVRER Cas9, which are underlined and in bold.

[0612] In some embodiments, the engineered SpCas9 variants are capable of recognizing protospacer-adjacent motif (PAM) sequences flanked by 3'H (non-G PAM) (see Tables 1A-1D; Figure 24 ). In some embodiments, the SpCas9 variant recognizes NRNH PAM (wherein R is A or G and H is A, C, or T). In some embodiments, the non-G PAM is NRRH, NRTH, or NRCH (see, e.g., Miller, SM et al., Continuous evolution of SpCas9 variants compatible with non-G PAMs, Nat. Biotechnol. (2020), the contents of which are incorporated herein by reference in their entirety).

[0613] 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, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SpyMacCas9 domain, SpCas9d domain, or SpCas9n domain can bind to a nucleic acid sequence having an NAA PAM sequence.

[0614] The sequences of exemplary Cas9 A homologs of Spy Cas9 in S. maculatus with the native 5'-NAAN-3' PAM specificity are known in the art and described, for example, by Jakimo et al. (www.biorxiv.org / content / biorxiv / early / 2018 / 09 / 27 / 429654.full.pdf) and provided below.

[0615] SpyMacCas9

[0616]

[0617] In some embodiments, variant Cas9 protein comprises H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1218A mutations, so that the ability of polypeptide to cut target DNA or RNA is reduced. The ability of such Cas9 protein to cut target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind target DNA (e.g., single-stranded target DNA) is retained. As another non-limiting example, in some embodiments, variant Cas9 protein contains D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1218A mutations, so that the ability of polypeptide to cut target DNA is reduced. The ability of such Cas9 protein to cut target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind target DNA (e.g., single-stranded target DNA) is retained. In some embodiments, when the variant Cas9 protein comprises W476A and W1126A mutations or when the variant Cas9 protein comprises P475A, W476A, N477A, ​​D1125A, W1126A and D1218A mutations, the variant Cas9 protein does not effectively bind to the PAM sequence. Therefore, in some such cases, when such variant Cas9 proteins are used in the binding method, the method does not require a PAM sequence. In other words, in some embodiments, when such variant Cas9 proteins are used in the binding method, the method can include a guide RNA, but the method can be performed in the absence of a PAM sequence (and the specificity of the binding is therefore provided by the targeting fragment of the guide RNA). Other residues can be mutated to achieve the above effects (i.e., inactivate one or other nuclease parts). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986 and / or A987 can be changed (i.e., substituted). Furthermore, mutations other than alanine substitutions are also suitable.

[0618] In some embodiments, the CRISPR protein-derived domain of the base editor may include all or part of a Cas9 protein having a canonical PAM sequence (NGG). In other embodiments, the Cas9-derived domain of the base editor may adopt a non-canonical PAM sequence. Such sequences have been described in the art and are apparent to those skilled in the art. For example, the Cas9 domain incorporating non-canonical PAM sequences has been described in Kleinstiver, BP et al., “Engineered CRISPR-Cas9nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, BP et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015), each of which is incorporated herein by reference in its entirety.

[0619] Cas9 domains that reduce PAM exclusivity

[0620] Typically, Cas9 proteins, such as Cas9 from Streptococcus pyogenes (spCas9), require a typical NGG PAM sequence to bind to a specific nucleic acid region, where the "N" in "NGG" is adenosine (A), thymidine (T), or cytosine (C), and G is guanosine. This may limit the ability to edit the desired bases in the genome. In some embodiments, the base editing fusion proteins provided herein may need to be placed in a precise position, such as a region containing a target base located upstream of the PAM. See, for example, 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 incorporated herein by reference. Therefore, in some embodiments, any fusion protein provided herein may include a Cas9 domain capable of binding to a nucleotide sequence that does not include a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and are apparent to those skilled in the art. For example, Cas9 domains that bind to non-canonical PAM sequences have been described in Kleinstiver, BP et al. “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, BP et al. “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015), the entire contents of each of which are incorporated herein by reference.

[0621] High-fidelity Cas9 domain

[0622] Some aspects of the present disclosure provide a high-fidelity Cas9 domain. In some embodiments, the high-fidelity Cas9 domain is an engineered Cas9 domain that comprises one or more mutations compared to the corresponding wild-type Cas9 domain, which reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA. Without wishing to be bound by any particular theory, a high-fidelity Cas9 domain with reduced electrostatic interaction with the DNA sugar-phosphate backbone may have fewer off-target effects. In some embodiments, the Cas9 domain (e.g., a wild-type Cas9 domain) comprises one or more mutations that reduce the association between the Cas9 domain and the DNA sugar-phosphate backbone. In some embodiments, the Cas9 domain comprises one or more mutations that reduce the association of the Cas9 domain with the sugar-phosphate backbone of 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%.

