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 increased efficiency, and methods of using base editors comprising adenosine deaminase variants to alter mutations associated with glycogen storage disease type 1a (GSD1a).

Description

[Technical field]

[0001] Cross-references to related applications This application is filed on February 13, 2019, US Provisional Application No. 62 / 805,271, filed on May 23, 2019, US Provisional Application No. 62 / 852,228, filed on May 23, 2019, and filed on May 23, 2019. US Provisional Application No. 62 / 852,224 filed on July 19, 2019, US Provisional Application No. 62 / 876,354 filed on October 9, 2019, and US Provisional Application No. 62 / 912,99 filed on October 9, 2019 No. 2, US Provisional Application No. 62 / 931,722 filed November 6, 2019, US Provisional Application No. 62 / 941,569 filed November 27, 2019, and January 27, 2020 An international PCT application claiming priority and their benefits to the US Provisional Application No. 62 / 966,526, all of which are incorporated by reference in its entirety.

[0002] Incorporation by reference All publications, patents, and patent applications mentioned herein are specifically and individually indicated that each individual publication, patent, or patent application is incorporated by reference. to the same extent, it is incorporated by reference herein. Unless otherwise stated, publications, patents, and patent applications referred to herein are hereby incorporated by reference in their entirety. [Background technology]

[0003] Most known genetic diseases require correction of point mutations at target loci rather than stochastic destruction of the gene, to study the underlying cause of the disease or address this cause. . Current genome editing techniques utilizing clustered regularly interspaced short palindromic repeat (CRISPR) systems introduce double-stranded DNA disruption at target loci as the first step for gene correction. In response to double-stranded DNA disruption, intracellular DNA repair processes, in most cases non-homologous terminal bonds result in random insertions or deletions (indels) at the DNA cleavage site. Although most genetic diseases are due to point mutations, current approaches for point mutation correction are inefficient, typically due to cellular responses to dsDNA disruption, resulting in a large number at target loci. Induces random insertions and deletions (indels). Therefore, there is a need for improved genome editing that is much less undesirable products such as more efficient and stochastic insertions or deletions (indels), or translocations.

[0004] Glycogen Disease Type 1 (also known as GSD1 or Von Gierke's disease) is a hereditary disorder that causes deficiency of glycogen degradation and gluconeogenesis, and the accumulation of glycogen and lipids in tissues, leading to life-threatening hypoglycemia and lactic acid Acidosis is caused and potential CNS damage and prolonged liver and kidney complications (eg, steatosis, hepatic adenomas, and hepatocellular carcinoma).

[0005] There are two types of GSD1, type 1a (GSD1a) and type 1b (GSD1b), which are caused by different genetic mutations. GSD1a is caused by a mutation in the glucose-6-phosphatase (G6PC) gene and occurs in approximately 80% of patients with GSD1. In the US, approximately one in 100,000 newborns has GSD1a, with approximately 22% of patients having the inferior mutation Q347* and 37% of patients having the inferior mutation R83C.

[0006] No GSD1a approved drug therapy exists. Although liver transplantation is curative, no approved treatments exist, and current treatment regimens include the near-continuous consumption of cornstarch. If chronically not treated, the patient may develop severe lactic acidosis, progressing to renal failure, and death in infancy or childhood. GSD1a is a critical area of ​​medical needs that has not yet been addressed. Therefore, there is a need for novel compositions and methods for treating patients with GSD1a.

[0007] Incorporation by reference All publications, patents, and patent applications mentioned herein are specifically and individually indicated that each individual publication, patent, or patent application is incorporated by reference. to the same extent, it is incorporated by reference herein. Unless otherwise stated, publications, patents, and patent applications referred to herein are hereby incorporated by reference in their entirety. Summary of the invention

[0008] The present invention features compositions and methods for accurate correction of pathogenic amino acids using a programmable nucleobase editor. In particular, the compositions and methods of the present invention are useful in the treatment of glycogen disease type 1a (GSD1a). Therefore, the present invention provides 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). 化合物得合物。 化合物合物。 化合物合物合物。 化合物合物合物。 化合物合物合物。 化合物合物合�

[0009] In one embodiment, the present invention is a method of editing a G6PC polynucleotide comprising a single nucleotide polymorphism (SNP) associated with glycogen disease type 1a (GSD1a), wherein the G6PC polynucleotide is comprised of one or more of the G6PC polynucleotides. 合方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法分方法� One or more of the methods provide a method in which one or more target (target) base editors to result in the A·T to G·C modification of SNPs associated with GSD1a. In another embodiment, the present invention targets the base editor with an adenosine deaminase base editor 8 (ABE8) containing a polynucleotide programmable DNA-binding domain and an adenosine deaminase domain, or a polynucleotide encoding the base editor. A cell is provided that comprises one or more guide polynucleotides leading to alteration of SNPs from A·T to G·C associated with GSD1a. In another embodiment, the invention is a method of treating GSD1a in a subject, in which the subject comprises a polynucleotide programmable DNA-binding domain and an adenosine deaminase domain, or the base administer the polynucleotide encoding the editor and one or more guide polynucleotides that lead to the alteration of the SNPs from A·T to G·C associated with GSD1a. Provides a method that includes: In another aspect, the present invention is a method of generating hepatocytes or precursors thereof, wherein a) a polynucleotide programmable nucleotide into an induced pluripotent stem cell or hepatocyte precursor containing SNPs associated with GSD1a. A·T to G of the SNPs associated with GSD1a, including the binding domain and the adenosine deaminase domain, and polynucleotides encoding adenosine deaminase base editor 8 (ABE8) or adenosine deaminase base editor 8 (ABE8) targeting the base editor. · Provided are methods comprising introducing one or more guide polynucleotides leading to modification to C, and b) differentiating an induced pluripotent stem cell or hepatocyte precursor into hepatocytes.

[0010] In one embodiment, the present invention is a method of editing a glucose-6-phosphatase (G6PC) polynucleotide comprising a single nucleotide polymorphism (SNP) associated with glycogen disease type 1a (GSD1a), the G6PC polynucleotide 合物分合物合物合物合物合物合物物合物物物物物物物物物物物物物物物物物物物物物物物物物物物物物物物物物物。 8 (ABE8) is complexed with one or more guide polynucleotides, and the adenosine deaminase base editor 8 (ABE8) is inserted into the Cas9 or Cas12 polypeptide. Provided is a method in which one or more of the guide polynucleotides target the base editor to result in the A·T to G·C modification of the SNP associated with GSD1a do. In another embodiment, the invention is a method of treating glycogenic disease type 1a (GSD1a) in a subject, the adenosine deaminase base editor comprising an adenosine deaminase variant inserted into a Cas9 or Cas12 polypeptide. 8 (ABE8), or a polynucleotide encoding the base editor and one or targeting the adenosine deaminase base editor 8 (ABE8) to effect the A·T to G·C modification of the SNP associated with GSD1a A method is provided comprising administering multiple guide polynucleotides, thereby treating GSD1a in a subject. In yet another embodiment, the invention is a method of treating glycogenic disease type 1a (GSD1a) in a subject, the subject comprising an adenosine deaminase variant inserted into a Cas9 or Cas12 polypeptide, or A polynucleotide encoding the fusion protein and one or more guide polynucleotides that target the fusion protein to result in the A·T to G·C modification of a single nucleotide polymorphism (SNP) associated with GSD1a. 化合物合物合物合物。 The present invention provides a method comprising administering, thereby treating GSD1a in a subject.

[0011] In one embodiment, the present invention includes the treatment of glycogen disease type 1a (GSD1a), comprising an effective amount of adenosine deaminase base editor 8 (ABE8), which comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase variant domain. A pharmaceutical composition is provided for In some embodiments, the pharmaceutical composition is capable of targeting adenosine deaminase base editor 8 (ABE8) to result in an A·T to G·C modification of the SNP associated with GSD1a. It includes multiple guide polynucleotides. In another aspect, the invention provides a pharmaceutical composition for the treatment of glycogen disease type 1a (GSD1a), comprising an effective amount of any of the cells provided herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0012] In another embodiment, the invention is a kit for the treatment of glycogen disease type 1a (GSD1a), and includes an adenosine deaminase base editor 8 (ABE8) containing a polynucleotide programmable DNA binding domain and an adenosine deaminase domain. Provided is a kit containing one or more guide polynucleotides that can target adenosine deaminase base editor 8 (ABE8) to result in the A·T to G·C modification of SNPs associated with GSD1a. . In yet another aspect, the present invention provides a kit for the treatment of glycogenic disease type 1a (GSD1a) and comprises any of the cells provided herein.

[0013] In some embodiments, contacting occurs in a cell, eukaryotic cell, mammalian cell, or human cell. In some embodiments, the cells are in vivo. In some embodiments, the cells are ex vivo. In some embodiments, the cell is a hepatocyte, a hepatocyte precursor, or an iPSc-derived hepatocyte. In some embodiments, the cell expresses a G6PC polypeptide. In some embodiments, the cell or hepatocyte precursor is from a subject with GSD1a. In some embodiments, the subject is a mammal or a human. In some embodiments, the hepatocyte or hepatocyte precursor is a mammalian or human cell. In some embodiments, the cell of interest is delivered to the polynucleotide encoding adenosine deaminase base editor 8 (ABE8) or the adenosine deaminase base editor 8 (ABE8) and the one or more guide polynucleotides. It will be done.

[0014] In various embodiments of the above aspects 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, alteration from A·T to G·C in SNPs associated with glycogen disease type 1a (GSD1a) changes glutamine (Q) to non-glutamine (X) amino acids. In one embodiment, alteration from A·T to G·C in SNPs associated with glycogen disease type 1a (GSD1a) changes arginine (R) to non-arginine (X) in the G6PC polypeptide. In one embodiment, the SNP associated with GSD1a results in expression of a 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, base editor correction replaces the non-glutamine amino acid (X) at position 347 with glutamine. In another embodiment, base editor correction replaces the non-arginine amino acid (X) at position 83 with arginine. In one embodiment, the A·T to G·C modification in the SNP associated with GSD1a results in the expression of a G6PC polypeptide that terminates early at amino acid position 347 or encodes cysteine ​​at position 83. In some embodiments, the modification in the SNP is one or more of Q347X and / or R83C.

[0015] In various embodiments of the foregoing aspects or any other aspects of the invention described herein, the adenosine deaminase variant is a flexible loop, an alpha helix region, or an unstructured portion of a Cas9 or Cas12 polypeptide. or inserted within the solvent-contactable portion. In some embodiments, the adenosine deaminase variant is flanked by N-terminal and C-terminal fragments of the Cas9 or Cas12 polypeptide. In some embodiments, the fusion protein or adenosine deaminase base editor 8 (ABE8) is a structure NH <000000> -[N-terminal fragment of Cas9 or Cas12 polypeptide]-[adenosine deaminase variant]-[C-terminal fragment of Cas9 or Cas12 polypeptide]-COOH, where each statement of "]-[" is set to any linker be. In one embodiment, the C-terminus of the N-terminus fragment or the N-terminus of the C-terminus fragment comprises part of a flexible loop of the Cas9 or Cas12 polypeptide. In one embodiment, the flexible loop comprises an amino acid adjacent to the target nucleobase. In some embodiments, one or more guide polynucleotides induce a fusion protein or adenosine deaminase base editor 8 (ABE8) to lead to 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 deamination of the target nucleobase ameliorates the symptoms of GSD1a. In one embodiment, the target nucleobase is 1-20 nucleobases separate from the PAM sequence in the target polynucleotide sequence. In one embodiment, the target nucleobase is upstream of the PAM sequence from 2 to 12 nucleobases.

[0016] In one embodiment, the N-terminal or C-terminal fragment of the Cas 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, and 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 contains the RuvC domain. In one embodiment, the Cas9 or Cas12 polypeptide comprises a partial or complete deletion in one or more structural domains, and the deaminase is a partial or complete deletion of the Cas9 or Cas12 polypeptide. It is inserted in position. In one embodiment, the deletion is within the RuvC domain, the deletion is within the HNH domain, or the deletion is within the RuvC domain and the C-terminal domain, and the L-I domain and the HNH domain. or cross-link the RuvC domain and the L-I domain.

[0017] 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 a Cas9 polypeptide. In some embodiments, the Cas9 polypeptide is Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), or a variant thereof. In some embodiments, the Cas9 polypeptide includes the following amino acid sequence (see Cas9 sequence): TIFF0007631209000001.tif171164 (single underlined: HNH domain; double underlined: RuvC domain; (Cac9 reference sequence), or corresponding region thereof.

[0018] In some embodiments, the Cas9 polypeptide comprises a deletion of amino acids 1017-1069 or its corresponding amino acids in numbering in the Cas9 polypeptide reference sequence, or the Cas9 polypeptide is numbering in the Cas9 polypeptide reference sequence In the Cas9 polypeptide comprises deletions of amino acids 792-872 or its corresponding amino acids, or the Cas9 polypeptide comprises deletions of amino acids 792-906 or its corresponding amino acids numbered 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, this flexible loop is positioned at positions 530-537, 569-579, 686-691, 768-793, 943-947, 1002-1040, 1052-1077, 1232 Includes regions selected from the group consisting of ~1248, and 1298-1300, or amino acid residues at their corresponding amino acid positions. In some embodiments, the deaminase is numbered in the Cas9 reference sequence, amino acid positions 768-769, 791-792, 792-793, 1015-1016, 1022-1023, 1026-1027, 1029-1030, 1040-1041 , 1052-1053, 1054-1055, 1067-1068, 1068-1069, 1247-1248, or 1248-1249, or inserted between their corresponding amino acid positions. In some embodiments, the deaminase is numbered in the Cas9 reference sequence, or amino acid positions 768-769, 792-793, 1022-1023, 1026-1027, 1040-1041, 1068-1069, or 1247-1248, or is inserted between the corresponding amino acid positions. In some embodiments, the deaminase is numbered in the Cas9 reference sequence, or amino acid positions 1016-1017, 1023-1024, 1029-1030, 1040-1041, 1069-1070, or 1247-1248, or the corresponding amino acids thereof. It is inserted between positions. In some embodiments, the adenosine deaminase variant is inserted into the Cas9 polypeptide at the locus identified in Table 10A. In some embodiments, the N-terminal fragment includes amino acid residues 1-529, 538-568, 580-685, 692-942, 948-1001, 1026-1051, 1078-1231, and / or of the Cas9 reference sequence 1248-1297, or their corresponding residues. In some embodiments, the C-terminal fragment includes amino acid residues 1301-1368, 1248-1297, 1078-1231, 1026-1051, 948-1001, 692-942, 580-685, and / or 538-568, or their corresponding residues.

[0019] In some embodiments, the Cas9 polypeptide is nickase, or the Cas9 polypeptide is nuclease-inactive. In some embodiments, the Cas9 polypeptide is a modified SpCas9, having specificity for or 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 for the modified PAM 5'-NGC-3' Has specificity.

[0020] In various embodiments, the polynucleotide programmable DNA binding domain is an altered Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof. In various embodiments of the above aspects or any other aspects of the invention depicted herein, the polynucleotide programmable DNA binding domain is configured to provide a modified protospacer adjacent motif (PAM) specificity. 合物合物用的合物用的合物用的合物用。 Includes modified SpCas9 having or having specificity for non-G PAM. In one embodiment, the modified SpCas9 has specificity for the nucleic acid sequence 5'-NGA-3'. In one embodiment, the modified SpCas9 has specificity for the nucleic acid sequence 5'-AGA-3' or 5'-GGA-3'. In one embodiment, the modified SpCas9 has specificity for the NGA PAM variant.

[0021] In various embodiments, the polynucleotide programmable DNA binding domain is Staphylococcus aureus Cas9 (SaCas9) or a variant thereof. In one embodiment, the SaCas9 has specificity for the nucleic acid sequence 5'-NNGRRT-3'. In one embodiment, the SaCas9 has specificity for the nucleic acid sequence 5'-GAGAAT-3'. In one embodiment, the SaCas9 has specificity for the NNGRRT PAM variant.

[0022] In various embodiments, the polynucleotide programmable DNA binding domain is a Cas12 polypeptide. In one embodiment, the adenosine deaminase variant is inserted into a 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, of BhCas12b 604-605, or 344-345, or the corresponding amino acid residues of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, or Cas12i;b) 147 and 148, 248 and 249, 299 and 300, 991 and 992, or 1031 and 1032, or the corresponding amino acid residues of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, or Cas12i; or c) of AaCas12b 157 and 158, 258 and 259, 310 and 311, 1008 and 1009, or 1044 and 1045, or the corresponding amino acid residues of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, or Cas12i. 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.

[0023] 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 an amino acid substitution D10A or its corresponding amino acid substitution. In some embodiments, the adenosine deaminase domain is capable of deamination of adenosine in deoxyribonucleic acid (DNA). In some embodiments, the adenosine deaminase domain is a monomer that includes an adenosine deaminase variant. In some embodiments, the adenosine deaminase domain is a heterodimer comprising a wild-type adenosine deaminase domain and an adenosine deaminase variant.

[0024] In some embodiments, the adenosine deaminase variant includes the amino acid sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD , the amino acid sequence comprises at least one modification. In some embodiments, the adenosine deaminase variant comprises modifications at amino acid positions 82 and / or 166 to the above sequence. In some embodiments, this at least one modification includes V82S, Y147T, Y147R, Q154S, Y123H, and / or Q154R for the above sequence. In some embodiments, this at least one modification is made up of Y147T+Q154R;Y147T+Q154S;Y147R+Q154S;V82S+Q154S;V82S+Y147R;V82S+Q154R;V82S+Y123H;I76Y+V82S;V82S+Y123H+ Y147T ;V82S+Y123H+Y147R;V82S+Y123H+Q154R;Y147R+Q154R+Y123H;Y147R+Q154R+I76Y;Y147R+Q154R+T166R;Y123H+Y147R+Q154R+I76Y;V82S+Y123H+Y147R+Q154R; and combinations of modifications selected from the group consisting of I76Y + V82S + Y123H + Y147R + Q154R. In some embodiments, this at least one modification is Y147T + Q154S for the above sequence.

[0025] In some embodiments, the adenosine deaminase variant comprises a C-terminal deletion beginning with 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 that comprises the TadA*8 adenosine deaminase variant domain. In some embodiments, the adenosine deaminase variant is an adenosine deaminase heterodimer containing 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. In some embodiments, the guide polynucleotide is a) GACCUAGGCGAGGCAGUAGG; b) CCAGUAUGGACACUGUCCAAA; c) CAGUAUGGACACUGUCCAAA; and d) AGUAUGGACACUGUCCAAAG. 合物分合物合物用。 Includes a nucleic acid sequence selected from the group.

[0026] In some embodiments, the one or more guide RNAs include CRISPR RNA (crRNA) and a small transcoding RNA (tracrRNA), the crRNA being complementary to the G6PC nucleic acid sequence containing the SNP associated with GSD1a. Contains a nucleic acid sequence. In some embodiments, adenosine deaminase base editor 8 (ABE8) forms a complex with a single guide RNA (sgRNA) containing a nucleic acid sequence complementary to a G6PC nucleic acid sequence containing an SNP associated with GSD1a. 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 variants include 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, adenosine deaminase base editor 8 (ABE8) is made up 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, ABE8.8-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, ABE8.18-m , ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8.23 -m, ABE8.24-m, ABE8.1-d, ABE8.2-d, ABE8.3-d, ABE8.4-d, ABE8.5-d, ABE8.6-d, ABE8.7-d , ABE8.8-d, ABE8.9-d, ABE8.10-d, ABE8.11-d, ABE8.12-d, ABE8. 13-d, ABE8.14-d, ABE8.15-d, ABE8.16-d, ABE8.17-d, ABE8.18-d, ABE8.19-d, ABE8.20-d, ABE8.21- It is selected from the group consisting of d, ABE8.22-d, ABE8.23-d, or ABE8.24-d.

[0027] In some embodiments, adenosine deaminase base editor 8 (ABE8) includes the following sequences or fragments thereof having adenosine deaminase activity: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD Includes or becomes essential.

[0028] In some embodiments, the gRNA includes the following sequence: GUUUAGAGCUAGAAAUAGCAAGUUAAAGGCUAGUCCGUUAUCAACUGAAAAGUGGCACCGAGUCGGUGCUUUU Includes scaffolds having

[0029] In some embodiments, the gRNA is the following sequence: GUUUAGUACUCUGUAAUGAAAAACAGAAUCUAAAAAGAGCAAAAAUGCCGUGUUAUCUCGUCAACUUGGCGAGAUUUU Includes scaffolds having

[0030] In certain embodiments, provided herein is a base editor that includes an adenosine deaminase base editor 8 (ABE8) complexed with one or more guide polynucleotides, the adenosine deaminase base editor 8 (ABE8) comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, one or more of the guide polynucleotides target the base editor to target the base editor to A-600-11 SNPs associated with GSD1a. It is a base editor that brings about changes from T to G·C. In some embodiments, the adenosine deaminase variant comprises a V82S and / or a T166R modification. In some embodiments, the adenosine deaminase variant further comprises one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, and Q154R. In some embodiments, the base editor domain comprises an adenosine deaminase heterodimer comprising a wild-type adenosine deaminase domain and an adenosine deaminase variant. In some embodiments, the adenosine deaminase variant is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, It is a truncated TadA8, which has deleted 17, 18, 19, or 20 N-terminal amino acid residues. In some embodiments, the adenosine deaminase variant is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, It is a truncated TadA8, which has deleted 17, 18, 19, or 20 C-terminal amino acid residues. 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 either modified protospacer flanking motif (PAM) specificity or specificity for non-G PAM. In some embodiments, the polynucleotide programmable DNA binding domain is nuclease-inactive Cas9. In some embodiments, the polynucleotide programmable DNA binding domain is Cas9 nickase.

[0031] In one embodiment, provided herein are one or more guide RNAs, as well as the following sequences: EIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSL FELENGRKRMLASAKFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSGG SGGSGGSGGMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGH FLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEN PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLP EKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLI HDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSE EVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEGADKRTADGSEFESPKKKRKV* (wherein, the bold sequence indicates a sequence derived from Cas9, the italic sequence indicates a linker sequence, and the underlined sequence indicates a bipartite nuclear localization sequence). A polynucleotide programmable DNA binding domain containing; MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD 合方法分合物分合物合物用82 and / or 166合物用方法合物用方法合物用方法合物用方法合物用方法合物用方法合物用方法合物用方法合物用方法合物用方法合物用方法合物用方法合物用方法合物用方法合物用方法合物用方法合物用方法合物用方法合物用方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方方 It is a system that targets the base editor and results in the A·T to G·C modification of SNPs associated with GSD1a.

[0032] In some embodiments, a cell is provided that comprises any one of the base editor systems described above. In some embodiments, the cell is a human cell or a mammalian cell. In some embodiments, the cells are ex vivo, in vivo, or in vitro.

[0033] The description and examples of this specification illustrate in detail the embodiments of the present disclosure. It is to be understood that the present disclosure is not limited to the particular embodiments described herein, and may therefore vary. Those skilled in the art will recognize that there are numerous variations and modifications of this disclosure that fall within its scope.

[0034] The practice of some of the embodiments disclosed herein is within the skill of those skilled in the art, unless otherwise indicated, of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, unless otherwise specified. genomics, and conventional techniques of recombinant DNA are used. For example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al.eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A. See Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0035] Although various features of the present disclosure may be described in the context of a single embodiment, these features may be provided separately or in any suitable combination. Conversely, while this disclosure may be described herein in the context of separate embodiments for clarity, the disclosure may also be implemented in a single embodiment. Section headings used herein are for collection purposes only and should not be construed as limiting the subject matter described.

[0036] Features of the present disclosure are specifically described in the appended claims. A better understanding of the features and advantages of the present disclosure can be obtained by referring to the following detailed description showing exemplary embodiments in which the principles of this disclosure are utilized, and taking into account the accompanying drawings described below. It must be.

[0037] Definition The following definitions supplement the definitions of the art and are intended to cover this application and are not attributed to related or unrelated matters, such as patents or applications relating to common ownership. Although any methods and materials similar or equivalent to those described herein can be used in carrying out the tests of this disclosure, the preferred materials and methods are described herein. Accordingly, the terms used herein are for the purposes of describing particular embodiments only and are not intended to be limiting.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of many terms used in the present invention: 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); ).

[0039] In this application, the use of the singular form includes the plural form unless otherwise noted. As used herein, the singular forms "a", "an", and "the" include multiple referents unless the context explicitly indicates that they are not. It won't. In this application, the use of "or" is understood to mean "and / or" unless otherwise stated, and to be comprehensive. Furthermore, the use of the term "including" and other forms (eg, "includes", "includes", and "included") is non-limiting.

[0040] As used in this specification and claims, the terms "comprising" (and any form thereof, such as "comprise" and "comprises"), "having" ("having" ("having" ("having" ("having" ("having" ("having" ("having" ("having" ("having" ("having" ("having" ("having" ("having" ("having" ("having" ("having" ("having" ("having" ("having" ("having" ("having") ("having" ("having" ("having" ("having" ("having" ("having") ("having" ("having" ("having" ("having" ("having") ("having" ("having" ("having" ("having" ("having") ("having" ("having" ("having" ("having") ("having have) and "has"), "including" (any form thereof, such as "include" and "includes") or "containing." (All forms thereof, such as "contains" and "contains") are comprehensive or open to do not exclude additional, undescribed elements or method steps. Any embodiment discussed herein can be practiced with respect to any method or composition of the present disclosure, and vice versa. Additionally, the compositions of the present disclosure can be used to accomplish the methods of the present disclosure.

[0041] The term "about" or "approximately" means that, as determined by one skilled in the art, is within an acceptable error range for a particular value, which means that the value is It is measured or determined, ie, depends in part on the limitations of the measurement system. For example, "about" may mean that, according to practice in the art, it is within a standard deviation of within one or more than one. 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 regard to biological systems or processes, the term can mean a value within the same order, eg within five or even two times. If a particular value is listed in the application and claims, unless otherwise stated, the term "about" is estimated, meaning that it is within an acceptable error range for that particular value. Should be.

[0042] The ranges provided herein are understood to be shorthand for all values ​​within this range. For example, the range of 1-50 is 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 It is understood that any number, combination or subrange of numbers from the group consisting of 47, 48, 49, or 50 is included.

[0043] References to "some embodiments", "some embodiment", "one embodiment", or "other embodiment" in the specification include the particular features, structures, and descriptions described in connection with that embodiment. Or it means that the properties are included in at least some of the embodiments of the present disclosure, but not necessarily all of them.

[0044] "Adenosine deaminase" refers to a polypeptide or fragment thereof capable of catalyzing the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine, or deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (eg, genetically engineered adenosine deaminase, evolved adenosine deaminase) can be from any organism, such as a bacteria.

[0045] 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 (eg, a human, chimpanzee, gorilla, monkey, bovine, dog, rat, or mouse). In some embodiments, the deaminase or deaminase domain is not naturally occurring. 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. For example, the deaminase domains are described in International PCT Application No. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is The entirety is incorporated by reference herein. Similarly, Komor, A.C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N.M., et al., “Programmable base editing.” of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, A.C., et al., “Improved base surprise prevention and bacteriaophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017)), and Rees, H.A., et al., “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 by reference herein. .

[0046] The wild-type TadA(wt) adenosine deaminase is the following sequence (also known as the TadA reference sequence): MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD has

[0047] In some embodiments, the adenosine deaminase includes the following sequence: MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG LHDPTAHAEI MALRQGGLVM QNYRLIDATL YVTFEPCVMC AGAMIHSRIG RVVFGVRNAK TGAAGSLMDV LHYPGMNHRV EITEGILADE CAALLCYFFR MPRQVFNAQK KAQSSTD Includes modifications in (also known as TadA*7.10).

[0048] In some embodiments, TadA*7.10 includes at least one modification. In some embodiments, TadA*7.10 includes modifications at amino acids 82 and / or 166. In certain embodiments, variants of the sequence referenced above include one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. Modified Y123H is also referred to herein as H123H (modified H123Y in TadA*7.10, returned to Y123H (wt). In other embodiments, the variant of the TadA*7.10 sequence is 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 Includes combinations of modifications selected from the group:

[0049] In other embodiments, the invention is directed to residues 149, 150, 151, 152, 153, 154, 155, 156, or to the corresponding mutation in TadA*7.10, TadA reference sequence, or another TadA. Provided are adenosine deaminase variants containing deletions (eg, TadA*8) that include deletions at the C-terminus starting at 157. In other embodiments, the adenosine deaminase variant is a TadA (eg, TadA*8) monomer containing one or more of the following modifications: TadA*7.10, a TadA reference sequence, or the corresponding in another TadA Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R for mutations. In other embodiments, the adenosine deaminase variant is directed against a corresponding mutation 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 a monomer containing a combination of modifications selected from the group I76Y + V82S + Y123H + Y147R + Q154R.

[0050] In yet other embodiments, the adenosine deaminase variant is the following modifications Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R for the corresponding mutation in TadA*7.10, TadA reference sequence, or another TadA. It is a homodimer containing two adenosine deaminase domains (eg, TadA*8) each having one or more of the following. In other embodiments, the adenosine deaminase variant is directed against a corresponding mutation 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 homodimers containing two adenosine deaminase domains (eg, TadA*8) each having a combination of modifications selected from the group I76Y + V82S + Y123H + Y147R + Q154R.

[0051] In other embodiments, the adenosine deaminase variant includes the wild-type TadA adenosine deaminase domain and the following modifications for the corresponding mutation in TadA*7.10, TadA reference sequence, or another TadA is a heterodimer containing an adenosine deaminase variant domain (eg, TadA*8) containing one or more of T166R and / or Q154R. In other embodiments, the adenosine deaminase variant is directed against the wild-type TadA adenosine deaminase domain and the corresponding mutation in TadA*7.10, 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; 合方法合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合

[0052] In other embodiments, the adenosine deaminase variant includes the following modifications Y147T, Y147R, Q154S, Y123H, V82S, T166R for the TadA*7.10 domain and the corresponding mutation in TadA*7.10, TadA reference sequence, or another TadA and / or heterodimers containing an adenosine deaminase variant domain (eg, TadA*8) containing one or more of Q154R. In other embodiments, the adenosine deaminase variant includes the following modifications to the TadA*7.10 domain and the corresponding mutation in TadA*7.10, 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; Or a heterodimer containing an adenosine deaminase variant domain (eg, TadA*8) containing a combination of I76Y + V82S + Y123H + Y147R + Q154R.

[0053] In one embodiment, the adenosine deaminase is the following sequence or fragment thereof having adenosine deaminase activity: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD is TadA*8 which contains or becomes essentially from the

[0054] In some embodiments, TadA*8 is truncated. In one embodiment, the truncated TadA*8 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 for the full-length TadA*8. , 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues are deleted. In some embodiments, the truncated TadA*8 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, for the full-length TadA*8. 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues are deleted. In some embodiments, the adenosine deaminase variant is full-length TadA*8.

[0055] In certain embodiments, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from one of the following: Staphylococcus aureus (S. aureus) TadA: MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN Bacillus subtilis (B. subtilis) TadA: MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE Salmonella typhimurium (S. typhimurium) TadA: MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV Shewanella putrefaciens (S. putrefaciens) TadA: MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEAACSAQLSRFFKRRRDEKKALKLAQRAQGIE Haemophilus influencerenzae F3031 (H. influencerenzae) TadA: MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTΑΗAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK Caulobacter crescentus (C. crescentus) TadA: MRTDESEDQDHRMMRLAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI Geobacter sulfurreducens (G. sulfurreducens) TadA: MSSLKKTPIRDDAYWMGKAIREAAKAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP TadA*7.10 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0056] "Adenosine Deaminase Base Editor 8 (ABE8) Polypeptide" or "ABE8" is the reference sequence of: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD 合方法合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物。 Means a base editor as defined herein, including adenosine deaminase variants containing modifications at amino acid positions 82 and / or 166. In some embodiments, ABE8 includes further modifications to the reference sequence as described herein.

[0057] "Adenosine Deaminase Base Editor 8 (ABE8) Polynucleotide" refers to a polynucleotide encoding ABE8.

[0058] "Dailing" is referred to herein as providing a patient or subject with one or more compositions described herein. By way of example, but not limited to, administration of the composition, e.g., injections, include intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection or intramuscular (i.m. ) can be performed by injection. One or more such routes may be used. Parenteral administration can be performed, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration can be by oral route.

[0059] "Agent" means any small molecule compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

[0060] "Modification" is a change (eg, an increase or decrease) in the structure, expression level, or activity of a gene or polypeptide as detected by known methods of standard techniques such as those described herein. It means. As used herein, modifications are made to change in the sequence of a polynucleotide or polypeptide, or to change in expression level (e.g., 25% change, 40% change, 50% change, or higher change). Includes:

[0061] "Improve" means reducing, suppressing, attenuating, reducing, arresting, or stabilizing the onset or progression of the disease.

[0062] "Analog" means a molecule that is not identical, but has similar functional or structural features. For example, a polynucleotide or polypeptide analog retains the biological activity of the corresponding naturally occurring polynucleotide or polypeptide, and furthermore functions of the analogue compared to naturally occurring polynucleotide or polypeptide. It has specific modifications that enhance it. Such modifications may increase the affinity, efficiency, specificity, protease-resistant or nuclease-resistant, membrane permeability, and / or half-life of an analogue without altering ligand binding, for example. be. The analogs may contain non-natural nucleotides or amino acids.

[0063] "Base Editor (BE)" or "Nucleobase Editor (NBE)" refers to an agent that binds to a polynucleotide and has nucleobase modification activity. In various embodiments, the base editor includes a nucleobase-modified polypeptide (eg, a deaminase) and a nucleic acid-programmable nucleotide binding domain, together with a guide polynucleotide (eg, a guide RNA). In various embodiments, the agent comprises a protein domain having base editing activity, ie, a domain capable of modifying a base (eg, A, T, C, G, U) within a nucleic acid molecule (eg, DNA). It is a biomolecular complex. In some embodiments, the polynucleotide programmable DNA binding domain is fused or linked to a deaminase domain. In one embodiment, the agent is a fusion protein that comprises a domain having base editing activity. In another embodiment, the protein domain having base editing activity is linked to the guide RNA (eg, via an RNA-binding domain fused to the RNA-binding motif on the guide RNA and to the deaminase). In some embodiments, the domain having base editing activity is capable of deamination of a base within a nucleic acid molecule. In some embodiments, the base editor is capable of deamination of one or more bases within a DNA molecule. In some embodiments, the base editor is capable of deamination of adenosine (A). In some embodiments, the base editor is an adenosine base editor (ABE).

[0064] In some embodiments, the base editor clones the adenosine deaminase variant (eg, TadA*8) into a scaffold containing Cas9 (eg, spCAS9 or saCAS9) of the circular permutant and a bipartite nuclear localization sequence. is generated by doing so (eg ABE8). The cyclic substituted Cas9 is known in the art and is described, for example, in Oakes et al., Cell 176, 254-267, 2019. Exemplary cyclic substitutions are shown below, where the bold sequences represent sequences derived from Cas9, the italic sequences represent linker sequences, and the underlined sequences represent two-partial nuclear localization sequences indicates. CP5 (and MSP "NGC=Pam Variant with mutations Regular Cas9 likes NGG" PID=protein interaction domain, and "D10A" nickase): TIFF0007631209000002.tif170163

[0065] In some embodiments, ABE8 is selected from the base editors from Table 7 or Table 9 below. In some embodiments, ABE8 includes an adenosine deaminase variant that evolved from TadA. In some embodiments, the adenosine deaminase variant of ABE8 is the TadA*8 variant described in Table 7 or Table 9 below. In some embodiments, the adenosine deaminase variant comprises one or more of the modifications selected from the group Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R (e.g. It is TadA*8). In various embodiments, ABE8 is 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 a TadA*7.10 variant (eg, TadA*8) having a combination of modifications selected from the group I76Y + V82S + Y123H + Y147R + Q154R. In some embodiments, ABE8 is a monomer construct. In some embodiments, ABE8 is a heterodimeric construct. In some embodiments, the adenosine deaminase base editor 8 (ABE8) includes the sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD Includes:

[0066] In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-related (eg, Cas or Cpf1) enzyme. In one embodiment, the base editor is a catalytically dead Cas9 (dCas9) fused to the deaminase domain. In one embodiment, the base editor is Cas9 nickase (nCas9) fused to the deaminase domain. Details of the base editor are listed in International PCT Application No. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US 2016 / 058344 (WO 2017 / 070632), each of which is in its entirety. It is incorporated by reference herein. Komor, A.C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N.M., et al., “Programmable base editing of A. T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, A.C., et al., “Improved base surprise prevention and bacteriaophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity”Science Advances 3:eaao4774 (2017), and Rees, H.A., et al., “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 by reference herein.