[0623] In some embodiments, any Cas9 fusion protein provided herein comprises one or more of N497X, R661X, Q695X and / or Q926X mutations, or corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, any Cas9 fusion protein provided herein comprises one or more of N497A, R661A, Q695A and / or Q926A mutations, or corresponding mutations in any amino acid sequence provided herein. In some embodiments, the Cas9 domain comprises a D10A mutation, or a corresponding mutation in any amino acid sequence provided herein. Cas9 domains with high fidelity are known in the art and are apparent to those skilled in the art. For example, Cas9 domains with high fidelity have been described in Kleinstiver, BP et al., “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.” Nature 529, 490-495 (2016); and Slaymaker, IM et al., “Rationally engineered Cas9 nucleases with improved specificity.” Science 351, 84-88 (2015), the entire contents of each of which are incorporated herein by reference.

[0624] In some embodiments, the improved 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 super-accurate Cas9 variant (HypaCas9). The improved Cas9eSpCas9 (1.1) contains an alanine substitution that weakens the interaction between the HNH / RuvC groove and the non-target DNA strand, preventing strand separation and cutting at off-target sites. Similarly, SpCas9-HF1 reduces off-target editing by alanine substitutions that disrupt the interaction of Cas9 with the DNA phosphate backbone. HypaCas9 contains mutations in the REC3 domain (SpCas9 N692A / M694A / Q695A / H698A) that increase Cas9 proofreading and target discrimination. All three high-fidelity enzymes produce fewer off-target edits compared to wild-type Cas9.

[0625] Exemplary high-fidelity Cas9s are provided below.

[0626] High-fidelity Cas9 domain mutations relative to Cas9 are shown in bold and underlined.

[0627]

[0628]

[0629] Fusion proteins containing a nuclear localization sequence (NLS)

[0630] In some embodiments, the fusion protein provided herein further comprises one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, such as nuclear localization sequences (NLS). In one embodiment, a bipartite NLS is used. In some embodiments, the NLS comprises an amino acid sequence that promotes the import of proteins comprising the NLS into the nucleus (e.g., by nuclear transport). In some embodiments, any fusion protein 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 the nCas9 domain or the dCas9 domain. In some embodiments, the NLS is fused to the N-terminus of a deaminase. In some embodiments, the NLS is fused to the C-terminus of a deaminase. In some embodiments, the NLS is fused to the C-terminus of a deaminase. In some embodiments, the NLS is fused to the fusion protein through one or more joints. In some embodiments, the NLS is fused to the fusion protein in the absence of a joint. In some embodiments, the NLS comprises the amino acid sequence of any NLS sequence provided or mentioned herein. Additional nuclear localization sequences are known in the art and are apparent to those skilled in the art. For example, Plank et al., describe NLS sequences in PCT / EP2000 / 011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, the NLS comprises an amino acid sequence selected from the group consisting of: PKKKRKVEGADKRTADGSEFESPKKKRKV, KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRKPKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0631] In some embodiments, the NLS is present in a linker or is flanked by a linker, such as those described herein. In some embodiments, the N-terminal or C-terminal NLS is a bipartite NLS. A bipartite NLS comprises two basic amino acid clusters separated by a relatively short spacer sequence (thus bipartite-2, whereas a monopartite NLS is not). The NLS KR[PAATKKAGQA]KKKK of nucleoplasmin is a prototype of the ubiquitous bipartite signal: two basic amino acid clusters separated by a spacer of approximately 10 amino acids. The sequence of an exemplary bipartite NLS is as follows:

[0632] PKKKRKVEGADKRTADGSEFESPKKKRKV

[0633] In some embodiments, the fusion protein comprising adenosine deaminase, napDNAbp (e.g., Cas9 domain) and NLS does not comprise a linker sequence. In some embodiments, there is a linker sequence between one or more domains or proteins (e.g., adenosine deaminase, Cas9 domain or NLS). In some embodiments, the general structure of an exemplary Cas9 fusion protein with adenosine deaminase and Cas9 domain comprises any of the following structures, wherein NLS is a nuclear localization sequence (e.g., any NLS provided herein), NH2 is the C-terminus of the N-terminal fusion protein, and COOH is the C-terminus of the fusion protein:

[0634] NH2-NLS-[adenosine deaminase]-[Cas9 domain]-COOH;

[0635] NH2-NLS[Cas9 domain]-[adenosine deaminase]-COOH;

[0636] NH2-[adenosine deaminase]-[Cas9 domain]-NLS-COOH; or

[0637] NH2-[Cas9 domain]-[adenosine deaminase]-NLS-COOH.