[0067] By way of example, the adenine base editor (ABE) used in the base editing compositions, systems, and methods described herein is the nucleic acid sequence (8877 base pairs) provided below (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: 10.1038 / nbt.4172.). Also included are polynucleotide sequences having at least 95% or more identity to the ABE nucleic acid sequence. ATATGCCAAGTACGCCCCTATTGACGTCAATGACGGTAAATGGCCGCCTGGCATTATGCCCAGTACAT GACCTTATGGGACTTTCCTACTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTGACTCACGGGGATTTG ACGTCAATGGGAGTTGTTTTGGCACCAAAATCAACGGGACTTTTCCAAAATGTCGTAACAACTCCGCCCC ATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGT CAGATCCGCTAGAGATCCGCGCCGCTAATACGACTCACTATAGGGAGAGCCGCCACCATGAAACGGACA GCCGACGGAAGCGAGTTCGAGTCACCAAAGAGAAGCGGAAAGTCTGAAGTCGAGTTTAGCCACGAGT ATTGGATGAGGCACGCACTGACCCTGGCAAAGCGAGCATGGGATGAAAGAGAAGTCCCGTGGGCGCCGT GCTGGTGCAACAATAGAGTGATCGGAGAGGGATGGAACAGGCCAATCGGCCGCCACGACCTACCGCA CACGCAGAGATCATGGCACTGAGGCAGGGAGGCCTGGTCATGCAGAATTACCGCCTGATCGACCC TGTATGTGACACTGGAGCCATGCGTGATGCGCAGGAGCAATGATCGGAAGAGTGGT GTTCGGAGCACGGGACGCCAAGACCGGCGCAGCAGGCTCCCTGATGGGTGCTGCACCACCCCCGGCATG AACCACCGGGTGGAGATCACAGAGGGAATCCTGGCAGACGAGTGCGCCGCCCTGCTGAGCGATTTCTTTA GAATGCGGAGACAGGATCAAGGCCCAGAAGAAGGCACAGAGCTCCCACCGACTCTGGAGGATCTAGCGG AGGATCCTCTGGAAGCGAGACACCAGGCACAAGCGAGTCCGCCACACCAGAGAGAGCTCCGGCGGCTCCC GGAGGATCCTCTGAGGTGGAGTTTTTCCCACGAGTACTGGATGAGACATGCCCTGACCCTGGCCAAGAGGG CACGCGATGAGAGGGAGGTGCCTGTGGGAGCCGTGGTGGTGGTGGAACAATAGAGTGATCGGCGAGGCTG GAACAGAGCCATCGGCCTGCACGACCCAACAGCCATGCCGAAATTATGGCCCTGAGACAGGGCGCCTG GTCATGCAGAACTACAGACTGATTGACGCCACCCCTGTACGTGACATTCGAGCCTTGCGTGATGTGCGCCG GCGCCATGATCCACTCTAGGATCGGCCGCGTGGTGTGTTGGCGTGAGGAACGCAAAAACCGGCGCCGCAGG CTCCCTGATGGACGTGCTGCACTACCCGGCATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCA GATGAATGTGCCGCCCTGCTGTGCTATATTTCTTCGGATGCCTAGACAGGTGTTCAATGCTCAGAAGAAGG CCCAGAGCTCCACCGACTCCGGAGGATCTAGCGGAGGCTCCTGGCTCTGAGACACCTGGCACAAGCGA GAGCGCAACACCTGAAAGCAGCGGGGGCAGCGGGGTCAGACAAGAGTACAGCATCGGCCTGGCC ATCGGCACCAACTCTGTGGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGG TGCTGGGCAACACCGACCGGCACAGCATCAAGAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGA AACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAGAAGATACACCACGGAAGAACCGGGATCTGC TATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCCACAGACTGGAAGAGT CCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGC CTACCACGAGAAGTACCACACATCTACCAGAGAAGAACTGGTGGACAGCACAAGGCCGAC CTGCGGCTGATCTATCTGGCCCTGGCCACATGATCAAGTTCCGGGCCACCTTCCTGATCGAGGGCGACC TGAACCCGAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGA GGAAAACCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAAGAGCAGA CGGCTGGAAAATCTGATCGCCCAGCTGCCGGCGAGAAGAGAATGGCCTGTTCGGAAACCTGATTGCCC TGAGCCTGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGCCGAGGATGCCAAACTGCAGCTGAG CAAGGACCCTACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTT CTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCA AGGCCCCCTGAGCGCCCTATGATCAAGAGATACGACGAGCACCACACCAGGACCTGACCCTGCTGAAAGC TCTCGTGCGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCC GGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGG ACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAA CGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATCTGCGGGCAGGAAGATTTTTTAC CCATTCCTGAAGGAAACCGGGAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACGTGGCC CTCTGGCCAGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAA CTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAG AACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGC TGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCGCCTTCCTGAGCGGGAGCAGAAAAGGC CATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAG AAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCCTGGGCACAT ACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGA AGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCC CACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGCAGGCTGAGCC GGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGG CTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAA GCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCATTA AGAAGGCATCCTGCAGACAGTGAAGGTGGTGGGGACGAGCTCGTGAAAGTGGGCCGGCACAAGCCGA GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGA ATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCCGTGGAAAACA CCCAGCTGCAGAACGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGATATGTACGTGGACCAGGA ACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGAC TCCATCGAACAAGGTGCTGACCAGAAGCGAAGAACCGGGGCAAGAGAGCGACAACGTGCCCTCCGAAG AGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTT CGACAATCTGACCAAGGCCGAGAGAGGCGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAG CTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCGGATGAACACTAAGTACG ACGAGAATGAAGCTGATCCGGGAAGTGAAGTGAAAGTGATCACCCCTGAAGTCCAAGCTGGTGTCCGAATTTCCG GAAGGATTTCCAGTTTTTACAAAGTGCGCGAGATCAACAACTACCACGCCACGCCACGCCTACCTGAAC GCCGTCGTGGGAACCGCCCTGATCAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACA AGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTT CTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCTGGCCAACGGCGAGATCCGGAAGCGG CCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGCCGGGGATTTTGCCACCGTGC GGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAA AGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAGAAAGAGGACTGGGACCCAGAAGAAG TACGGCGGCTTCGACAGCCCACCGTGGCCTATTCTGTGCTGGTGGGTGGCCAAAGTGGAAAGGCAAGT CCAAGAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAA TCCCATCGACTTTCTGGAAGCCAAGGCTACAAGAGTGAAAAGGACCTGATCATCAAGCTGCCTAAG TACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGGCGAACTGCAGAAGGGAA ACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGG CTCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATC GAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCT ACAACAAGCACCGGGATAAGCCATCAGAGAGCAGGCCGAGAGAATATATCATCCACCTGTTTACCCTGACCAA TCTGGGAGCCCCCTGCCCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCAAA GAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCCTGTCTC AGCTGGGAGGTGACTCTGGCGGCTCAAAAGAACCGCCGACGCAGCGAATTCGAGCCCAAGAGAAGAG GAAAGTCTAACCGGTCATCATCACACCATTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTT CTAGTTGCCAGCCATCTGTTGTTGCCCCCCGTGCCTTGCCCCCCGTGCCTTGACCCTGGAAGGTGCCAC TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGT GGGGTGGGCAGGACAGCAAGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGGGATGCGGTGGGCT CTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGATACCGTCGACCTCTAGCTAGAGCTTGGCGTA ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCAACATACGAGCCGGA AGCATAAAGTGTAAAGCCTAGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGC CCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGGGGAGAGGCGGGGGG TTTGCGTATTGGGCGCTCTTCCGCTTCGCTCACTGACTCGCTGCGCTCGCTCGGTCGTTCGGGCGA GCGGTATCAGCTCACTCAAAGGCGTAATACGGTTATCCCACAGAATCAGGGGATAACGCAGGAAAGAACA TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAGGCCGCGTTGCTGGCGTTTTTTCCATAGGCT CCGCCCCCTGACGAGCATCACAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA AGATACCAGGCGTTTCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGAT ACCTGTCCGCCTTCTCCCTTCGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCCGTTCAGCCGACCGCTGCGCCTTA TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTA ACAGGATTAGCAGAGCGAGTATGTAGGCGGTGCTACAGAGTTCTGAAGTGGTGGCCTAACTACGGCTA CACTAGAAGAACAGTATTGGTATCTGCGCTCTGAAGCCAGTTACCTTCGGAAAAGAGTTGGTAGC TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTGCAAGCAGATTACGCGCA GAAAAAGGATCTCAAGAAGATCCTTTGATCTTTCTACGGGGTCTGACACTCAGTGGAACGAAAACTC ACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAATGA AGTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTAC GATACGGGAGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCCACGCTCACCGGCTCCA GATTTATCAGCAATAAACCAGCCAGCCGGAAGGCCGAGCGAGAAGTGGTCCTGCAACTTATCCGCCT CCATCCAGTCTAATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTGCGCAACGT TGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTGGTATGGCTTCATCAGCTCCGGTTCC CAACGATCAAGGCGAGTTACATGATCCCCATGTTGTGCAAAAAGCGGTTAGCTCCCTTCGGTCCTCCGA TCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTAC TGTCATGCCATCCGTAAGATGCTTTCTGTGACTGGTGAGTACTCAACCAAGTCATCTGAGAATAGTGT ATGCGGCGACCGAGTTGCTCTTGCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAA AAGTGCTCATCATGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTCCAGCGTTCTGGGTGA GCAAAAACAGGAAGGCAAAATGCCGCAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATAC TCTTCCTTCCAATATTATTGAAGCATTATCAGGTTATTGTCTCATGAGCGGATACATATTGAATG TATTTAGAAAATAAACAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGA TCGGGAGATCGATCCCCCGATCCCTAGGGTCGACTCTCAGTACAATCTGCTGCTGATGCCGCATAGTTAA GCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAAC AAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGAT GTACGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAGTTATATTAGTAATCAATTACGGGGTCAT TAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCGCCTGGCTGACCGCC CAACGACCCCGCCATGCCATGTCAATAATGACGTATGTTCCATGTAACGCCAATAGGGACTTTCCAT TGACGTCAATGGGTGGAGTATTACGGTAAACTGCCACTTGGCAGTACATCAAGTGTATC

[0068] "Base editing activity" means acting to chemically modify a base within a polynucleotide. In one embodiment, the first base is converted to a second base. In one embodiment, the base editing activity is a cytidine deaminase activity, eg, an activity that converts the target C·G to T·A. In another embodiment, the base editing activity is an adenosine or adenine deaminase activity, eg, an activity that converts A·T to G·C. In another embodiment, the base editing activity is a cytidine deaminase activity, eg, an activity that converts target C·G to T·A, and an adenosine or adenine deaminase activity, eg, an activity that converts A·T to G·C. be. In some embodiments, base editing activity is assessed by editing efficiency. Base editing efficiency can be measured by any suitable means, eg, by Sanger sequencing or next-generation sequencing. In some embodiments, base editing efficiency is measured by the percentage of total sequencing reads that resulted in nucleobase conversion by the base editor, for example, the total number of target A.T base pairs converted to G.C base pairs Measured by the rate of sequencing reads. In some embodiments, base editing efficiency is measured by the percentage of total cells that resulted in the base editor when base editing is performed on a population of cells.

[0069] The term "base editor system" refers to a system for editing a nucleobase of a target nucleotide sequence. In various embodiments, the base editor system includes (1) a polynucleotide-programmable nucleotide-binding domain (eg, Cas9); (2) a deaminase domain (eg, adenosine deaminase) for deamination of said nucleobase; and ( 3) Contains one or more guide polynucleotides (eg, guide RNAs). 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.

[0070] In some embodiments, the base editor system may include multiple base editing components. For example, the base editor system may include multiple deaminases. In some embodiments, the base editor system may include one or more adenosine deaminases. In some embodiments, a single guide polynucleotide can be utilized to target different deaminases to target nucleic acid sequences. In some embodiments, a pair of guide polynucleotides may be utilized to target different deaminases to target nucleic acid sequences.

[0071] The deaminase domain and polynucleotide programmable nucleotide binding components of the base editor system may be covalently or non-covalently associated with each other, or by any combination of their associations and interactions. For example, in some embodiments, the deaminase domain may 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 linked to the deaminase domain. In some embodiments, the polynucleotide-programmable nucleotide-binding domain may be targeted to the target nucleotide sequence by non-covalently interacting or associating with the deaminase domain. For example, in some embodiments, the deaminase domain may interact with, associate, or form a complex with additional heterologous portions or domains that are part of a polynucleotide-programmable nucleotide-binding domain Additional heterologous portions or domains may be included. In some embodiments, the further heterologous moiety may bind, interact, associate, or form a complex with the polypeptide. In some embodiments, the further heterologous moiety may bind, interact, associate, or form a complex with the polynucleotide. In some embodiments, the further heterologous moiety may be associated with the guide polynucleotide. In some embodiments, the further heterologous moiety may be attached to a polypeptide linker. In some embodiments, the further heterologous moiety may be attached to a polynucleotide linker. This further heterologous portion may be a protein domain. In some embodiments, the further heterologous portion includes K-homologous (KH) domain, MS2 coat protein domain, PP7 coat protein domain, SfMu Com coat protein domain, sterylalpha motif, telomerase Ku binding motif and Ku protein, telomerase It can be an Sm7 binding motif and an Sm7 protein, or an RNA recognition motif.

[0072] The base editor system may further include a guide polynucleotide component. It should be understood that the components of the base editor system may be associated with one another by covalent, non-covalent interactions, or by any combination of these associations and interactions. In some embodiments, the deaminase domain may be targeted to the target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the deaminase domain may interact with, associate, or form a complex with a portion or segment of a guide polynucleotide (eg, a polynucleotide motif). It may include a domain (eg, a polynucleotide binding domain, such as an RNA-binding protein or a DNA-binding protein). In some embodiments, the further heterologous portion or domain (eg, a polynucleotide binding domain, such as an RNA-binding protein or a DNA-binding protein) can be fused or linked to the deaminase domain. In some embodiments, the further heterologous moiety may bind, interact, associate, or form a complex with the polypeptide. In some embodiments, the further heterologous moiety may bind, interact, associate, or form a complex with the polynucleotide. In some embodiments, the further heterologous moiety may be attached to the guide polynucleotide. In some embodiments, the further heterologous moiety may be attached to a polypeptide linker. In some embodiments, the further heterologous moiety may be attached to a polynucleotide linker. This further heterologous portion may be a protein domain. In some embodiments, the further heterologous portion includes K-homologous (KH) domain, MS2 coat protein domain, PP7 coat protein domain, SfMu Com coat protein domain, sterylalpha motif, telomerase Ku binding motif and Ku protein, telomerase It can be an Sm7 binding motif and an Sm7 protein, or an RNA recognition motif.

[0073] In some embodiments, the base editor system may further include inhibitors of the base excision repair (BER) component. It should be understood that the components of the base editor system may be associated with one another by covalent, non-covalent interactions, or by any combination of these associations and interactions. Inhibitors of the BER component may include BER inhibitors. In some embodiments, the inhibitor of the BER can be a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of BER may be an inosine BER inhibitor. In some embodiments, the inhibitor of the BER can be targeted to the target nucleotide sequence by a polynucleotide-programmable nucleotide-binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to an inhibitor of the BER. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to the deaminase domain and the inhibitor of the BER. In some embodiments, the polynucleotide programmable nucleotide binding domain may target an inhibitor of the BER to a target nucleotide sequence by non-covalent interaction or association with an inhibitor of the BER. For example, in some embodiments, the inhibitor of the BER component may interact with, associate, or conjugate with, or conjugate with, additional heterologous portions or domains that are part of a polynucleotide-programmable nucleotide-binding domain. It may include additional heterologous portions or domains that may be formed.

[0074] In some embodiments, the inhibitor of the BER may be targeted to the target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the inhibitor of the BER may interact with, associate, or form a complex with, part or segment of the guide polynucleotide (eg, a polynucleotide motif). It may include portions or domains (eg, polynucleotide binding domains such as RNA-binding proteins or DNA-binding proteins). In some embodiments, additional heterologous portions or domains of the guide polynucleotides (eg, polynucleotide binding domains, such as RNA-binding proteins or DNA-binding proteins) can be fused or linked to an inhibitor of the BER. In some embodiments, the further heterologous moiety may bind, interact, associate, or form a complex with the polynucleotide. In some embodiments, the further heterologous moiety may be attached to the guide polynucleotide. In some embodiments, the further heterologous moiety may be attached to a polypeptide linker. In some embodiments, the further heterologous moiety may be attached to a polynucleotide linker. This further heterologous portion may be a protein domain. In some embodiments, the further heterologous portion includes K-homologous (KH) domain, MS2 coat protein domain, PP7 coat protein domain, SfMu Com coat protein domain, sterylalpha motif, telomerase Ku binding motif and Ku protein, telomerase It can be an Sm7 binding motif and an Sm7 protein, or an RNA recognition motif.

[0075] The term "Cas9" or "Cas9 domain" refers to an RNA comprising a Cas9 protein or a fragment thereof (eg, a protein containing an active, inactive, or partially active DNA cleavage domain of Cas9, and / or a gRNA-binding domain of Cas9). Refers to a derived nuclease. Cas9 nucleases are sometimes referred to as casnl nucleases or CRISPR (clustered regularly interspaced short palindromic repeat) binding nucleases. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, metastatic factors, and conjugation plasmids). The CRISPR cluster includes a spacer, a sequence complementary to the preceding mobile element, and a target invasion nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In the type II CRISPR system, correct processing of pre-crRNA requires small transcoding RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. tracrRNAs serve as a guide to the processing of pre-crRNAs by ribonuclease 3. Cas9 / crRNA / tracrRNA then cleaves a linear or circular dsDNA target complementary to the spacer with an endonuclease. The target strands that are not complementary to the crRNA are first cleaved by an endonuclease and then adjusted to 3'-5' by an exonuclease. In nature, DNA binding and cleavage typically requires proteins and both RNAs. However, a single guide RNA ("sgRNA" or simply "gRNA") can be engineered to incorporate both crRNA and tracrRNA aspects into a single RNA species. See, e.g., 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 by reference herein). I want to be Cas9 recognizes short motifs (PAM or protospacer flanking motifs) in CRISPR repeats to help distinguish between self-vs. The sequences and structures of Cas9 nucleases are known to those skilled in the art (eg, "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J. , Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 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 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 in adaptive bacterial immunology.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337 See:816-821 (2012) (the entire contents of which are incorporated by reference herein). Cas9 orthologs have been described in a variety of species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will become apparent to those skilled in the art based on this disclosure, and as such Cas9 nucleases and sequences, Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunocy systems (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated by reference herein, Cas9 sequences from the organisms and loci.

[0076] An exemplary Cas is Streptococcus pyogenes Cas9 (spCas9), which is the amino acid sequence: TIFF0007631209000003.tif166164 (Single underline: HNH domain; Double underline: RuvC domain)

[0077] Nuclease-inactivated Cas9 proteins may be referred to interchangeably with "dCas9" proteins (nuclease "inactivated" Cas9) or catalytically inactive CAs9. Methods for generating Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains are known (e.g., Jinek et al., Science. 337:816-821(2012); Qi et al., “Repurposing See CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28;152(5):1173-83, the entire contents of each of which are incorporated by reference herein. I want to be.) For example, the DNA cleavage domain of Cas9 is known to include the following two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to gRNA, and the RuvC1 subdomain cleaves the strand non-complementary. Mutations within these subdomains may suppress the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of S. 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 (eg, inactivated) DNA cleavage domain, ie, Cas9 is a nickase called the "nCas9" protein ("Niccase" Cas9). be. In some embodiments, a protein comprising a fragment of Cas9 is provided. For example, in some embodiments, the protein comprises one of the following two Cas9 domains: (1) the gRNA-binding domain of Cas9; (2) the DNA cleavage domain of Cas9. In some embodiments, a protein containing Cas9 or a fragment thereof is referred to as a "Cas9 variant." The Cas9 variant shares homology with Cas9 or a fragment thereof. For example, the 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% They are identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the Cas9 variant is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, compared to wild-type Cas9. 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, It can have 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes. In some embodiments, the Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical to the corresponding fragment of wild-type Cas9. are 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% identical, at least about 99.5% identical; Or includes fragments of Cas9 (eg, gRNA-binding or DNA-cleaving domains) that are at least about 99.9% identical. 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 of the corresponding wild-type Cas9 amino acid length %, 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%.

[0078] In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, It can be 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0079] 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). ATGGATAAGAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGGGGGTGATCACTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTAT CAAAAAAATCTTATAGGGCTCTTTATTTGGCAGTGGAGAGAGAGCGGAAGCGACTCGTCCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTGTTATCTACAGGAGATTT TTTCAAATGAGATGGCGAAAGTAGATGATAGTTCTTCATCATCGACTTGAAGAGTCATTTTTGGTGGAAGAGAAGAAGCATGAACGTCATCCTATTTTGGAAATATAGTAGATGAAGTTGCTTAT CATGAGAAATATCCAACTATCATCATCTGCGAAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTGAG GGAGATTTAAATCCTGATAATAGTGTGGACAAACTATTATCCAGTTGGTACAAATCTACAATCAATCAATTATTGAAGAAACCCTATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTGC ACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAGAAATGGCTTGTTGGGAATCTCATTGCTTTGTCATGTCATGGGATTGACCCCTAATTTTAAATCAAATTTG ATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTATACGATGATGATTTAGATAATTGGCGCAAATTGGAGATCAATATGCTGATTTGGCAGCTAAGAATTTATCAGATGCT ATTTTACTTCAGATATCCTCAGAGAGTAAATAGTGAAATAACTAAGGCTCCCCCTATCAATGATTAAGCGCTACGATGAACATCAAGACTTAGCTTTTAAAAGCTTTTAGTTCGACAACAA CTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGG TACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTGACAACGGCTCTATTCCCATCAAATTCACACTTGGGTGAGCTGCATGCTATTTGGAGAAGAAGAAG ACTTTTATCCATTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAA GAAACAATTACCCCATGGAATTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAAATTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAAACATAGTTG CTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAATGCGAAAACCAGCATTTCTTCAGGTGAACAGAGAAAGCCATTGTTGATTTACTCTCAAAACAAAA TCGAAAAGTAACCGTTAAGCAATTAAAGAGAATTATTCAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGCGCCTACCATGATTTGCTAA AAATTAAAGATAAAGATTTTTTGGATAATGAGAAAATGAAATGAAGATATTGTTTTTAACATGATTGACCCTTATTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATGCTCAC CTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGGACGTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTGAAA TCAGATGGTTTTGCCAATCGCAATTTATGCAGCTGATCCATGATGATAGTTGACATTTAAGAGATATTCAAAGAAGCAGGTGTCTGGACAAGGCCATAGTTACATGAACAGATTGCTAACTT AGCTGGCAGTCCTGCTATTAAAAAGGTATTTACAGACTGTAAAAATTGTTGATGAACTGGTCAAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGAACTC AAAAGGCCAGAAAATTCGCGAGAGAGCGTATGAAACGAATCGAAGAGGTATCAAGAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCCAATTGCAAAATGAAAGCTCTATC TATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATGTTTAAGTGATTATGTCGATCACAAAGTTTCATACAAGTTTCATACAAGTTTCATCAATAGACAATAAGGTA CTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAAGATGAAAACTATTGGAGACAACTTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTTGATAA TTTAACGAAAGCTGAACGTGGAGTTTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTGGATAGTCGCATGAATACTA AATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATTAGTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATGCC CATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAACTTGAATCGGAGTTGTGTGATTATAAAGTTGTTGGTGATTATAAAGTTGTTGTAAAATGATTGCTAAGTCTGAGCAA GAAATAGGCAAAGCAACCGCAAAATATTTCTTTATACATGAACTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGG AGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGCCCCAAGTCAAGTCAATATTGTCAAGAAAACAGAGAGAGAGGCGGATTCTCCAAGGAGTCAATTTTTACCAA AAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTGGTTGCTAAGGTGGAAAAGGGAAATCGAAG AAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAGTTCCTTGAAAAAATCCGATTGACTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAGACTTAATCATTAAACTA CCTAAATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTTTATAGCTAGTCA TTATGAAAGTTGAAGGTAGTCCAGAGAAGAAGAAAAACAATTGTTGTGGAGCATAAGCATAAGCATTTAGATGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAG ATGCCAATTTAGATAAAGTTCTTAGTGCATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAAATATATAT TTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTAGATGCCACTCATCAATCCACTGGTCTTATATGAAACACGCATTGATGAGGTGACTGA TIFF0007631209000004.tif168164 (Single underline: HNH domain; Double underline: RuvC domain)

[0080] In some embodiments, wild-type Cas9 corresponds to or includes the following sequences of nucleotides and / or amino acids: ATGGATAAAAAGTATTCTATGGTTTAGACATCGGCACTAATTCCGTTGGGCTGTCATAACCGATGAATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAA AAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACAAGAAATTTTTTAGCAA TGAGATGGCCAAAGTTGACGATTCTTTCTCACCGTTGGAAGAGTCCTTCTTGGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAGT ACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTGGCTCTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATC CGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATCTTAGCGCCCGCCTCTCTAAATCC CGACGGCTAGAAAACCTGATCGCACAATTACCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTGACACCAAATTTTAAGTCGAACTTCGAAGATGCCAAA TTGCAGCTTAGTAAGGACACGTACGATCTCGACAATCTACTGGCACAAATTGGAGAATCAGTATGCGGACTTATTTTGGCTGCCAAAAACCTTAGCGATGCAATCCCTATCTGACATACTGAGA GTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACATGAACATCAAGACTTGACACCTCAAGGCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTC TTTGATCAGTCGAAAAACGGGTACGGTTATGACGGGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCCATATTGAGAAGATGGATGGGGACGGAAGAGTTGCTTGTAAAACTCAATCG CGAAGATCTACTGCGAAAGCAGCGGACTTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATATGAAGGCAGGAGGATTTTTTATCCGTTCCAAAGACAATCGTGAAAA GATTGAGAAAATCCTAACCTTTTCGCATACCTTATGTGGGACCCTGCCGAGGGAACTCTCGGTTCGCATGGACAAGAAAGTCCGAAGAAACGATTACTCCCATGGAATTTGGAAGTTGTCG ATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGAGGATGACCAACTTTGACAAGAATTTGAACGAAAAAGTATTGCCTAAGCAGTTTACTATTACGAGAGTGTACAATGAACTCACGAAAG TTAAGTATGTCACTGAGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAGAAGAAGCAATAGTAGATCTGTTATCAAGACCGCAAAGTGACAGTTAAGCAATTGAAGAGGACTATTTAAG AAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCCTCCTAAAGATAATTAAGATAAGATAAGGACTTCCTGGATAACGAGAGAATGAAGAT ATCTTAGAAGATATAGTGTTGACTCTTACCCTTTGAAGATCGGGAAATGATTGAGGAAAAACATACGCTCACCTGTTCGGAACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGC TGGGGACGATTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTATGCAGCTGATCCATGATGACTCTTTA ACCTTCAAAGAGGATATACAAAGGCACAGTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTGCTGGTTCGCCAGCCATCAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGA GCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGATAGAGAGGTATTAAAGA ACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCCAATTGCAGAACGAGAAACTTGCAAAATGGAAGGGACATGTATGTTGATCAGGATAAACCGTTTAT CTGATTACGACGTCGATCACATTGTACCCCCAATCCCTTTTTGAAGGACGAATTCGAATAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGA AAATGAAGAACTATTGGCGCAGCTCCTAAATGCGAAACTGATAACGCAAGAAGTTCGATAACTTAACTAAGCTGAGAGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTC GTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGAGATAGATTCCCGAATGAATACGAAATACGAGAGAGAGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAATTGGTGTCGGAC TTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTAATGCCGTCGTAGGGACCGCACTCATTAAGAATACCCCGAAGCTAGAAAGTGAGTTTGTG TATGGTGATTACAAAGTTATATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCCAAATACTTCTTTATATGAATTTCTTAAGACGGAAATCACTCTGGCA AACGGAGAGATACGCAAACGACCTTTAATGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCAAGTCAACATAGTAAAGAA AACTGAGGTGCAGACCGGAGGTTTTCAAAGGAATCGATTCTCCAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGGACCGAAAAAGTACGGTGGCTTCGATAGCCTACAGTTGCCTAA TTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAACTGAAGTCAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCGTCTTTTGAAAAGAACCCCATCGACTTCCTGGGCGA AAGGTTACAAGGAAGTAAAAGGATCTCATAATTAAACTACCAAGTATAGTCTGTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGAGCTTCAAAGGGGAACGAACTCGCACTAC CGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAGAGAAGCAACTTTTGTTGAGCAGCAAAACATTATCTCGACGAAATCATAGAGCAAA ATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGCCAATCTGGACAAAGTATTAAGCGCATACAAGCAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCC AACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACAACGATAGATCGCAAACGATAGATCGCAAACGATACACTTCCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCACGGGATTATATGAAACTCGGATA GATTTGTCACAGCTTGGGGTGACGGATCCCCCAAGAGAGAAGAGGAAAGTCTCGAGCGACTACAAGACCATGACGGTGATTATAAAGATCATGACAAGGATGACGATGACAAGGCTGCAGGA TIFF0007631209000005.tif167162 (Single underline: HNH domain; Double underline: RuvC domain)

[0081] In some embodiments, wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI reference sequence: NC_002737.2 (nucleotide sequence: as follows); and Uniprot reference sequence: Q99ZW2 (amino acid sequence: as follows) do. ATGGATAAGAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGGGGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTAT CAAAAAAATCTTATAGGGCTCTTTATTATTGACAGTGGAGAGAGAGCGGAAGCGACTCGTCCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTGTTATCTACAGGAGATTT TTTCAAATGAGATGGCGAAAGTAGATGATAGTTCTTCATCATCGACTTGAAGAGTCATTTTTGGTGGAAGAGAAGAAGCATGAACGTCATCCTATTTTGGAAATATAGTAGATGAAGTTGCTTAT CATGAGAAATATCCAACTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATGCGCATATGATTAAGTTTCGTGGTCATTTTTGAG GGAGATTTAAATCCTGATAATAGTGTGGACAAACTATTATCCAGTTGGTACAAACCTACAATCAATCAATTATTGAAGAAAACCCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGC ACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTGGGAATCTCATTGCTTTGTCATGGGTTGACCCCCTAATTTTAAATCAAATTTG ATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTATACGATGATGATTTAGATAATTGGCGCAAATTGGAGATCAATATGCTGATTTGGCAGCTAAGAATTTATCAGATGCT ATTTTACTTCAGATATCCTCAGAGAGTAAATACTGAAATAACTAAGGCTCCCCCTATCAATGATGAACGCTACGATGAACATCAAGACTTAGCTTTTAAAAGCTTTTAGTTCGACAACAA CTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGG TACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTGACAACGGCTCTATTCCCATCAAATTCACACTTGGGTGAGCTGCATGCTATTTGGAGAAGAAGAAG ACTTTTATCCATTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAA GAAACAATTACCCCATGGAATTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAAATTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAAACATAGTTG CTTTATGAGTATTTTACGGTTTATAACGAATTAGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTCAGGTGAACAGAGAAAGCCATTGTTGATTTACTCTCAAAACAAAA TCGAAAAGTAACCGTTAAGCAATTAAAGAGAATTATTCAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATAGATTTAATGCTTCATTAGGTACCATGATTTGCTAAAA AATTATTAAAGATAAAGATTTTTTGGATAATGAGAGAAAATGAAATGATATTGTTTTTAACATGACC TCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGGACGTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTGAAAT CAGATGGTTTTGCCAATCGCAATTTATGCAGCTGATCCATGATGATAGTTGACATTTAAGAGAGAGATTCAAAAGCAAGTGTCTGGACAAGGCGATAGTTACATGAACATATGCAAATTTA GCTGGTAGCCCTGCTATTAAAAAGGTATTTACAGACTGTAAAAGTTGTTGATGAATGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGAAC TCAAAAGGCCAGAAAATTCGCGAGAGAGCGTATGAAACGAATCGAAGAGGTATCAAGAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTC TCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATTAATCGTTTAAGTGATTATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGAATAAGG TCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAAGATGAAAACTATTGGAGACAACTTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTGAT AATTTAACGAAAGCTGAACGTGGAGTTTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTGGATAGTCGCATGAATACT AAATACGATGAAAATGATAAACTTATTCGAGAGAGGTTAAAGTGATTACCTTAAAATTAGTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATGC CCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCCAAAACTTGAATCGGAGTTGTGTGATTATAAAGTTGTTGTGATTATAAAGTTGTTGTAAAATGATTGCTAAGTCTGAGCA AGAAATAGGCAAAGCAACCGCAAAATATTTCTTTATACATGAACTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTG GAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGCCCCAAGTCAAGTCAAGTCAATATTGTCAAGAAACAGAGAGAGGCGGATTCTCCAAGGAGTCAATTTTACCA AAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAA GAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAGTTCCTTGAAAAAATCCGATTGACTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAGACTTAATCATTAAACT ACCTAAATATAGTCTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTTTAGCTAGTC ATTATGAAAGTTGAAGGTAGTCCAGAGAAGAAGAAAAACAATTGTTGTGGAGCATAAGCATAAGCATTATTAGATGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTAGCAG ATGCCAATTTAGATAAAGTTCTTAGTGCATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAAATATATAT TTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTAGATGCCACTCATCAATCCACTGGTCTTATATGAAACACGCATTGATGAGGTGACTGA TIFF0007631209000006.tif168164 (SEQ ID NO: 1. Single underline: HNH domain; Double underline: RuvC domain)

[0082] In some embodiments, Cas9 refers to Cas9 from: or Cas9 from any other organism: 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 (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (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).

[0083] In some embodiments, Cas9 is derived from Neisseria meningitidis (Nme). In some embodiments, Cas9 is Nme1, Nme2, or Nme3. In some embodiments, the PAM interaction domains for Nme1, Nme2, or Nme3 are respectively N 4 GAT, N 4 CC, and N 4 is a CAAA (see, e.g., Edraki, A., et al., A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing, Molecular Cell (2018). The exemplary Neisseria meningitidis Cas9 protein Nme1Cas9 (NCBI reference: WP_002235162.1; type II CRISPR RNA-guided endonuclease Cas9) has the following amino acid sequence: 1 maafkpnpin yilgldigia svgwamveid edenpiclid lgvrvferae vpktgdslam 61 arrlarsvrr ltrrrahrll rarrllkreg vlqaadfden glikslpntp wqlraaaldr 121 kltplewsav llhlikhrgy lsqrkneget adkelgallk gvadnahalq tgdfrtpael 181 alnkfekesg hirnqrgdys htfsrkdlqa elillefekqk efgnphvsgg lkegietllm 241 tqrpalsgda vqkmlghctf epaepkaakn tytaerfiwl tklnnlrile qgserpltdt 301 Eatlmdepy rkskltyaqa rkllgledta ffkglrygkd naeastlmem kayhaisral 361 ekeglkdkks plnlspelqd eigtafslfk tdeditgrlk driqpeilea llkhisfdkf 421 vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee kiylppipad eirnpvvlra 481 lsqarkving vvrrygspar ihietarevg ksfkdrkeie krqeenrkdr ekaaakfrey 541 fpnfvgepks kdilklrlye qqhgkclysg keinlgrlne kgyveidhal pfsrtwddsf 601 nnkvlvlgse nqnkgnqtpy ​​eyfngkdnsr ewqefkarve tsrfprskkq rillqkfded 661 gfkernlndt ryvnrflcqf vadrmrltgk gkkrvfasng qitnllrgfw glrkvraend 721 rhhaldavvv acstvamqqk itrfvrykem nafdgktidk etgevlhqkt hfpqpweffa 781 qevmirvfgk pdgkpefeea dtpeklrtll aeklssrpea vheyvtplfv srapnrkmsg 841 qghmetvksa krldegvsvl rvpltqlklk dlekmvnrer epklyealka rleahkddpa 901 kafaepfyky dkagnrtqqv kavrveqvqk tgvwvrnhng iadnatmvrv dvfekgdkyy 961 lvpiyswqva kgilpdravv qgkdeedwql iddsfnfkfs lhpndlvevi tkkarmfgyf 1021 aschrgtgni nirhdldhk igkngilegi gvktalsfqk yqidelgkei rpcrlkkrp 1081 vr

[0084] Another exemplary Neisseria meningitidis Cas9 protein Nme2Cas9 (NCBI reference: WP_002230835; type II CRISPR RNA-guided endonuclease Cas9) has the following amino acid sequence: 1 maafkpnpin yilgldigia svgwamveid eeenpirlid lgvrvferae vpktgdslam 61 arrlarsvrr ltrrrahrll rarrllkreg vlqaadfden glikslpntp wqlraaaldr 121 kltplewsav llhlikhrgy lsqrkneget adkelgallk gvannahalq tgdfrtpael 181 alnkfekesg hirnqrgdys htfsrkdlqa elillefekqk efgnphvsgg lkegietllm 241 tqrpalsgda vqkmlghctf epaepkaakn tytaerfiwl tklnnlrile qgserpltdt 301 Eatlmdepy rkskltyaqa rkllgledta ffkglrygkd naeastlmem kayhaisral 361 ekeglkdkks plnlsselqd eigtafslfk tdeditgrlk drvqpeilea llkhisfdkf 421 vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee kiylppipad eirnpvvlra 481 lsqarkving vvrrygspar ihietarevg ksfkdrkeie krqeenrkdr ekaaakfrey 541 fpnfvgepks kdilklrlye qqhgkclysg keinlvrlne kgyveidhal pfsrtwddsf 601 nnkvlvlgse nqnkgnqtpy ​​eyfngkdnsr ewqefkarve tsrfprskkq rillqkfded 661 gfkecnlndt ryvnrflcqf vadhilltgk gkrrvfasng qitnllrgfw glrkvraend 721 rhhaldavvv acstvamqqk itrfvrykem nafdgktidk etgkvlhqkt hfpqpweffa 781 qevmirvfgk pdgkpefeea dtpeklrtll aeklssrpea vheyvtplfv srapnrkmsg 841 ahkdtlrsak rfvkhnekis vkrvwlteik ladlenmvny kngreielye alkarleayg 901 gnakqafdpk dnpfykkggq lvkavrvekt qesgvllnkk naytiadngd mvrvdvfckv 961 dkkgknqyfi vpiyawqvae nilpdidckg yriddsytfc fslhkydlia fqkdekskve 1021 fayyincdss ngrfylawhd kgskeqqfri stqnlvliqk yqvnelgkei rpcrlkkrp 1081 vr

[0085] In some embodiments, dCas9 corresponds to, or includes, or partially, or entirely, a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises a D10A and H840A mutation, or a corresponding mutation in another Cas9. In some embodiments, dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A): TIFF0007631209000007.tif168163 (single underlined: HNH domain; double underlined: RuvC domain).