[0638] Should be understood that fusion rotein described in the present disclosure can comprise one or more additional features.For example, in some embodiments, described fusion rotein can comprise inhibitor, cytoplasm localization sequence, export sequence, for example nuclear export sequence or other localization sequence, and can be used for dissolving, purifying or detecting the sequence tag of described fusion rotein.Suitable protein tag provided herein includes but is not limited to biotin carboxylase carrier protein (BCCP) label, myc label, calmodulin label, FLAG label, hemagglutinin (HA) label, polyhistidine label, also referred to as histidine label or His label, maltose binding protein (MBP) label, nus label, glutathione-S-transferase (GST) label, green fluorescent protein (GFP) label, thioredoxin label, S label, Softags (for example Softag 1, Softag 3), chain label, biotin ligase label, F1AsH label, V5 label and SBP label.Other suitable sequences are apparent to those skilled in the art.In certain embodiments, described fusion rotein comprises one or more His tags.

[0639] Vectors encoding CRISPR enzymes 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. The CRISPR enzyme can comprise an NLS 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 NLSs at the amino terminus and one or more NLSs at the carboxy terminus). When more than one NLS is present, each NLS can be selected independently of the others, such that a single NLS can be present in more than one copy and / or combined with one or more other NLSs present in one or more copies.

[0640] The CRISPR enzyme used in the method may comprise about 6 NLSs. An NLS is considered to be near the N- or C-terminus when the amino acid closest to the NLS is within about 50 amino acids of the polypeptide chain from the N- or C-terminus, such as within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 amino acids.

[0641] Nucleobase editing domain

[0642] Described herein is a base editor comprising a fusion protein comprising a polynucleoti...

Claims

1. A method of editing a glucose-6-phosphatase (G6PC) polynucleotide containing a single nucleotide polymorphism (SNP) associated with type 1a glycogen storage disease (GSD1a) in vitro, the method comprising contacting the G6PC polynucleotide with a base editor in a complex with one or more guide RNAs, in, One or more of the guide RNAs direct the base editor to achieve A·T to G·C changes of the SNP associated with GSD1a, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain is composed of the following amino acid sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV consists of LHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 3) or a fragment thereof lacking only the N-terminal methionine, the adenosine deaminase domain having one or more changes, wherein: a) The one or more changes are composed of: Changes of V82S, Y147T, Y147R, Q154R and Q154S selected from the group consisting of reference SEQ ID NO:3, or a combination of changes of reference SEQ ID NO:3: V82S and Y147R, V82S and Q154R, V82S and Q154S, V82S and Y123H, and V82S, Y123H and Y147T; the SNP associated with GSD1a results in the expression of a G6PC polypeptide with a Q347X mutation, wherein X represents a stop codon; and one or more of the guide RNAs include spacers composed of the nucleic acid sequence GACCUAGGCGAGGCAGUAGG; or b) The one or more changes are composed of: Changes of V82S, Y147T, Y147R and Q154S selected from the group consisting of reference SEQ ID NO:3, or a combination of changes of reference SEQ ID NO:3: Y147T and Q154S, Y147R and Q154S, V82G, Y147T and Q154S, and R20A, K21A, Y147T, and Q154S; The SNP associated with GSD1a results in the expression of the G6PC polypeptide with R83C mutation; and one or more of the guide RNAs include spacers composed of the nucleic acid sequences CAGUAUGGACACUGUCCAAA or AGUAUGGACACUGUCCAAA.

2. The method of claim 1, in, The contact is in the cell.

3. The method of claim 1, in, The polynucleotide programmable DNA binding domain is Streptococcus pyogenes Cas9 (SpCas9).

4. The method of claim 3, in, The polynucleotide programmable DNA binding domain is SpCas9 which is specific for non-G prototype spacer adjacent motifs (PAMs).