[0086] In some embodiments, the Cas9 domain comprises a D10A mutation and is at a corresponding position in either position 840 in the amino acid sequence provided above or in any of the amino acid sequences provided herein. The residue remains histidine.

[0087] In other embodiments, there is provided a dCas9 variant with mutations other than D10A and H840A, leading to, for example, nuclease-inactivated Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (eg, substitutions in the HNH nuclease subdomain and / or RuvC1 subdomain). In some embodiments, a variant or homolog of dCas9 is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical Alternatively, a variant or homolog is provided that is 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 variant of dCas9 includes about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, Variants are provided with about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or more shorter or longer amino acid sequences.

[0088] In some embodiments, the Cas9 fusion proteins provided herein include the full-length amino acid sequence of the Cas9 protein, for example, one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not include the full-length Cas9 sequence and only include one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and further suitable sequences of the Cas9 domains and fragments will be apparent to those skilled in the art.

[0089] It should be understood that additional Cas9 proteins (eg, nuclease-inactivated Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including this variant and homologue, are within the scope of this disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is nuclease-inactive Cas9 (dCas9). In some embodiments, the Cas9 protein is Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is nuclease active Cas9.

[0090] Exemplary, catalytically inert Cas9(dCas9): DKKYSIGLAIGTNS VGWAVIT DEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEEN PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQ LPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSE QEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK LPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0091] Exemplary catalytic Cas9 nickase (nCas9): DKKYSIGLAIGTNS VGWAVIT DEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEEN PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQ LPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSE QEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK LPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0092] Exemplary, catalytically active Cas9: DKKYSIGLDIGTNS VGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEEN PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKS DGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKL PKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD.

[0093] In some embodiments, Cas9 refers to Cas9 from archaeals (eg, nanoarchia) that make up the domains and kingdoms of single-cell prokaryotic organisms. In some embodiments, Cas9 is, for example, Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21 (This entire content is by reference). refers to CasX or CasY as described in (incorporated in the specification). Using genomic degradation metagenomics, many CRISPR-Cas systems, including Cas9, which was first reported in the archaeal domain of life, were identified. This branched Cas9 protein was found in rarely studied nanoarchia as part of the active CRISPR-Cas system. In bacteria, the two previously unknown systems: CRISPR-CasX and CRISPR-CasY were discovered, which belong to the most compact systems already discovered. In some embodiments, Cas9 refers to a CasX or a variant of CasX. In some embodiments, Cas9 refers to a CasY or a variant of CasY. It is to be understood that other RNA-induced DNA-binding proteins may be used as nucleic acid programmable DNA-binding proteins (napDNAbp) and are within the scope of this disclosure.

[0094] In certain embodiments, napDNAbp useful in the methods of the present invention include cyclic substitutions known in the art and described, for example, by Oakes et al., Cell 176, 254-267, 2019. . Exemplary cyclic substitutions are shown below, where the bold sequences represent sequences derived from Cas9, the italic sequences represent linker sequences, and the underlined sequences represent two-partial nuclear localization sequences indicates. CP5 (and MSP "NGC=Pam Variant with mutations Regular Cas9 likes NGG" PID=protein interaction domain, and "D10A" nickase): TIFF0007631209000008.tif172162

[0095] Non-limiting examples of polynucleotide-programmable nucleotide binding domains that can be incorporated into the base editor include CRISPR protein-derived domains, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs).

[0096] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein can be a CasX protein or a CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, 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 96%, at least 96%, at least 96%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97%, at least 97% %, at least 97%, at least 98%, at least 99%, or at least 99.5% identical. In some embodiments, napDNAbp is a naturally occurring CasX or CasY protein. In some embodiments, napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, with any CasX or CasY protein described herein. Contains an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical. It should be understood that Cas12b / C2c1, CasX, and CasY from other bacterial species may also be used in accordance with this disclosure.

[0097] Cas12b / C2c1 (uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2|C2C1_ALIAG CRISPR-associated endo-nuclease C2c1 OS = Alicyclobacillus acido-terrestris(strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) GN=c2c1 PE=1 SV=1 MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECDKTAEECKAELLERLRARQVENGHRGPAGSDDELLQLARQYYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGL GIAKAGNKPRWVRMREAGEPGWEEEKEKAETRKSADRTADVLRALADFGLKPLMRVYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQEYAKLVEQKNRFEQKNFVGQEHLVHLVNQLQDMK EASPGLESKEQTAHYVTGRALRGSDKVFEKWGKLAPDAPFDLYDAEIKNVQRRNTRRFGSHDLFAKLAEPEYQALWREDASFLTRYAVYNSILRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFG ERRHAIRFHKLLKVENGVAREVDDVTVPISMSEQLDNLLPRDPNEPIALYFRDYGAEQHFTGEFGGAKIQCRRDQLAHMHRRRGARDVYLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHP DDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPFFFPIKGNDNLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPVDAANHMTPD WREAFENELQKLKSLHGICSDKEWMDAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYAKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVY ALDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELINQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCTQEHNPEPFPWWLNKFVVEHTLDACPLRADDLIPTGEGEIFVS PFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRLRCDWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYERERGKKRRKVFAQEKLSEEEAELLVEADEAREKSVVLMRDPSGIINRGNWTRQKEFWSMV NQRIEGYLVKQIRSRVPLQDSACENTGDI

[0098] CasX (uniprot.org / uniprot / F0NN87;uniprot.org / uniprot / F0NH53) >tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx protein OS = Sulfolobus islandicus(strain HVE10 / 4) GN = SiH_0402 PE=4 SV=1 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRRVKLE VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0099] >tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS = Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4 SV=1 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAK VSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0100] Deltaproteobacteria CasX MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTT AGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDI IIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVT AEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQK WYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLS LETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASK SRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEY QITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0101] CasY (ncbi.nlm.nih.gov / protein / APG80656.1) >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacteriaium] MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPREIVSAINDDYVGLYGLSNFDDLYNAEKRNEEKVYSVLDFWYDCVQYGAVFSYTAPGLLKNVAEVRGGSYELTKTLKGSHLYDELQIDKVIKFLNKKEISRA NGSLDKLKKDIIDCFKAEYRERHKDQCNKLADDIKNAKKDAGASLGERQKKLFRDFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNRNRGEVLFNKLKEYAQKLDKNEGSLEMWEYIGIGNSGTAFSNFLGEGFLGRLRENKITELK KAMMDITDAWRGQEQEEELEKRLRILAALTIKLREPKFDNHWGYRSDINGKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEMINRFGESDTKEEAVVSSLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDVQE ALIKERLEAEKKKPKRKKSDAEDEKETIDFKELFPHLAKPLKLVPNFYGDSKRELYKKYKNAAIYTDALWKAVEKIYKSAFSSSLKNSFFDTDFDKDFFIKRLQKIFSVYRRFNTDKWKPIVKNSFAPYCDIVSLAENEVLYKPKQS RSRKSAAIDKNRVRLPSTENIAKAGIALARELSVAGFDWKDLLKKEEHEEYIDLIELHKTALALLLAVTETQLDISALDFVENGTVKDFMKTRDGNLVLEGRFLEMFSQSIVFSELRGLAGLMSRKEFITRSAIQTMNGKQAELLYIPHE FQSAKITTPKEMSRAFLDLAPAEFATSLEPESLSEKSLLKLKQMRYYPHYFGYELTRTGQGIDGGVAENALRLEKSPVKKREIKCKQYKTLGRGQNKIVLYVRSSYYQTQFLEWFLHRPKNVQTDVAVSGSFLIDEKKVKTRWNYDALTV ALEPVSGSERVFVSQPFTIFPEKSAEEEGQRYLGIDIGEYGIAYTALEITGDSAKILDQNFISDPQLKTLREEVKGLKLDQRRGTFAMPSTKIARIRESLVHSLRNRIHHLALKHKAKIVYELEVSRFEEGKQKIKKVYATLKKADVYSE IDADKNLQTTVWGKLAVASEISASYTSQFCGACKKLWRAEMQVDETITTQELIGTVRVIKGGTLIDAIKDFMRPPIFDENDTPFPKYRDFCDKHHISKKMRGNSCLFICPFCRANADADIQASQTIALLRYVKEEKKVEDYFERFRKLKN IKVLGQMKKI

[0102] The term "Cas12" or "Cas12 domain" refers to a Ca12 protein or a fragment thereof (eg, a protein containing an active, inactive, or partially active DNA cleavage domain of Cas12 and / or a gRNA-binding domain of Cas12). refers to RNA-induced nucleases containing Cas12 belongs to the Class 2, V-type CRISPR / Cas system. Cas12 nucleases may also be referred to as CRISPR (clustered regularly interspaced short palindromic repeat) binding nucleases. An example Bacillus hisashii Cas 12b (BhCas12b) Cas12 domain sequence is provided below: MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVD PNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYE KERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHT EKLKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKG KKLKSGIESLEIGLRVMSIDLGQRQAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELM YKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTE PGEVRRLEPGQRFAIDQLNHLNALKEDRLLKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEER SRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDG QTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSKQSMKRPAATKKAGQAKKKK.

[0103] Amino acid sequences with at least 85% or higher identity to the BhCas12b amino acid sequence are also useful in the methods of the present invention.

[0104] The terms "conservative amino acid substitution" or "conservative mutation" refer to the replacement of one amino acid with another amino acid having common properties. A functional method for defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins in homologous organisms (Schulz, G. E. and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag, New York (1979)). Such analysis allows the groups of amino acids within a group to be preferentially exchanged with one another, and therefore can be defined where they are most similar to each other in their influence on the overall protein structure (above) Schulz, G. E. and Schirmer, R. H.). Non-limiting examples of conserved mutations include, for example, lysine from amino acids arginine which can maintain a positive charge; aspartic acid from glutamic acid and vice versa; free -OH maintained threonine is threonine to serine; and free NH 2 Amino acid substitutions such as glutamine can be used to maintain asparagine.

[0105] As used interchangeably herein, the term "coding sequence" or "protein coding sequence" refers to a segment of a polynucleotide encoding a protein. This region or sequence is bounded by the start codon near the 5' end, and the region near the 3' end is bounded by the stop codon. The coding sequence is also called an open reading frame.

[0106] As used herein, the term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In one embodiment, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (eg, genetically engineered adenosine deaminases, evolved adenosine deaminases) can be from any organism, such as a bacteria. In some embodiments, the adenosine deaminase is derived from bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influencezae, or Caulobacter crescentus.

[0107] 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 a human, chimpanzee, gorilla, monkey, bovine, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is not naturally present. 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. For example, the 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 hereby incorporated by reference in its entirety. is incorporated into. Also, the contents of Komor, A.C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 ( 2016), Gaudelli, N.M., et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017), Komor, A.C., et al., “Improved base surprise repair inhibition and bacteriaophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), and Rees, H.A., et al., “Base editing: precision chemistry on the genome and transcriptome of living cells." Nat Rev Genet. 2018 Dec;19(12):770-788. doi: See also 10.1038 / s41576-018-0059-1.

[0108] "Detection" refers to identifying the presence, absence or amount of an analyte to be detected. In one embodiment, sequence changes in the polynucleotide or polypeptide are detected. In another embodiment, the presence of an indel is detected.

[0109] A "detectable label" is a composition that, when linked to a molecule of interest, makes the latter detectable via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. It means. Useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, high electron density reagents, enzymes (for example, those commonly used in ELISA), biotin, digoxigenin, or hapten. Included.

[0110] "Disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue or organ. An example of a disease is glycogen disease type 1 (also known as GSD1 or Von Gierke's disease). In some embodiments, GSD1 is type 1a (GSD1a).

[0111] "Effective amount" means the amount required to ameliorate symptoms of the disease compared to untreated patients. The effective amount of the active compound(s) used to practice the invention for the therapeutic treatment of a disease will vary depending on the administration mode, age, weight of the subject, and overall health status. Ultimately, your attending physician or veterinarian will determine the appropriate amount and dose. Such an amount is called an "effective" amount. In one embodiment, the effective amount is the amount of the base editor of the present invention sufficient to introduce the modification into a gene of interest (eg, G6PC) within a cell (eg, in vitro or in vivo). In one embodiment, an effective amount is the amount of base editor necessary to achieve a therapeutic effect (eg, reducing or controlling GSD1a or its symptoms or conditions). Such therapeutic effects need not be sufficient to alter G6PC in all cells of the subject, tissue or organ, and approximately 1%, 5%, 10% of the cells present in the subject, tissue or organ; Just change the G6PC at 25%, 50%, 75% or more. In one embodiment, the effective amount is sufficient to ameliorate one or more symptoms of GSD1a.

[0112] "Fragment" means a portion of a polypeptide or nucleic acid molecule. This portion includes at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference nucleic acid molecule or polypeptide. The fragments may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0113] "Glucose-6-phosphatase (G6PC) polypeptide" means a polypeptide or fragment thereof having at least about 95% amino acid sequence identity to NCBI accession number AAA16222.1. In a particular embodiment, the present invention provides methods for editing G6PC polynucleotides containing single nucleotide polymorphisms (SNPs) associated with glycogen disease type 1a (GSD1a). In one embodiment, alteration from A·T to G·C in the SNP associated with GSD1a changes glutamine (Q) to a non-glutamine (X) amino acid in the G6PC polypeptide. In another embodiment, alteration from A·T to G·C in the SNP associated with GSD1a changes arginine (R) to non-arginine (X) in the G6PC polypeptide. In one embodiment, the SNP associated with GSD1a results in expression of a 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, base editor correction replaces glutamine at position 347 with a non-glutamine amino acid (X). In another embodiment, base editor correction replaces the arginine at position 83 with a non-arginine amino acid (X). In certain embodiments, the G6PC includes one or more modifications compared to the following reference sequence: In certain embodiments, the G6PC associated with GSD1a comprises one or more mutations selected from Q347X and R83C. Exemplary G6PC amino acid sequences from Homo Sapiens are provided below: 1 MEEGMNVLHD FGIQSTHYLQ VNYQDSQDWF ILVSVIADLR NAFYVLFPIW FHLQEAVGIK 61 LLWVAVIGDW LNLVFKWILF GQRPYWWVLD TDYYSNTSVP LIKQFPVTCE TGPGSPSGHA 121 MGTAGVYYVM VTSTLSIFQG KIKPTYRFRC LNVILWLGFW AVQLNVCLSR IYLAAHFPHQ 181 VVAGVLSGIA VAETFSHIHS IYNASLKKYF LITFFLFSFA IGFYLLKGL GVDLLWTLEK 241 AQRWCEQPEW VHIDTTPFAS LLKNLGTLFG LGLALNSSMY RESCKGKLSK WLPFRLSSIV 301 ASLVLLHVFD SLKPPSQVEL VFYVLSFCKS AVVPLASVSV IPYCLAQVLG QPHKKSL

[0114] "Glucose-6-phosphatase polynucleotide" means a polynucleotide encoding a G6PC polypeptide. Exemplary G6PC nucleotide sequences from Homo Sapiens are provided below (GenBank:U01120.1): 1 ATAGCAGAGC AATCACCACC AAGCCTGGAA TAACTGCAAG GGCTCTGCTG ACATCTTCCT 61 GAGGTGCCAA GGAAATGAGG ATGGAGGAAG GAATGAATGT TCTCCATGAC TTTGGGATCC 121 AGTCAACACA TTACCTCCAG GTGAATTACC AAGACTCCCA GGACTGGTTC ATCTTGGTGT 181 CCGTGATCGC AGACCTCAGG AATGCCTTCT ACGTCCTT CCCCATCTGG TTCCATCTTC 241 AGGAAGCTGT GGCATTAAA CTCCTTGGG TAGCTGTGAT TGGAGACTGG CTCAACCTCG 301 TCTTTAAGTG GTAAGAACCA TATAGAGAGG AGATCAGCAA GAAAGAGGC TGGCATTCGC 361 TCTCGCAATG TCTGTCCATC AGAAGTTGCT TTCCCCCAGGC TATTCAGGAA GCCACGGGCT 421 ACTCATGCTT CCAACCCCCTC TCTCTGACTT TGGATCATCT ACATAAAGGG GGAAGACAGA 481 AAAATCCTA CCAGTGAGTT GAAATACAG GAAAGCCTAT TTCATATGGG TTAAAGGGTA 541 GGACAGTTGA ATTTCGTGAA AAGTCTGAGT TATATAGGCT TTGAGCAAAG AGTTTTATTA 601 GTATGAAGCA GAAGAGGTAA CATAAGAAA GATGTATGGGGGCCAGGCATG GTGGCTCACA 661 CCTGTAATCC CAGCACTTTG GGAGGCCGAG GTGGGCGAAT CACTCCTGGG TGAACTCAGG 721 AGTTCAAGAC CAGCCTGGGC AACATGGCGA AACTCCATCT CTACAAAAAC ATTACGAAAAA 781 TTAGCTGGGC GTGTTGGTGC TGTAGTCCCA GCTACTCAGG AGGCTGAGGT GAGAGGCGGA 841 GGAGGTTGCA GTGAGTCAAG ATCATGCCAC TGCACTCCAG CCTGGGCAAC AGAGTAAGAC 901 CCTGTCTCAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACACAC 961 AGATGATTCA ACAGCCTGTT TTGTGGGTA ATGAAAAGTC ACCCTGGGAA CTGGGCTCCA 1021 GCCTCGTTC TGCCACCCAC CAACTACATG TCCTTGGCAA GTCATATCAA TTATCTGAGT 1081 TTCTGTTTTA TAATCTACAA ATAGGTTATC TCTGGCAGCT TAATATAAT CAGGGTTAAC 1141 ATTTATTAAA CAGTGTGTGC CAGTCCATGT GCTATGTGCT TTTCTGTGAG GTAGTTACTG 1201 CTATTTACAG AAACAGTAGA TGCAGAGACC AAGGTGCTGA GTTAAATGAT TAGGCCAACA 1261 AGGTTAGTAC ATGCCGAGCC AGGATGGAAG CCCAGGTAGG CAGGCTGGCT TCCGCGCAA 1321 TGCTCTTATG AACTATGTTA CGTCCAGTGC TGATAAACTG ACTCTCTGGGGGA 1381 AAGCCCTGAG TTTAGCATTT GCCAATTTCT ATCACGTAAA CATTCCCATT CTGGCCACTT 1441 TCTTTCTTTC TTTCTTTGT TTGTTGT GAGATGGAGT CTCGCACTGT TGCCTGGCTG 1501 GAGTGCAATG GTGCAATCTC AGCTCACTGC AACCTCTGCC TCTCCGGTTC AAGTGATTCT 1561 CCTGCCTCAG CCTCCCAAGT AGCTGGGATT ACAGGTGCCC GCCACCATGC CCAGCTAATT 1621 TTTTTTGTAT TTTTAGTAGA GACATGGTTT CACTATGTTG ACTAGGCTGG TCTCGAACTC 1681 CTGACCTCAT GATCTGCCTG CCTTGGCCTC CCTAAGTGCT AGGATTACAG GCGTGAGCCA 1741 CTACACCCAG CCGCATGATT CTAAAAAATA AAAGATGAA GTGTTATCC AAACATCTGA 1801 TCTCCATTGA AGAACCATGC AATCTCTCTG GGTTGATAGA GGCCAGAGTT AGTGGCTCTC 1861 CCTGATTTCG GTGAGAAATC ACTATTCCAC CATCACGGGA TAAAGGCAT CCTGACTGGC 1921 GGTTGACACC TATTTCCACA GTGAAAGATA TATCTAGTAC TTTTAAAGGG GAAGTGGTTT 1981 GTCTGAGATA CTCTGTTTCA AAGTAGAGAG GATACAGAAC AAGCATCTGA AGCTATATAC 2041 ATCCTTACAG AGAGCAATTC TGATGGAAAT GCAGGCCATG TTTCCCTGGGGGCTCGT 2101 CCTAGGGCT GGAGTGCATT CTCTGATGTC AGAGGAAATG CAAGATTCCC TGAGGCCTGA 2161 GGGAACCCAT GGTATATGCA AGTCCAAGTT TCAAACTGTA GTTCCATATG CATTCTTCCA 2221 GGACAAATAC TTCTTGAGGT TAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGTCACATA GCTGCCATTT TATGGATTTC 2281 AGGATTTT TTTTTT TTTGAGATGG AGTCTTGCTC TGTCACCCAG CCTGTAGTGC 2341 AGTGGCATAA TCTCGGCTCA CGGCAACCTC CGCCTCCCCAG GTTCAAGCGA TTCTCTTGCC 2401 TTAGCCTCCC GAGTAGCTGG GATTACAGTC ACGCACCACC ACATCTGGCT AATTCTTTAT 2461 ATTTTTTGGT AGAAACGGTG TTTCACCATG TTGGCCAGGC TGGTCTCAAA CTCCTGACCT 2521 CATGTGATCT GCCTGCCTTG GCCTCCAAA GTGCTGAGAT TACAGGTGTG AGCCACCGCG 2581 CCTGCCTGGA GTTCAGAATC TTGGGCTTCA TTATTTGTGT TTAAATAGAT CATACAGTCA 2641 GGCACGGTGG CTCATGCCTG TAATCCCAGC ACTTTGGGAG GCTGAGGTGG GAGATTGCC 2701 TGAGTTCAGG AGATGGAGAC CAGCCTGGGC AACATGGTGA AACCCCCGTCT CTACTAAAAA 2761 TACAAAAACT AGCTGGATGT GGTGGCACAC ACCTGTAGTC CCAGCTATTC AGGAGGCTGA 2821 GGTGGGAGGA TCCCAGGAGG TAGAGGTCAC AATGAGCCGA GATTGCGCCA CTGCACTCCA 2881 GGCTGGGTTA CTGAGCCAGA TCCTGTCTCA AAAAAAAAAAAAGATAATACA TTCAAACAGT 2941 TCAAAATGCA AAAGTTACAT ACATAAGGAA GTGTCATGAA ATATCTCCCCT CTCACACCTTC 3001 TCCCCAGCCA CCCAGTTCTC CCTTCTAGAG GCAACATGTG AAATCCTTCT CAGGCTACAC 3061 TCTTCTTGAA GGTGTAGGCT TTGGCAAAA GCATTCATTC AGTAACCCCA GAAACTTGTT 3121 CTGTTTTCC ATAGGATTCT CTTTGGACAG CGTCCATACT GGTGGTTTT GGATACTGAC 3181 TACTACAGCA ACACTTCCGT GCCCTGATA AAGCAGTTCC CTGTAACCTG TGAGACTGGA 3241 CCAGGTAAGC GTCCCAGCCC CTGCAGACAG AAGCTGAGTG GACCTCGTTT ACCTGTTATG 3301 GATGAAACTG ACCTTGAGGG GACATGAGGA GAGCCATTCC TTTGTACTTT TGTCATGCTC 3361 TTCAATTGGC ACAAATTAAT TCACTTCTGC AATACTTCC TGAATAGCAC AGTAGTATTG 3421 GAAATCTGCC TATTACAGAA CCTGGATGGA GTCCAGAGAG GCACGGGCAT CCATGGGCAA 3481 AGGGCTCGTG AGAGTCACCG CCCTGCAGCG CTGTGTCCTG AGAAAGGAGG GGCAGAAGC 3541 CTGAGCTTCT GGGGGTCCT CCCAATGGCC TGCCCACTG GATGTGCCCT CCTGAGCTGA 3601 CCGTCCAATC CCTTGCCTC TCTGTGCCTA CGTTTTATTA GTTACAGCCA GATGGTTACT 3661 GTCAAATCAA ATGATAGATT TCATTTTCAG TATGTAATAG GAAGCCCCTC CCTCACCCTA 3721 AAGTCTCAGC TGCCCTCTAA GACTAGTACT CTCTAAGGTA CTAGTATCCC TTCCCAGAG 3781 ACCCTTTCCC TGACCCCAAA ACTAGGGAAG GTCCCCTTAGT TATTTGCTCT CACAGACCAC 3841 GCATTACCT CAGAGCATAT TCACTCATTC AGCTGTTACT TACCAAGCAC CTACTGGGAG 3901 CTATACACTG TTCTATGTGC TAGGGATACC TCTGTCAGTG AACAACACAG ACAAAAGAT 3961 CCCTGCCTT GTGGAGCTGA AATCTGAATA GAGGAGGTGA AATATACAA AATTATAATA 4021 AATAAGTAAA CTAGGCCAGT TGTGGTTGCT CATGCCTGTA ATCCCAGCAC TTTGGGAAGC 4081 CAAGGTAGGT AGATCACCTG AGGTCAGGAG TTCAAAACCA GCCTGGCCAA CATTGCAAAAA 4141 TCCTGTCTTT ACTAAAAATG GAAAATTGG TCAGGCGTGA TGCACACGC CTGTAGTCTC 4201 AGCTACCTGG GAGGCTGAGG CAGGAGAATC GCTTGAACCT GGGAGGCAGA GGTTGCAGTG 4261 AACCGAGATC GGACCACTGC ACTCCAGCCT GAATGACAGA ACGACTCT GTCTCAAAAA 4321 AAAAGTAAAC TATTAATG TAGGATAGGC CAGCACGGT GGCTCACCCT GTAATCCCAG 4381 CACTTTGGGA GGCTGAGGCG GGTGGATCAC CTGAGGTGAG GAGTTCAAGA CCAGCCTGGC 4441 CAACATGGCA AACCCTGTC TCTACTAAAAA ATACAAAAT TAGCTGGGTG TCCTGGTGCA 4501 TGCCTGTAAT CTGAGCTACT CAGGAGGCTA AGGCAGGAGA ATCGCTTGAA CCTGGGAGGT 4561 GGTGAGCCAA GATTGCGCCA TTGCACTCCA GCCTGGGCGA CAAAATGAGA CACCATCTGA 4621 AAAAAAAAAA AAAATATATA TATATATACA CACACACACA CACACACACA CACACACACA 4681 TATAATACTA GAAATGATT GTTTATAGGC AAAAAAAAAAAAGAAGA AGAAGAAGAAGAA 4741 AAGGAAAGGA GAAGGAAAGA AGGACCAAAC ATCTTTTGTA GAAATATGTT TGCTTTCATC 4801 ATAACAGCTT GTTATCAAGG ATGAATTTCT CCCTGAAATT AATGGAGGCA CAGACTGGAA 4861 AGTTTAAAGT GGCTTTAAGA GGTTATTTTA TTTAGTCCTC TGTCTTAATA GAAGCAAATT 4921 ATTATCTG CTCCCTTAGGT AGAGTAGCTA AGGCTCAGAA AGTAGGCCGGGGCGCGTGGC 4981 TCACGCCTGT AATCCTAGCA CTTTGGGAGG CCAACGCAGG TGGATCACCT GAGGTCAGGA 5041 GTTGAGACC AGCCTGGCCA ACATGGTGAA ACCTCGTCAC TAATAAAAAAAAAAC 5101 TTAGCCAGGC ATGGTGGCGGGCGCCTGTAA TCCCAGCTAC CCAGGAGGCT GCGGCAGGAG 5161 AATCACTTCA ACCCGGGAGG CAGAGGTTGC AGTGAGCTGA AATCAACACCA CTGCACTCCA 5221 GCCTTGGTGA CAGAGAAAGA TTCTGTCAGG AAAAAAAAAAAAAAAGTTTAA ATGAATTACC 5281 CAAGGTATAT AATTGTTAGT GTTAGAAGGA AGAGAAGGG AGGGAAG GAAGGGAGAA 5341 AGAAAGGGAA GGAGGAAGG AGGGAGGAA GAAGCCTTT ATTTATCTAT GGGTTCCCT 5401 GGAAAGCAGG CTGAAATGGA GATTCACGTG CAGGAGTTTA GATACTCTGG GGAACTATAC 5461 TTGTAGAAGG GAAGGAACAGGCA GAAGGAGGCA GAAGGAGAGG TCCGGTTGTG ATTCTGCCTC 5521 ATCCAACCCC ACAGCGAGCT CTGAAGCTGGGATGGCTCC TCAGAGTTGG TCCAAGTTGG 5581 GACAAGGGAA TCAGACCCTG GGGAGAGCGT AACCTTGATC AAGGCGACTC TCTTTAGCCC 5641 AGGGCAATGC CAGGAGAAGG CTGAGAGCAG AAGCCATCT ACCATCACAC TCTCAACAGC 5701 TACGAAATAA GTCCTGCAGT TCAGGAGGGA GGTCTGGGCG GCACATCTCA GGACCCTCTA 5761 TCTCTCAGGG TAGAGGAATT AAGAATGGGA TGGGAACCAG ACGGGCCATG GTGGCTCACA 5821 CCTATAATCC CAACACTTTG GGAGGCCAAG GGTAGGAGGA TTGCTTGAGC CCAAGAGTTC 5881 AAAACCAGCC TGGGCAAAAA CAATCAAACAAAAAACAAAAACACATTTAA AAAATTTGCT 5941 GTGTGTGGTGCACCT GTGGTCCCAG CTACTCAGGGGCTGAGGTGGGAGATTGC 6001 TTGAGTCCAG GAGGTCGAGG CTGCAGTGAG CTATGATCAT GGCACTGCAT TGCAGCCTAG 6061 GAGACAAAGC AAGACACTGT CTCTAAAAAAA ACAAAAAACATAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAC 6121 CGGTTGCAAG CAGGGTTAAA TAGCGTGGTC AGAGTAGGAC TCACTGAGAA TATGAGATCT 6181 GAGTCAAGTC TTCAAGGATG TGAGGAAGTA AGTTTCTGGC AGAAGAGCTG TGAAGGGCTG 6241 TCTGGCCAGA GAAGATTGCA ATGCAAAAGC CCTGAGGTGG GAACGTGTTT GGTGTGTTTA 6301 AAGGAAAGCA ATGAGGCCAG TGTAGCCAGA ACAGAGTGTG CAAGGAGAGA AGGAACAGAA 6361 GATGTGGAGG GCAGATCAGT TTGTAATTGT ACGCCCAGTA TGCTGATTCT TTGTGTAATC 6421 TCCAGACTGT ATTAAACTGC AAGAGCAGGG CCCCTCTCTG GCTTTGCTCA TCATTGTATT 6481 CCCAGAGCCT TGCACAATGC TTGGTGCATA GGAGATGGAA ATTTGTTAAA TAAATGAATT 6541 ATGGATAACG AATGGATGGT AAGATGGGTG GATGGGGTGAACGG ATGGGG 6601 GGTGAATGGA TGGATGAATG GGTAGATGGG TGGATAGGGGGGGGGGGGGGCTGGGTA 6661 GATGATGCAC TGTCTCCCAG ATGAGGACCT TTTCACCTTT ACTCCATTCTGCC 6721 CTTTAGGGAG CCCCCTCTGGC CATGCCATGG GCACAGCAGG TGTATACTAC GTGATGGTCA 6781 CATCTACTCT TTCCATCTTT CAGGGAAAGA TAAAGCCGAC CTACAGATTT CGGTAAGAAC 6841 TCACCACTGG GGTGTAGGTGGGCA GGAGGCAGCT CTCTCTGTAG CTGACACACC 6901 ACGTATTCTT CCTCACATCC CCCTAGCCG CTCCCACACC TGGGCAGCCG CTGATTAAGA 6961 GTTGTGGCAC TTTGGATAGG GATAAACCTC AGAGTCAGGG AATGTTGG CTGAAAGGGA 7021 TCCAGTAGTG CAATCCGTTG TTTACAGAT AAGGAAACAA AGCCCAACAC CATGAAGGGA 7081 CTTATAAAAA TAAGGTAGTG AAGTAGCAGC AGGGCTTAAA TAAAACCCA TGTCTGTACC 7141 AACCAGAG TCACCCATCC AGGTTAAAAT AACCAGAGAGAA ACAGAAGATA TTCCTACTAC 7201 AGAGAATTCC GGGTGTGCAG CCACAGTGCA AATCCTTTT ATTTT TTGAGATGCA 7261 GTCTCGCTCT GTCATCCAGG CTGAAGTGCA GTGGCACGAT CATGTCTCGC TGCAACCTCT 7321 GCCTCCCAGG CTCAAGCGAT CCTCCACCT CAGCCATCTG AGTAGCTGGG ACCACAGGCC 7381 ACACACCA CCCAGCTAAT TTCTCGTATC TTTTTGTAGA GACAGAGTTC TGCTATGTTG 7441 CCCAGGCTCA GGCTGGTCTT GATCTCAAGC AATTGGCTTG CCTCAGCCTC CTAAATATT 7501 GGGATTACAG GCATGAGCCA CCGCGCCAGC CATGCAAATC CTTAATTATC AAACAGATAA 7561 AATAGGGAAG TTAAAATTCA TATACACAAG GGTTAACCAC TTGCCACAGG CATTTTTTTT 7621 TTTTTTGA GACGGAATCT CGCTCTGTTG CCCAGGCTGG AGTGCAGTGG CGCCATCTCG 7681 CCTCACTGCA ACCTCCGCTT CCTGGGTTCA AGCTATTCTT CTGCCTCAGC CTACCGAGTA 7741 GCTGGGACTA CAGGCACGTG CCACCACC TGGCTAATTT TTTTTT AGTAGAGATG 7801 GGTTTCACC ATATTGGCCA GGCTGGTCTT GAACTCCTGA CCTAGTGATC CATCCGCCTC 7861 AGCCTCCCAA AGTGCTGGGA TTGCAGGCAT GAGCCACCGC GCCTGGCCTT TTTTTTTTTT 7921 TTTTGAGACG GAGTTTTGCT CTTGTTGCCC AGGCTAGAGT GCAGTGGCGC AGTCTCGGCT 7981 CACTGTAACC TCCACCT GAGTTCAAGC AATTCTCCTG CCTCAGCCTC TCAATAGCT 8041 GGGATTACAG GCGTGAGCCA CCCCACCTGG CTAATTTTTGT AATTTTTTT TTAGTAGAGA 8101 TGGGTTTCA CCTGTTGATC AGGCTGGTCT CAAACTCCTG ACCTCAAGTG ATCACCCAC 8161 CTCGGCCTCC CAAAGTGCTG GGATTACAAG CATAAGCCAC CGTGCCTGGT CAATTTGAT 8221 CTTTTTTAAA GAGACAGGGG TCTTGCTATG TTGCCCAGAC TAGTCTTGAA CTCCTGGCCT 8281 CAAGTGATCC TCTCACCTCG GCCTCCAAA GTATTGGGAT TACAGGTCTG AGCCGCTGCA 8341 CCCAGCCCC AACAGGCATC TTTGGACTTT TGAGTACTGG CTTTAATTTA CAAAAATTCC 8401 ACTGAGAGCA CCTAAGTTG CCAGGCTCCA ACATTTCTGC AGGGCTGTT TTCTTTGCTG 8461 AAGGATCTGC ACCTGTGTTC TGTTATGGTT GCCTCTTCTG TTGCAGGTGC TTGAATGTCA 8521 TTTTGTGGTT GGGATTCTGG GCTGTGCAGC TGAATGTCTG TCTGTCACGA ATCTACCTTG 8581 CTGCTCATTT TCCTCATCAA GTTGTTGCTG GAGTCCTGTC AGGTATGGGC TGATCTGACT 8641 CCTTCCTTC TCCCCAAAC CCATTCCGT TTCTCTCCCT AATCAGGACA AAATCCCAGC 8701 ATTCCAGCCA CATCCTGTGTGT GTAATCAGTA CTGTTAGCAT TTCTGTGGGT TGAAAGTCAA 8761 GAATGAGCAA CTTGAAATGA TTAATTTTCTA TAAGAGTGCC CAGATCTATA GAATGAATTG 8821 TGTAGAAGTT ACCATACATC AAATTAACGC ACCAAATTGA ATTAGCTTGA AATCTCAGAG 8881 CTTTTACAA TCTTTATTC TTACTGGTCT TCAACAGGCC CTAATTTACT TTTCAGGGAA 8941 TCTGCCAAAT TTAACAAATT AACACGATGT CCTAGGAAAG CTGTTCATTT AAATACATTC 9001 ATTTGCAAAC CTAATAGATA ACTGCAGTTG ATCTCTTTA TAGGTTCAGA GTTTTGAATA 9061 TGTTTTTT TGTTTTTT TTTTGAGATG GAGTCTCGCT CTGTGACCCA GGCTAGAGTG 9121 CAGTGGTGCG ATCTCGGCTC ACTGCAAGCT CCACCTCCTG GGTTCACGCC ATTCTCCTGC 9181 CTCAGCCTCT CCGAGTAGCT GGGACTACAG GCGCCCGCCA CCATGCCCGG CTAATTTTTTT 9241 GTATTTTTAG CAGAGACGGG GTTTCACCGT GGTCTTGATC TCCTGACCTC GTGATCCGCC 9301 CGCCTCGGCC TCCCAAAGCG CTGGGATTAC AAGGGTGAGC CACCGCACCC TGCCTGAATA 9361 TGTGTTTTCT TAGATCCAAT TAACAAGGGT AAGACAAGAT TTAAGTTAAG CATAAGAAAG 9421 ATTTTGTGG AGGCACTGGA ATATAAGACC TTAACAAAAC TGTGGAATTT CTCCCCCTGGA 9481 GATTTGTAAG AACGGAACAT AGCAGCATTC AAAGAAGAAT GTTGAGAACA AGGGAGATAA 9541 TGGTTTCATG GTAATCACAA AAGTAACACA GCATTTAGTA CTGGGTTCCA TGTTGAGGA 9601 AGAACCTGGA AGCCATATCA CATGAAAAAC CTGGGAATGT TTAGGTTAGA GAGAATAACT 9661 GTGTTCAAAT GTGTGACAGA GGGACTAGAT TCATCACTTA CTAACTCCTG CAGAAAGAAC 9721 TGAGAAAAAT AGACAGTATT AGAGGGGGAC CAGTTTCACA CAGACAAGGA AGAACTATTC 9781 AGCAATCAAT TCCGTTCAAA GATAAAATGG ACTGTTATAG TGGGGTGAG CTCCCTACCT 9841 CTGAGGGTAT TTCAAGTAGA GATAGGAGGA CCTCCTGGTA GGAAATTTGC ATACGGTGGG 9901 AGATTGTACG TGATATGGCA CCTCCATCTG AAAGAGTCTA TATTGAGGGC AGGCTGGAGT 9961 CACACATGGG AATAAGCCAG GCGACCCTCC CATCTGCCAT CTGTGATTTA ATTCCACAGT 10021 CGCAGAACGG ATGGCATGTC ACCCACTCCT CCAAACCCAC CTCTAGCAAA GGTCCCAAAT 10081 CCTTCCTATC TCTCACAGTC ATGCTTTCT CCACTCAGGC ATTGCTGTTA CAGAAACTTT 10141 CAGCCACATC CACAGCATCT ATAATGCCAG CCTCAAGAAA TATTTTCTCA TTACCTTCT 10201 CCTGTTCAGC TTCGCCATCG GATTTTATCT GCTGCTCAAG GGACTGGGTG TAGACCTCCT 10261 GTGGACTCTG GAGAAAGCCC AGAGGTGGTG CGAGCAGCCA GAATGGGTCC ACATTGACAC 10321 CACACCCTTT GCCAGCCTCC TCAAGAACCT GGGCACGCTC TTTGGCCTGGGCTGCTCT 10381 CAACTCCAGC ATGTACAGGG AGAGCTGCAA GGGGAAACTC AGCAAGTGGC TCCCATTCCG 10441 CCTCAGCT ATTGTAGCCT CCCTCGTCCT CCTGCACGTC TTTGACTCCT TGAAACCCCC 10501 ATCCCAAGTC GAGCTGGTCT TCTACGTCTT GTCCTTGC AAGAGTGCGG TAGTGCCCT 10561 GGCATCCGTC AGTGTCATCC CCTACTGCCT CGCCCAGGTC CTGGGCCAGC CGCACAAGAA 10621 GTCGTTGTAA GAGATGTGGA GTCTTCGGTG TTTAAAGTCA ACAACCATGC CAGGGATTGA 10681 GGAGGACTAC TATTTGAAGC AATGGGCACT GGTATTGGA GCAAGTGACA TGCCATCCAT 10741 TCTGCCGTCG TGGAATTAAA TCACGGATGG CAGATTGGAG GGTCGCCTGG CTTATTCCCA 10801 TGTGTGACTC CAGCCTGCCC TCAGCACAGA CTCTTTCAGA TGGAGGTGCC ATATCACGTA 10861 CACCATATGC AAGTTTCCCG CCAGGAGGTC CTCCTCTC TACTTGAATA CTCACAAG 10921 TAGGGAGCTC ACTCCCACTG GAACAGCCCA TTTTATCTTT GAATGGTCTT CTGCCAGCCC 10981 ATTTTGAGGC CAGAGGTGCT GTCAGCTCAG GTGGTCCT TTTACAATCC TAATCATATT 11041 GGTAATGTT TTTGAAAAGC TAATGAAGCT ATTGAGAAAG ACCTGTTGCT AGAAGTTGGG 11101 TTGTTCTGGA TTTTCCCCCTG AAGACTTACT TATTCTCCG TCACATATAC AAAGCAAGA 11161 CTTCCAGGTA GGCCAGCTC ACAAGCCCAG GCTGGAGATC CTAACTGAGA ATTTTTCTACC 11221 TGTGTTCATT CTTACCGAGA AAGGAGAAA GGAGCTCTGA ATCTGATAGG AAAGAAGGC 11281 TGCCTAAGGA GGAGTTTTTA GTATGTGGCG TATCATGCAA GTGCTATGCC AAGCCATGTC 11341 TAAATGGCTT TAATTATATA GTAATGCACT CTCAGTAATG GGGGACCAGC TTAAGTATAA 11401 TTAATAGATG GTTAGTGGGG TAATTCTGCT TCTAGTATT TTTTTACTGT GCATACATGT 11461 TCATCGTATT TCCTTGGATT TCTGAATGGC TGCAGTGACC CAGATATTGC ACTAGGTCAA 11521 AACATTCAGG TATAGCTGAC ATCTCCTCTA TCACATTACA TCATCCTCCT TATAAGCCCA 11581 GCTCTGCTTT TTCCAGATTC TTCCACTGGC TCCACATCCA CCCCACTGGA TCTTCAGAAG 11641 GCTAGAGGGC GACTCTGGTG GTGCTTTGT ATGTTTCAAT TAGGCTCTGA AATCTTGGGC 11701 AAAATGACAA GGGGAGGCC AGGATTCCTC TCTCAGGTCA CTCCAGTGTT ACTTTTAATT 11761 CCTAGAGGT AAATATGACT CCTTTCTCTA TCCCAAGCCA ACCAAGAGCA CATTCTTAAAA 11821 GGAAAAGTCA ACATCTTCTCTCTCTCTCTCTCTCTTTTTTTT TTTTGAG ACAGGGTCTC ACTATGTTGC 11881 CCAGGCTGCT CTTGAATTCC TGGCTCAAG CAGTCCTCC ACCCTACCAC AGCGTCCCGC 11941 GTAGCTGGGA CTACAGGTGC AAGCCACTAT GTCCAGCTAG CCAACTCCTC CTTGCCTGCT 12001 TTTTTTT TTTCTTTTT TGAGACGGCG CACCTATCAC CCAGGCTGGA GTGGAGTGGC 12061 ACGATCTTGG CTCACTGCAA CCTCTTCCTC CTGGTTCAAG CGATTCTCAT GTCTCAGCCT 12121 CCTCAGTAGC TAGACTACC GGCGTGCACC ACCATGCCAG GCTAATTTTT ATATTTTTAG 12181 AATTTTAGAA GAGATGGGAT TTCATCATGT TGGCCAGGCT GGTCTCGAAC TCCTGACCTC 12241 AAGTGATCCA CCTGCCTTGG CCTCCCAAGG TGCTAGGATT ACAGGCATGA GCCACCGCAC 12301 CGGGCCCTCC TTGCCTGTTT TTCAATCTCA TCTGATATGC AGAGTATTTC TGCCCCACCC 12361 ACCTACCCC CAAAAAAAGC TGAAGCCTAT TTATTTGAAA GTCCTTGTTT TTGCTACTAA 12421 TTATATAGTA TACCATACAT TATCATTCAA AACACCATC CTGCTCATAA CATCTTTGAA 12481 AAGAAAATA TATATGTGCA GTATTTATT AAAGCAACAT TTTATTTAAG AATAAGTCT 12541 TGTTAATTAC TATATTTTAG ATGCAATGTG ATCTGAAGTT TCTAATTCTG GCCCAACTAA 12601 ATTTCTAGCT CTGTTTCCCCT AAACAATAA TTTGGTTTCCT CTGTGCCTGC ATTTTCCCT 12661 TGGAGAAGAA AAGTGCTCTC TCTTGAGTTG ACCGAGAGTC CCATTAGGGA TAGGGAGACT 12721 TAAATGCATC CACAGGGGCA CAGGCAGAGT TGAGCACATA AACGGAGGCC CAAAATCAGC 12781 ATAGAACCAG AAAGATTCAG AGTTGGCCAA GAATGAACAT TGGCTACCAG ACCAAAGTC 12841 AGCATGAGTT GCTCTATGGC ATCAAATTGC AACTTGAGAG TAGATGGGCA GGTCACTAT 12901 CAAATTAAGC AATCAGGGCA CACAAGTTGC AGTAACACAA CAAGACTAGG CCAGCTCTGG 12961 AATCCAGTAA CTCAGTGTCA GCAAGGTTTT GGGTTATAGT TCAAGAAAGT CTAAACAGAG 13021 CCAGTCACAG CACCAAGGAA TGCTCAAGGG AGCTATTGCA GGTTTCTG CTAAGAGATT 13081 TATTTCATCC TGGGTGCAGG GTTCGACCTC CAAAGGCCTC AAATCATCAC CGTATCAATG 13141 GATTTCCTGA GGGTAAGCTC CGCTATTTCA CACCTGAACT CCGGAGTCTG TATATTCAGG 13201 GAAGATTGCA TTCTCCTACT GGATTTGGGC TCTCAGAGGG CGTTGTGGGA ACCAGGCCCC 13261 TCACAGAATC AAATGGTCCC AACCAGGGAG AAAGAAATA GTCTTTT TTTTTAAT 13321 AGAGATGGGG GTCTCACTAT GCTGCCCAGG CTGGTCTTGA ACTCCTGGGT TCAAGTGATC 13381 CTCCTGCCTC AGCCTCCAA AGTGCTGGGA TTACAGTGTG AGCCACTGCG CTTGGCCAGA 13441 AATGGTTTTG ATCTGTCTGA ACTGAACCCT ACTGCTTAGG CATAGCCCA TCCTTGATAA 13501 TCTATTTGCT CCCAAGGACC AAGTCCAAGA TCCTTACAAG AAAGGTCTGC CAGAAAGTAA 13561 ATACTGCCCC CACTCCCTGA AGTTATGAG GTTGATAAGA AACATAACA GATAAAGTTT 13621 ATTGAGTGCT AACTTTA