5. The method of claim 4, in, The SpCas9 is specific for the nucleic acid sequence 5'-NGA-3', wherein N is A, T, C or G.

6. The method according to claim 4 or 5, in, The SpCas9 is specific for the prototype spacer adjacent motif (PAM) composed of the nucleic acid sequences 5'-AGA-3' or 5'-GGA-3'.

7. The method of claim 4, in, The SpCas9 is specific for the nucleic acid sequence NGA, wherein N is A, T, C or G.

8. The method of claim 1, in, The polynucleotide programmable DNA binding domain is Staphylococcus aureus Cas9 (SaCas9).

9. The method of claim 8, in, SaCas9 has specificity for the nucleic acid sequence 5'-NNGRRT-3' of the prototype spacer adjacent motif (PAM), where N is A, T, C or G, and R is A or G.

10. The method of claim 9, in, SaCas9 is specific for the prototype spacer adjacent motif (PAM) composed of the nucleic acid sequence 5'-GAGAAT-3'.

11. The method of claim 8, in, SaCas9 is specific for the prototype spacer adjacent motif (PAM) composed of the nucleotide sequence NNGRRT, where N is A, T, C or G, and R is A or G.

12. The method of claim 1, in, The polynucleotide programmable DNA binding domain is nuclease-inactive.

13. The method of claim 12, in, The polynucleotide programmable DNA binding domain is a nick enzyme.

14. The method of claim 13, in, The nickase comprises the following amino acid sequence: DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKS DGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKDLIIKL PKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD.

15. The method of claim 1, in, The adenosine deaminase domain is capable of deaminating adenosine in deoxyribonucleic acid (DNA).

16. The method of claim 1, in, The base editor comprises a single adenosine deaminase domain.

17. The method of claim 1, in, The base editor further comprises a second adenosine deaminase domain, wherein the second adenosine deaminase domain is a wild-type adenosine deaminase domain.

18. The method of claim 1, in, The adenosine deaminase domain consists of the following sequences: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

19. A cell, comprising: a base editor or a polynucleotide encoding the base editor, in, The base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain is composed of the following amino acid sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTA HAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGS LMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD(SEQ ID NO:3) or a fragment thereof lacking only the N-terminal methionine, the adenosine deaminase domain has one or more changes, and one or more guide RNAs that guide the base editor to implement A·T to G·C alterations of SNPs associated with GSD1a, wherein: a) the one or more changes consist of: Changes of V82S, Y147T, Y147R, Q154R and Q154S selected from the group consisting of reference SEQ ID NO:3, or a combination of changes of reference SEQ ID NO:3: V82S and Y147R, V82S and Q154R, V82S and Q154S, V82S and Y123H, and V82S, Y123H and Y147T; the SNP associated with GSD1a results in the expression of a glucose-6-phosphatase (G6PC) polypeptide with a Q347X mutation, wherein X represents a stop codon; and one or more of the guide RNAs include spacers composed of the nucleic acid sequence GACCUAGGCGAGGCAGUAGG; or b) The one or more changes are composed of: Changes of V82S, Y147T, Y147R and Q154S selected from the group consisting of reference SEQ ID NO:3, or a combination of changes of reference SEQ ID NO:3: Y147T and Q154S, Y147R and Q154S, V82G, Y147T and Q154S, and R20A, K21A, Y147T, and Q154S; The SNP associated with GSD1a results in the expression of the G6PC polypeptide with R83C mutation; and one or more of the guide RNAs include spacers composed of the nucleic acid sequences CAGUAUGGACACUGUCCAAA or AGUAUGGACACUGUCCAAA.

20. The cell of claim 19, in, The cells are hepatocytes or hepatocyte precursors.

21. The cell of claim 19, in, The cells express the G6PC polypeptide.

22. The cell of claim 19, in, The cells are from individuals suffering from GSD1a.

23. The cell of claim 19, in, The cells are mammalian cells.

24. The cell of claim 19, in, The cells are human cells.

25. The cell of claim 19, in, The A·T to G·C alteration of the SNP associated with GSD1a results in the replacement of the stop code at position 347 of the amino acid sequence of the G6PC with glutamine or the replacement of the cysteine ​​at position 83 of the amino acid sequence of the G6PC with arginine.

26. The cell of claim 19, in, The polynucleotide programmable DNA binding domain is Streptococcus pyogenes Cas9 (SpCas9).

27. The cell of claim 26, in, The polynucleotide programmable DNA binding domain is SpCas9 which is specific for non-G prototype spacer adjacent motifs (PAMs).

28. The cell of claim 27, in, The SpCas9 is specific for the prototype spacer adjacent motif (PAM) composed of the nucleic acid sequence 5'-NGA-3', wherein N is A, T, C or G.

29. The cell of claim 27 or 28, in, The SpCas9 is specific for the prototype spacer adjacent motif (PAM) composed of the nucleic acid sequences 5'-AGA-3' or 5'-GGA-3'.

30. The cell of claim 29, in, The SpCas9 is specific for the prototype spacer adjacent motif (PAM) composed of the nucleic acid sequence NGA, where N is A, T, C or G.

31. The cell of claim 19, in, The polynucleotide programmable DNA binding domain is Staphylococcus aureus Cas9 (SaCas9).

32. The cell of claim 31, in, SaCas9 is specific for the prototype spacer adjacent motif (PAM) composed of the nucleic acid sequence 5'-NNGRRT-3', wherein N is A, T, C or G and R is A or G.

33. The cell of claim 32, in, SaCas9 is specific for the prototype spacer adjacent motif (PAM) composed of the nucleic acid sequence 5'-GAGAAT-3'.

34. The cell of claim 31, in, SaCas9 is specific for the prototype spacer adjacent motif (PAM) composed of the nucleic acid sequence NNGRRT, where N is A, T, C or G, and R is A or G.

35. The cell of claim 19, in, The polynucleotide programmable DNA binding domain is nuclease-inactive.

36. The cell of claim 19, in, The polynucleotide programmable DNA binding domain is a nick enzyme.

37. The cell of claim 36, in, The nickase comprises the following amino acid sequence: DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKS DGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKDLIIKL PKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD.

38. The cell of claim 19, in, The adenosine deaminase domain is capable of deaminating adenosine in deoxyribonucleic acid (DNA).

39. The cell of claim 19, in, The base editor comprises a single adenosine deaminase domain.

40. The cell of claim 19, in, The base editor further comprises a second adenosine deaminase domain, wherein the second adenosine deaminase domain is a wild-type adenosine deaminase domain.

41. The cell of claim 19, in, The adenosine deaminase domain consists of the following sequences: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

42. The cell of claim 19, in, The scaffolds of the one or more guide RNAs are from the following sequences composition: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAAAGGCUAGUCCGUUAUCAACUUGAAAAAAGUGGCACCGAGUCGGUGCUUUUU.

43. The cell of claim 19, in, The scaffolds of the one or more guide RNAs are from the following sequences composition: GUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAAAAGGCAAAAAUGCCGUGUUAUCUCGUCAACUUGUUGGCGAGAUUUUUUU.

44. Use of a base editor or polynucleotide encoding the base editor in the manufacture of a drug for the treatment of type 1a glycogen storage disease (GSD1a) in an individual, in, The treatment includes administering to the individual: The base editor or polynucleotide encoding the base editor, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain is composed of the following amino acid sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO:3) or a fragment thereof lacking only the N-terminal methionine, the adenosine deaminase domain has one or more changes, and one or more guide RNAs that guide the base editor to implement the A·T to G·C alteration of SNPs associated with GSD1a, wherein: a) The one or more changes are composed of: Changes of V82S, Y147T, Y147R, Q154R and Q154S selected from the group consisting of reference SEQ ID NO:3, or a combination of changes of reference SEQ ID NO:3: V82S and Y147R, V82S and Q154R, V82S and Q154S, V82S and Y123H, and V82S, Y123H and Y147T; the SNP associated with GSD1a results in the expression of a glucose-6-phosphatase (G6PC) polypeptide with a Q347X mutation, wherein X represents a stop codon; and one or more of the guide RNAs include spacers composed of the nucleic acid sequence GACCUAGGCGAGGCAGUAGG; or b) The one or more changes are composed of: Changes of V82S, Y147T, Y147R and Q154S selected from the group consisting of reference SEQ ID NO:3, or a combination of changes of reference SEQ ID NO:3: Y147T and Q154S, Y147R and Q154S, V82G, Y147T and Q154S, and R20A, K21A, Y147T, and Q154S; The SNP associated with GSD1a results in the expression of the G6PC polypeptide with R83C mutation; and one or more of the guide RNAs include spacers composed of the nucleic acid sequences CAGUAUGGACACUGUCCAAA or AGUAUGGACACUGUCCAAA.

45. The use of claim 44, in, The individual is a mammal.

46. ​​The use of claim 44, in, Treatment of the GSD1a comprises delivering the base editor or polynucleotide encoding the base editor and the one or more guide RNAs to the cells of the individual.