[0115] "Guide RNA" or "gRNA" refers to a polynucleotide that can be specific for a target sequence and is capable of complexing with a polynucleotide-programmable nucleotide-binding domain protein (eg, Cas9 or Cpf1). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). A gRNA may exist as a complex of two or more RNAs, or may exist as a single RNA molecule. GRNAs that exist as a single RNA molecule are sometimes referred to as single guide RNAs (sgRNAs), whereas "gRNAs" are guide RNAs that exist as single molecules or as complexes of two or more molecules. is used interchangeably to refer to. Typically, gRNAs present as a single RNA species include (1) domains that share homology with the target nucleic acid (eg, directing binding of the Cas9 complex to the target); and (2) Cas9 proteins It contains two domains, domains that bind to In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and includes a stem-loop structure. For example, in some embodiments, domain (2) is a tracrRNA as provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated by reference herein. They are the same or homologous. Other examples of gRNAs (e.g. those containing domain 2) include US provisional patent application U.S.S.N.61 / 874,682 filed September 6, 2013, titled "Switchable Cas9 Nucleases and Uses Thereof" and September 6, 2013 It can be found in the US Provisional Patent Application No. 61 / 874,746 filed on the day, titled "Delivery System For Functional Nucleases," the entire contents of each of which are incorporated by reference in its entirety. In some embodiments, the gRNA includes two or more of domains (1) and (2) and may be referred to as "extended gRNA." The extended gRNA binds to two or more Cas9 proteins and binds to the target nucleic acid in two or more clearly different regions, as described herein. The gRNA contains nucleotide sequences complementary to the target site, which mediate the binding of the nuclease / RNA complex to the target site, providing sequence specificity of the nuclease:RNA complex. As will be appreciated 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 the DNA polynucleotide sequence. In RNA, uracil base pairs with adenine and replaces thymine during DNA transcription.

[0116] "Heterodimers" are, for example, variants of the wild-type TadA domain and TadA domain (e.g., TadA*8) or two variant TadA domains (e.g., TadA*7.10 and TadA*8 or two TadA*8 domains), It refers to a fusion protein containing two domains.

[0117] "Hybridization" refers to hydrogen bonds between complementary nucleobases and can be Watson-Crick, Hoogsteen or inverse Hoogsteen hydrogen bonds. For example, adenine and thymine are complementary nucleobases that form hydrogen bonds to form pairs.

[0118] The term "inhibitor of base repair" or "IBR" refers to a protein capable of inhibiting the activity of a nucleic acid repair enzyme, such as a base excision repair (BER) enzyme. In some embodiments, the IBR is an inhibitor of inosine base excision repair. Exemplary inhibitors of base repair include APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4PDG, UDG, hSMUGl, and hAAG inhibitors. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is a catalytically inert EndoV or a catalytically inert hAAG. In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, the base repair inhibitor is a catalytically inactive EndoV or a catalytically inactive hAAG.

[0119] In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI refers to a protein capable of inhibiting the base excision repair enzyme of uracil-DNA glycosylase. In some embodiments, the UGI domain comprises wild-type UGI or a fragment thereof. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to the UGI or UGI fragment. In some embodiments, the base repair inhibitor is an inhibitor of inosine base excision repair. 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 inosing lycosylases (eg, alkyl adening lycosylases (AAG)) can bind to inosine, but create an abasic site. neither can remove inosine, thereby blocking newly formed inosine portions from the DNA damage / repair mechanisms sterically. In some embodiments, the catalytically inactive inosine-specific nuclease is capable of binding to inosine in the nucleic acid, but does not cleave the nucleic acid. Non-limiting examples of representative catalytically inactive inosine-specific nucleases include catalytically inactive alkyl adenosinglycosylases (AAG nucleases) (eg, from humans) and (eg, from E. coli) ) catalytically inactive endonuclease V (EndoV nuclease). In some embodiments, the catalytically inactive AAG nuclease comprises an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0120] "Increase" means a positive change of at least 10%, 25%, 50%, 75%, or 100%.

[0121] "Intein" is a fragment of a protein that can excise itself and link the remaining fragments (exteins) with peptide bonds in a process known as protein splicing. Inteins are also known as "protein introns." The process by which an intein excises itself and links the rest of the protein is referred to herein as "protein splicing" or "intein-mediated protein splicing." In some embodiments, the intein of the precursor protein (an intein-containing protein prior to intein-mediated protein splicing) is derived from two genes. Such inteins are referred to herein as split inteins (eg, split inteins-N and split inteins-C). For example, in cyanobacteria, DnaE, ​​the catalytic subunit a of DNA polymerase III, is encoded by two separate genes dnaE-n and dnaE-c. The intein encoded by the dnaE-n gene may be referred to herein as "intein N." The intein encoded by the dnaE-c gene may be referred to herein as "intein C."

[0122] Other intein systems may also be used. By way of example, synthetic inteins are described based on intein pairs of dnaE inteins, i.e. Cfa-N (eg, split intein-N) and Cfa-C (eg, split intein-C) (eg, incorporated herein by reference Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5). 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. 可以上述方法号分) (as described in US Pat. No. 8,394,604, incorporated by reference.

[0123] Exemplary nucleotide and amino acid sequences of inteins are provided.

[0124] DnaE Intein-N DNA: TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTCTGCCAATCGGGAAGATTGGGAAACGGATAGAATGCACAGTTTACTGTCGATAACAATGGTAACATGGTAACATTACTCAGCCAGTTGCCCAGTGGCACGACCGGGGA GAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATGAAGGACCACAAATTTATGACAGTCGGCCAGATGGCCAGATGCTGCCTATAGACGAAATCTTTGAGCGAGAGAGTTGGACCTCATGCGAGTTGACAACCTTCTAAT

[0125] DnaE Intein-N Protein: CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDR GEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN

[0126] DnaE Intein-C DNA: ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAACGTTATGA TATTGGAGTCGAAAGAGAGATCACAACTTTGCTGAAGAACGGATTCATAGCTTCTAAT

[0127] Intein-C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN

[0128] Cfa-N DNA: TGCCTGTCTTATATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCCTATTGGAAAGAGAGAATTGTCGAAGAGAATTGAATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTACACACAGACCCATTGCTCAATGGCACAATCGCG GCGAACAAGAAGTATTGAGTACTGTCTCGAGGATGGAAGCATCATACGAGCAACTAAAGATCATAAATTCATGACGGGCAGATGTTGCCATAGATGAGATGAGATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGCCA

[0129] Cfa-N protein: CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP

[0130] Cfa-C DNA: ATGAAGAGGACTGCCGATGGATCAGAGTTGAATCTCCCAAGAGAGAGAAGAGGAAAGTAAGATAATATCTCGAAAAGTCTTGGTACCCAAAATGTCTATGGAGTGGAGAAAGAACGGTCTCGTAGCCAGCAAC

[0131] Cfa-C protein: MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN

[0132] Intein N and intein C can be fused to the N-terminus of split Cas9 and the C-terminus of split Cas9, respectively. For example, in some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of the divided Cas9, i.e., N--[N-terminal portion of the divided Cas9]-[Intein-N] --Forms the structure of C. In some embodiments, intein-C is fused to the N-terminus of the C-terminal portion of the divided Cas9, i.e., N-[intein-C]-[C-terminal portion of the divided Cas9]-C form the structure of The mechanism of intein-mediated protein splicing for ligating proteins (eg, split Cas9) where inteins are fused is eg Shah et al., Chem Sci. 2014; 5(1 ):446-461, known in the art. Methods for designing and using inteins are known in the art and are described, for example, by WO2014004336, WO2017132580, US20150344549 and US20180127780, each of which is incorporated by reference in its entirety.

[0133] The term "isolated", "purified", or "biologically pure" refers to a substance in which, when found in its natural state, the associated components have been removed to varying degrees. refers to. "Isolation" refers to the original source of availability or the degree of separation from the surrounding environment. "Purification" indicates a higher degree of separation than isolation. A "purified" or "biologically pure" protein is sufficient to ensure that other substances do not substantially affect the biological properties of the protein or cause other harmful consequences. It has been removed. That is, the nucleic acid or peptide of the present invention is substantially free of cellular material, viral material or medium when produced by recombinant DNA technology, or chemical precursor when synthesized chemically. If it is substantially free of other chemicals, it has been purified. 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 one band in an electrophoresis gel. For proteins that can undergo modifications, eg, phosphorylation or glycosylation, different modifications can result in different isolated proteins that can be purified separately.