47. The use of claim 46, in, The cells are hepatocytes or hepatocyte precursors.

48. The use of claim 44, in, The polynucleotide programmable DNA binding domain is Streptococcus pyogenes Cas9 (SpCas9).

49. The use of claim 48, in, The polynucleotide programmable DNA binding domain is SpCas9 which is specific for non-G prototype spacer adjacent motifs (PAMs).

50. The use of claim 49, in, The SpCas9 is specific for the prototype spacer adjacent motif (PAM) composed of the nucleic acid sequence 5'-NGA-3', wherein N is A, T, C or G.

51. The use of claim 49, in, The SpCas9 is specific for the prototype spacer adjacent motif (PAM) composed of the nucleic acid sequences 5'-AGA-3' or 5'-GGA-3'.

52. The use of claim 49, in, The SpCas9 is specific for the prototype spacer adjacent motif (PAM) composed of the nucleic acid sequence NGA, where N is A, T, C or G.

53. The use of claim 44, in, The polynucleotide programmable DNA binding domain is Staphylococcus aureus Cas9 (SaCas9).

54. The use of claim 53, in, SaCas9 is specific for the prototype spacer adjacent motif (PAM) composed of the nucleic acid sequence 5'-NNGRRT-3', wherein N is A, T, C or G and R is A or G.

55. Use according to claim 53 or 54, in, SaCas9 is specific for the prototype spacer adjacent motif (PAM) composed of the nucleic acid sequence 5'-GAGAAT-3'.

56. The use of claim 53, in, SaCas9 is specific for the prototype spacer adjacent motif (PAM) composed of the nucleic acid sequence NNGRRT, where N is A, T, C or G, and R is A or G.

57. The use of claim 44, in, The polynucleotide programmable DNA binding domain is nuclease-inactive.

58. The use of claim 44, in, The polynucleotide programmable DNA binding domain is a nick enzyme.

59. The use of claim 58, in, The nickase comprises the following amino acid sequence: DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKS DGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKDLIIKL PKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD.

60. The use of claim 44, in, The adenosine deaminase domain is capable of deaminating adenosine in deoxyribonucleic acid (DNA).

61. The use of claim 44, in, The base editor comprises a single adenosine deaminase domain.

62. The use of claim 44, in, The base editor further comprises a second adenosine deaminase domain, wherein the second adenosine deaminase domain is a wild-type adenosine deaminase domain.

63. A method of producing hepatocytes in vitro, the method comprising: (a) Introducing induced pluripotent stem cells or hepatocyte progenitor cells containing single nucleotide polymorphisms (SNPs) associated with type 1a glycogen storage disease (GSD1a), and a base editor or a polynucleotide encoding the base editor, in, The base editor comprises a polynucleotide programmable nucleotide binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain is composed of the following amino acid sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO:3) or a fragment thereof lacking only the N-terminal methionine, the adenosine deaminase domain has one or more changes, and one or more guide RNAs that guide the base editor to implement the A·T to G·C alteration of the SNP associated with GSD1a, wherein: i) The one or more changes are composed of: Changes of V82S, Y147T, Y147R, Q154R and Q154S selected from the group consisting of reference SEQ ID NO:3, or a combination of changes of reference SEQ ID NO:3: V82S and Y147R, V82S and Q154R, V82S and Q154S, V82S and Y123H, and V82S, Y123H and Y147T; the SNP associated with GSD1a results in the expression of a glucose-6-phosphatase (G6PC) polypeptide with a Q347X mutation, wherein X represents a stop codon; and one or more of the guide RNAs include spacers composed of the nucleic acid sequence GACCUAGGCGAGGCAGUAGG; or ii) The one or more changes are composed of: Changes of V82S, Y147T, Y147R and Q154S selected from the group consisting of reference SEQ ID NO:3, or a combination of changes of reference SEQ ID NO:3: Y147T and Q154S, Y147R and Q154S, V82G, Y147T and Q154S, and R20A, K21A, Y147T, and Q154S; The SNP associated with GSD1a results in the expression of the G6PC polypeptide with R83C mutation; and one or more of the guide RNAs include spacers consisting of the nucleic acid sequences CAGUAUGGACACUGUCCAAA or AGUAUGGACACUGUCCAAA; and (b) differentiation of the induced pluripotent stem or hepatocyte progenitor cells into hepatocytes.

64. The method of claim 63, in, The hepatocyte progenitor cells were obtained from individuals suffering from GSD1a.

65. The method of claim 63, in, The hepatocytes are mammalian cells.

66. The method of claim 63, in, The induced pluripotent stem cells of step (a) express the G6PC polypeptide with the Q347X mutation, where X represents the stop codon.

67. The method of claim 63, in, The induced pluripotent stem cells of step (a) express the G6PC polypeptide with R83C mutation.

68. The method of claim 63, in, The adenosine deaminase domain is capable of deaminating adenosine in deoxyribonucleic acid (DNA).

69. The method of claim 63, in, The base editor comprises a single adenosine deaminase domain.

70. The method of claim 63, in, The base editor further comprises a second adenosine deaminase domain, wherein the second adenosine deaminase domain is a wild-type adenosine deaminase domain.

71. A pharmaceutical composition for the treatment of type 1a glycogen storage disease (GSD1a), comprising: Effective amount of base editor, in, The base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain is composed of the following amino acid sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD(SEQ ID NO:3) or a fragment thereof lacking only the N-terminal methionine, the adenosine deaminase domain has one or more changes, and one or more guide RNAs that guide the base editor to implement the A·T to G·C alteration of SNPs associated with GSD1a, wherein: a) The one or more changes are composed of: Changes of V82S, Y147T, Y147R, Q154R and Q154S selected from the group consisting of reference SEQ ID NO:3, or a combination of changes of reference SEQ ID NO:3: V82S and Y147R, V82S and Q154R, V82S and Q154S, V82S and Y123H, and V82S, Y123H and Y147T; the SNP associated with GSD1a results in the expression of a glucose-6-phosphatase (G6PC) polypeptide with a Q347X mutation, wherein X represents a stop codon; and one or more of the guide RNAs include spacers composed of the nucleic acid sequence GACCUAGGCGAGGCAGUAGG; or b) The one or more changes are composed of: Changes of V82S, Y147T, Y147R and Q154S selected from the group consisting of reference SEQ ID NO:3, or a combination of changes of reference SEQ ID NO:3: Y147T and Q154S, Y147R and Q154S, V82G, Y147T and Q154S, and R20A, K21A, Y147T, and Q154S; The SNP associated with GSD1a results in the expression of the G6PC polypeptide with R83C mutation; and one or more of the guide RNAs include spacers composed of the nucleic acid sequences CAGUAUGGACACUGUCCAAA or AGUAUGGACACUGUCCAAA.

72. A pharmaceutical composition for the treatment of type 1a glycogen storage disease (GSD1a), comprising an effective amount of the cell of any one of claims 19 to 43.

73. The pharmaceutical composition of claim 71 or 72, further comprising a pharmaceutically acceptable excipient.

74. A kit for the treatment of type 1a glycogen storage disease (GSD1a), the kit comprising a base editor, in, The base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain is composed of the following amino acid sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD(SEQ ID NO:3) or a fragment thereof lacking only the N-terminal methionine, the adenosine deaminase domain has one or more changes, and one or more guide RNAs that guide the base editor to implement A·T to G·C alteration of SNPs associated with GSD1a, wherein: a) The one or more changes are composed of: Changes of V82S, Y147T, Y147R, Q154R and Q154S selected from the group consisting of reference SEQ ID NO:3, or a combination of changes of reference SEQ ID NO:3: V82S and Y147R, V82S and Q154R, V82S and Q154S, V82S and Y123H, and V82S, Y123H and Y147T; wherein the SNP associated with GSD1a results in the expression of a glucose-6-phosphatase (G6PC) polypeptide with a Q347X mutation, wherein X represents a stop codon; and one or more of the guide RNAs include spacers composed of the nucleic acid sequence GACCUAGGCGAGGCAGUAGG; or b) The one or more changes are composed of: Changes of V82S, Y147T, Y147R and Q154S selected from the group consisting of reference SEQ ID NO:3, or a combination of changes of reference SEQ ID NO:3: Y147T and Q154S, Y147R and Q154S, V82G, Y147T and Q154S, and R20A, K21A, Y147T, and Q154S; The SNP associated with GSD1a results in the expression of the G6PC polypeptide with R83C mutation; and one or more of the guide RNAs include spacers composed of the nucleic acid sequences CAGUAUGGACACUGUCCAAA or AGUAUGGACACUGUCCAAA.

75. A kit for the treatment of type 1a glycogen storage disease (GSD1a), the kit comprising the cells according to any one of claims 19 to 43.

76. A base editor system comprising a base editor in a complex with one or more guide RNAs, in, The base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain is composed of the following amino acid sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD(SEQ ID NO:3) or a fragment thereof lacking only the N-terminal methionine, the adenosine deaminase domain has one or more alterations, wherein one or more of the guide RNA guides the base editor to achieve A·T to G·C alterations of the SNP associated with GSD1a, and wherein: a) The one or more changes consist of a combination of changes or changes selected from the following reference SEQ ID NO: 3: V82S and Y147R, V82S and Q154R, V82S and Q154S, V82S and Y123H, and V82S, Y123H and Y147T; the SNP associated with GSD1a results in the expression of a glucose-6-phosphatase (G6PC) polypeptide with a Q347X mutation, wherein X represents a stop codon, and one or more of the guide RNAs include spacers composed of the nucleic acid sequence GACCUAGGCGAGGCAGUAGG; or b) The one or more changes are composed of: Changes of V82S, Y147T, Y147R and Q154S selected from the group consisting of reference SEQ ID NO:3, or a combination of changes of reference SEQ ID NO:3: Y147T and Q154S, Y147R and Q154S, V82G, Y147T and Q154S, and R20A, K21A, Y147T, and Q154S; The SNP associated with GSD1a results in the expression of the G6PC polypeptide with R83C mutation; and one or more of the guide RNAs include spacers composed of the nucleic acid sequences CAGUAUGGACACUGUCCAAA or AGUAUGGACACUGUCCAAA.

77. The base editor system of claim 76, in, The base editor further comprises a second adenosine deaminase domain, wherein the second adenosine deaminase domain is a wild-type adenosine deaminase domain.

78. The base editor system of claim 76, in, The polynucleotide programmable DNA binding domains are Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), or Streptococcus pyogenes Cas9 (SpCas9).

79. The base editor system of claim 78, in, The prototype spacer adjacent motif of the polynucleotide programmable DNA binding domain is SpCas9 which is specific for the non-G prototype spacer adjacent motif (PAM).

80. The base editor system of claim 78, in, The polynucleotide programmable DNA binding domain is nuclease inactive Cas9.

81. The base editor system of claim 78, in, The polynucleotide programmable DNA binding domain is a Cas9 nickase.

82. A base editor system comprising one or more guide RNAs and fusion proteins comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, the polynucleotide programmable DNA binding domain consisting of the following sequences: EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKDLIIKLPKYSLFE LENGRKRMLASAKFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSGGSG GSGGMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAHMIKFRGHFLIEGDLN PDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEG MRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKL ITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEGADKRTADGSEFESPKKKRKV, and the adenosine deaminase domain is composed of the following amino acid sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD(SEQ ID NO:3) or a fragment thereof lacking only the N-terminal methionine, the adenosine deaminase domain has one or more alterations, wherein one or more of the guide RNA guides the base editor to achieve A·T to G·C alterations of the SNP associated with GSD1a, and in: a) The one or more changes are composed of: Changes of V82S, Y147T, Y147R, Q154R and Q154S selected from the group consisting of reference SEQ ID NO:3, or a combination of changes of reference SEQ ID NO:3: V82S and Y147R, V82S and Q154R, V82S and Q154S, V82S and Y123H, and V82S, Y123H and Y147T; the SNP associated with GSD1a results in the expression of a glucose-6-phosphatase (G6PC) polypeptide with a Q347X mutation, wherein X represents a stop codon; and one or more of the guide RNAs include spacers composed of the nucleic acid sequence GACCUAGGCGAGGCAGUAGG; or b) The one or more changes are composed of: Changes of V82S, Y147T, Y147R and Q154S selected from the group consisting of reference SEQ ID NO:3, or a combination of changes of reference SEQ ID NO:3: Y147T and Q154S, Y147R and Q154S, V82G, Y147T and Q154S, and R20A, K21A, Y147T, and Q154S; The SNP associated with GSD1a results in the expression of the G6PC polypeptide with R83C mutation; and one or more of the guide RNAs include spacers composed of the nucleic acid sequences CAGUAUGGACACUGUCCAAA or AGUAUGGACACUGUCCAAA.

83. A cell comprising the base editor system according to any of claims 76 to 82.

84. The cell of claim 83, in, The cells are mammalian cells.

85. The cell of claim 83, in, The cells are in vivo or in vitro.

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