[0134] "Isolated polynucleotide" means a nucleic acid (eg, DNA) that does not contain a gene adjacent to the gene in the natural genome of the organism from which the nucleic acid molecule of the present invention is derived. Thus, the term is, for example, integrated into a vector; autonomously replicating plasmid or virus; integrated into the genomic DNA of a prokaryotes or eukaryotes; or separate from other sequences. 合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物。 Includes recombinant DNA present as a molecule (eg, a cDNA or genomic or cDNA fragment produced by PCR or restriction endonuclease digestion). Additionally, the term includes RNA molecules transcribed from DNA molecules, as well as recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0135] "Isolated polypeptides" refer to the polypeptides of the invention separated from the associated components in their natural state. Typically, the polypeptide is isolated when it is at least 60% free by weight from the proteins and natural organic molecules to which it is associated in its native state. Preferably, the preparation is at least 75% by weight, more preferably at least 90%, and even more preferably at least 99%, the preparation is a polypeptide of the present invention. Isolated polypeptides of the present invention can be obtained, for example, by extracting from natural sources, by expression of recombinant nucleic acids encoding such polypeptides; or by chemically synthesizing proteins. Purity can be determined by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0136] As used herein, the term "linker" is a type of two molecules or portions (e.g., two components of a protein complex or ribonuclear complex, or two domains of a fusion protein, e.g., a polynucleotide-programmable DNA binding domain It can refer to a covalent linker (eg, covalent), non-covalent linker, chemical group, or molecule that links (eg, dCas9) with a deaminase domain (eg, adenosine deaminase)). The linker can connect different components or different parts of the base editor system. For example, in some embodiments, the linker can connect the guide polynucleotide binding domain of the polynucleotide-programmable nucleotide binding domain and the catalytic domain of the deaminase. In some embodiments, the linker is capable of binding a CRISPR polypeptide and a deaminase. In some embodiments, the linker is capable of connecting Cas9 and deaminase. In some embodiments, the linker is capable of connecting dCas9 and deaminase. In some embodiments, the linker is capable of connecting nCas9 and deaminase. In some embodiments, the linker is capable of connecting the guide polynucleotide and the deaminase. In some embodiments, the linker is capable of connecting the deamination moiety of the base editor system and the polynucleotide-programmable nucleotide binding moiety. In some embodiments, the linker is capable of connecting the RNA-binding portion of the deamination component of the base editor system with the polynucleotide-programmable nucleotide-binding component. In some embodiments, the linker is capable of binding the RNA-binding portion of the deamination moiety of the base editor system to the RNA-binding portion of the polynucleotide-programmable nucleotide-binding moiety. The linker is located between or sandwiched between two groups, molecules, or other moieties, and is linked to each via covalent or non-covalent interactions, thus linking the two. . In some embodiments, the linker can be an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker may be a DNA linker. In some embodiments, the linker may be an RNA linker. In some embodiments, the linker can include an aptamer capable of binding to a ligand. In some embodiments, the ligand can be a carbohydrate, peptide, protein, or nucleic acid. In some embodiments, the linker may include an aptamer derived from a riboswitch. Riboswitches derived from aptamers are theophylline riboswitch, thiamine pyrophosphate (TPP) riboswitch, adenosinecobalamin (AdoCbl) riboswitch, S-adenosylmethionine (SAM) riboswitch, SAH riboswitch, flavin mononucleotide (FMN) It can be selected from a riboswitch, a tetrahydrofolate riboswitch, a lysine riboswitch, a glycine riboswitch, a purine riboswitch, a GlmS riboswitch, or a Prequeosin 1 (PreQ1) riboswitch. In some embodiments, the linker may comprise an aptamer bound to a protein domain, such as a polypeptide or a polypeptide ligand. In some embodiments, the polypeptide ligand includes a K-homologous (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and It can be an Sm7 protein, or an RNA recognition motif. In some embodiments, the polypeptide ligand may be part of a base editor-based component. For example, the nucleobase editing moiety may include a deaminase domain and an RNA recognition motif.

[0137] In some embodiments, the linker can be an amino acid or multiple amino acids (eg, a peptide or protein). In some embodiments, the linker is about 5 to 100 amino acids in length, such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, It can be 19, 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or 90-100 amino acids. In some embodiments, the linker can be about 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 amino acids in length. Longer or shorter linkers are also contemplated.

[0138] In some embodiments, the linker connects the gRNA-binding domain of an RNA-programmable nuclease containing the Cas9 nuclease domain with the catalytic domain of a nucleic acid editing protein (eg, adenosine deaminase). In some embodiments, the linker connects dCas9 and the nucleic acid editing protein. For example, a linker is located between two groups, molecules, or other moieties, or adjacent by two groups, molecules, or other moieties, and is linked to each via a covalent bond, and thus two Connect the two. In some embodiments, the linker is an amino acid or multiple amino acids (eg, a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is from 5 to 200 amino acids in length, such as 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, It can be 130, 140, 150, 160, 175, 180, 190, or 200 amino acids. Longer or shorter linkers are also contemplated.

[0139] In some embodiments, the domains of the nucleobase editor are fused via a linker that contains the amino acid sequence of SGGSSGSETPGTSESATPESSGGSS, SGSSGSSGS, or GGSGSPGSPTSEEGTSESATPESSGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSETSESATPESGPGSEPATSGGSGGSGS. In some embodiments, the domain of the nucleobase editor is fused via a linker containing the amino acid sequence SGSETPGTSESATPES, which can also be referred to as the XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker is (SGGS) n ,(GGGS) n ,(GGGGS) n ,(G) n、 (EAAAK) n ,(GGS) n , SGSETPGTSESATPES, or (XP) n Includes motifs, or combinations of any of these, where n is independently an integer between 1 and 30 and 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.

[0140] In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGSSGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGSSGSETPGTESATPESSGGSSGSSGSSGS. In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGSSGSETPGTSESATPESSGGSSGSSGSSGSSGSSGSSGSSGETPGTSESATPESSGGSSGS. In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPATS.

[0141] "Marker" means any protein or polynucleotide having an altered expression level or activity associated with a disease or disorder.

[0142] As used herein, the term "mutation" refers to the fact that a residue in a sequence, eg, a nucleic acid or amino acid sequence, is replaced by another residue, or one or more residues in the sequence. refers to a deletion or insertion of Mutations are typically described herein by identifying the original residue, then identifying the location of the residue within the sequence, and identifying the newly substituted residue. . Various methods for making amino acid substitutions (mutations) provided herein are well known in the art, including, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Provided by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). In some aspects, the base editor of the present disclosure is intended to "intentional" in a nucleic acid (eg, a nucleic acid within the genome of interest) without generating a significant number of unintentional mutations, eg, a nonintentional point mutations. 「はおうかないますねですねですねですねですねですねですねですねですねですねですねですねですねですねですねですねですねですね。 English: "A mutation that was obtained" can be efficiently generated, for example. In some embodiments, the intended mutation occurs by a specific base editor (eg, an adenosine base editor) bound to a guide polynucleotide (eg, adenosine base editor) that is specifically designed to produce the intended mutation. It is a mutation.

[0143] Generally, mutations made or identified in a sequence (eg, the amino acid sequences described herein) are numbered in relation to a reference (or wild type) sequence, ie, a sequence that does not contain the mutation. . Those skilled in the art will readily understand how to determine the location of a mutation in amino acid and nucleic acid sequences relative to a reference sequence.

[0144] The term "non-conservative mutation" refers to amino acid substitutions between different groups, such as tryptophan to lysine or serine to phenylalanine. In this case, it is preferred that the non-conservative amino acid substitution does not interfere or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the wild-type protein.

[0145] The terms "nuclear localization sequence," "nuclear localization signal," or "NLS" refer to amino acid sequences that promote the transfer of proteins to the cell nucleus. Nuclear localization sequences are known in the art and are, for example, international PCT application PCT / EP 2000 / 011690 filed on November 23, 2000 and published on May 31, 2001 as WO / 2001 / 038547. 回施加分 et al., the contents of which are incorporated by reference in the disclosure of exemplary nuclear localization sequences. In another embodiment, the NLS is an optimized NLS, described, for example, by Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises an amino acid sequence selected from KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0146] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to compounds containing nucleobases and acidic moieties, such as nucleosides, nucleotides, or polymers of nucleotides. Typically, a polymeric nucleic acid, eg, a nucleic acid molecule containing three or more nucleotides, is a linear molecule in which adjacent nucleotides are linked together via a phosphodiester bond. In some embodiments, "nucleic acid" refers to an individual nucleic acid residue (eg, a nucleotide and / or nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide strand containing three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" may be used interchangeably to refer to a polymer of nucleotides (eg, a strand of at least three nucleotides). In some embodiments, "nucleic acid" includes RNA and single-stranded and / or double-stranded DNA. Nucleic acids may occur naturally, for example, in the context of genomes, transcripts, mRNAs, tRNAs, rRNAs, siRNAs, snRNAs, plasmids, cosmids, chromosomes, chromatids, or other naturally occurring nucleic acid molecules. On the other hand, a nucleic acid molecule may be, for example, a non-naturally occurring molecule, a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or a fragment thereof, or a synthetic DNA, RNA, DNA / RNA hybrid, or a non-naturally occurring nucleotide. Alternatively, it can be a non-naturally occurring molecule, including nucleosides. Additionally, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, such as analogs having apart from the phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems, purified if necessary, chemically synthesized, and the like. For chemically synthesized molecules, the nucleic acid may, where appropriate, include, for example, nucleoside analogs, such as chemically modified bases or sugars, and analogs with backbone modifications. Nucleic acid sequences are shown in the 5'-3' direction unless otherwise indicated. In some embodiments, the nucleic acid is a natural nucleoside (eg, adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (eg, 2-aminoadenosine, 2-thiothymidine, inosine , pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5- Methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanine, O(6)-methylguanine, and 2-thiocytidine); Chemically modified bases; Biology modified bases (eg, methylated bases); inserted bases; modified sugars (2'-e.g., fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (eg, (and includes phosphorothioate and 5'-N-phosphoroamidite bonds).

[0147] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" can be used interchangeably with "polynucleotide programmable nucleotide binding domains" and that guide nucleic acid or polynucleotides that guide napDNAbp into a specific nucleic acid sequence ( Refers to a protein that associates with a nucleic acid (eg, DNA or RNA), such as, for example, gRNA). 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 an RNA-binding domain that is programmable by the polynucleotide. In some embodiments, the nucleotide binding domain programmable by the polynucleotide is a Cas9 protein. The Cas9 protein can bind to a guide RNA that induces the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, such as nuclease active Cas9, Cas9 nickase (nCas9), or nuclease-inactive Cas9 (dCas9). Non-limiting examples of nucleic acid programmable DNA binding proteins include Cas9 (eg, dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. It can be mentioned. 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 / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse 1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Css1, Cssd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Type II It includes Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, their homologs, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins may not be specifically listed in this disclosure, but are within the scope of this disclosure. 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 divers 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 by reference herein.

[0148] The terms "nucleobase", "nitrogen base", or "base" are used interchangeably herein to refer to a nitrogen-containing biological compound that forms a nucleoside, where nucleoside is a component of a nucleotide. be. The ability of nucleobases to form base pairs and stack each other directly results in long helical structures such as ribonucleic acids (RNAs) and deoxyribonucleic acids (DNAs). The five nucleobases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) are called primary or standard bases. Adenine and guanine are derived from purines, while cytosine, uracil, and thymine are derived from pyrimidines. The DNA and RNA can also contain other (non-primary) bases that have been modified. Non-limiting exemplary modified nucleobases include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydromethylcytosine. Hypoxanthine and xanthine can be produced by the presence of a mutagen, both by deamination (replacement of an amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can result from deamination of cytosine. A "nucleoside" consists of one nucleobase and one pentasaccharide (either 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), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). It will be done. A "nucleotide" consists of one nucleobase, one pentasaccharide (either ribose or deoxyribose), and at least one phosphate group.

[0149] The terms "nucleobase editing domain" or "nucleobase editing protein" as used herein, from cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine) and adenine (or or alphabetical) (or thymidine). Refers to a protein or enzyme capable of catalyzing nucleobase modifications in RNA or DNA, such as deamination of 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 (eg, 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 may be a nucleobase editing domain that has been manipulated or evolved from a naturally occurring nucleobase editing domain. The nucleobase editing domain can be from any organism, such as a bacterial, human, chimpanzee, gorilla, monkey, bovine, dog, rat, or mouse.

[0150] As used herein, "acquiring" such as in "acquiring a drug" includes synthesising, purchasing, or otherwise obtaining the drug.

[0151] As used herein, "patient" or "subject" refers to a mammalian subject or individual who has been diagnosed with a disease or disorder, is at risk of causing or is suspected of having or having them. In some embodiments, the term "patient" refers to a mammalian subject with a greater than average chance of developing a disease or disorder. Exemplary patients include humans, non-human primates, cats, dogs, pigs, cows, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, guinea pigs) and books It may be another mammal that can benefit from the treatments disclosed in the specification. Exemplary human patients may be male and / or female.

[0152] "Patients in need of it" or "subjects in need of it" are used herein to diagnose a disease or disorder, such as, but not limited to, glycogenic disease type 1 (GSD1 or Von Gierke's disease). It is referred to as a patient who has been or suspected of having.

[0153] The terms "pathogenic mutation," "pathogenic variant," "disease cause mutation," "disease cause variant," "harmful mutation," or "predisposed mutation," are the terms that the individual is susceptible or predisposed to a particular disease or disorder. refers to a genetic change or mutation that increases the In some embodiments, the pathogenic mutation includes at least one wild-type amino acid in the protein encoded by the gene, which is replaced by at least one pathogenic amino acid.

[0154] 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 . The term refers to a protein, peptide or polypeptide of any size, structure or function. Typically, the protein, peptide, or polypeptide is at least 3 amino acids in length. A protein, peptide, or polypeptide can refer to an individual protein or a group of proteins. One or more amino acids in a protein, peptide, or polypeptide include, for example, carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for bonds, functionalizations, or other modifications, etc. It can be modified by the addition of a chemical entity of The protein, peptide, or polypeptide may also be a single molecule or a multimolecular complex. A protein, peptide, or polypeptide can be merely a fragment of a naturally occurring protein or peptide. The protein, peptide, or polypeptide can be naturally occurring, recombinant, or synthetic, or any combination thereof. As used herein, the term "fusion protein" refers to a hybrid polypeptide that contains protein domains from at least two different proteins. One protein can be located at the amino-terminal (N-terminal) portion or carboxy-terminal (C-terminal) protein of the fusion protein, thus forming an amino-terminal fusion protein or a carboxy-terminal fusion protein, respectively. The protein may contain different domains, such as the nucleic acid binding domain (eg, the gRNA-binding domain of Cas9 that induces binding of the protein to a target site) and the nucleic acid cleavage domain, or the catalytic domain of a nucleic acid editing protein. In some embodiments, the protein includes a proteinaceous moiety, eg, an amino acid sequence that constitutes a nucleic acid binding domain, and an organic compound, eg, a compound that can act as a nucleic acid cleavage agent. In some embodiments, the protein is complexed with a nucleic acid (eg, RNA or DNA) or associated with a nucleic acid. Any of the proteins provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers. Methods for the expression and purification of recombinant proteins are well known and described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). Included are those which are incorporated by reference in its entirety.

[0155] The polypeptides and proteins disclosed herein, including functional moieties and functional variants thereof, may include synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and are for example aminocyclohexanecarboxylic acids, norleucine, alpha-aminon-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxy Proline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carbon acid , 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyl-lysine, 6-hydroxylysine , ornithine, α-aminocyclopentanecarboxylic acid, aminocyclohexanecarboxylic acid, 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 are included. The polypeptides and proteins can be linked to post-translational modifications of one or more amino acids of the polypeptide construct. Non-limiting examples of post-translational modifications include phosphorylation, acylation including acetylation and formylation, glycosylation (including N-links and O-links), amidation, hydroxylation, methylation and ethylation alkylation including, ubiquitination, addition of pyrrolidone carboxylic acids, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation and iodolation.

[0156] The term "recombinant" as used herein in connection with proteins or nucleic acids refers to proteins or nucleic acids that do not exist in nature but are products of human engineering. For example, in some embodiments, the recombinant protein or nucleic acid molecule is at least one, at least two, at least three, at least four, at least five, at least as compared to any naturally occurring sequence. It includes an amino acid or nucleotide sequence containing six or at least seven mutations.

[0157] "Decreasing" means a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0158] "Reference" means a criteria or control condition. In one embodiment, the reference is a wild type or healthy cell. In other embodiments, but not limited to, the reference is exposed to a control vector that is not exposed to the test conditions or does not carry a placebo or conventional saline, medium, buffer, and / or a polynucleotide of interest. It is an untreated cell.

[0159] A "reference array" is a predefined array used as the basis for a sequence comparison. The reference sequence can be a subset or entirely of a particular sequence; for example, a segment 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 generally at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence is generally at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides or about 300 nucleotides or any integer around or between them. . 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 a wild-type protein.

[0160] The terms "RNA-programmable nuclease" and "RNA-induced nuclease" are used together with one or more RNAs that are not targets for cleavage (eg, bound or accompanying them). In some embodiments, the RNA-programmable nuclease, when complexed with RNA, may be referred to as a nuclease:RNA complex. Typically, bound RNA is called a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs, or as a single RNA molecule. GRNAs present as single RNA molecules can be referred to as single guide RNAs (sgRNAs), whereas "gRNAs" exist either as single molecules or as complexes of two or more molecules. It is used to be compatible to refer to RNA. Typically, gRNAs present as single RNA species include two domains: (1) domains that share homology with the target nucleic acid (eg, which directs binding of the Cas9 complex to the target), and (2) contain domains that bind to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and includes a stem-loop structure. For example, in some embodiments, domain (2) is provided in Jinek et ah, Science 337:816-821 (2012), whose entire contents are incorporated by reference herein. They are the same or homologous. Other examples of gRNAs (e.g. those containing domain 2) include US provisional patent application U.S.S.N.61 / 874,682 filed September 6, 2013, titled "Switchable Cas9 Nucleases and Uses Thereof" and September 6, 2013 It can be found in the US Provisional Patent Application No. 61 / 874,746 filed on the day, titled "Delivery System For Functional Nucleases," the entire contents of each of which are incorporated by reference in its entirety. In some embodiments, the gRNA includes two or more of domains (1) and (2) and may be referred to as "extended gRNA." For example, an extended gRNA will bind to two or more Cas9 proteins, as described herein, and will bind to the target nucleic acid in two or more clearly 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, providing sequence specificity of the nuclease:RNA complex.

[0161] In one embodiment, the RNA-programmable nuclease is a Cas9 endonuclease (CRISPR-related system) such as Cas9 (Csnl) from Streptococcus pyogenes (eg, "Complete genome sequence of an Ml strain of Streptococcus pyogenes." Ferretti J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C, Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 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., Pirzada Z.A., Eckert See M.R., Vogel J., Charpentier E., Nature 471:602-607 (2011)).

[0162] Because RNA-programmable nucleases (eg, Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins in principle target any sequence designated by the guide RNA. You can do it. Methods for using RNA-programmable nucleases such as Cas9 for site-specific cleavage (eg, to modify the genome) are known in the art (eg, Cong, L. et al., Multiplex genome). engineering using CRISPR / Cas systems. Science 339, 819-823 (2013), Mali, P. et ah, RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013), Hwang, W.Y. et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013), Jinek, M. et ah, RNA-programmed genome editing in human cells. eLife 2, e00471 (2013), Dicarlo, J.E. et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013), Jiang, W. et ah RNA-guided editing of bacteria genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (See 2013), the entire contents of each of these are incorporated by reference herein.

[0163] The term "single nucleotide polymorphism (SNP)" is a single nucleotide mutation that occurs at a particular position in the genome, where each mutation exists to some extent (eg >1%) that can be recognized within the population. For example, at a particular base position in the human genome, C nucleotides can appear in most individuals, but in a small number the position is occupied by A. This means that there is an SNP at this particular position and that the variation of two nucleotides, C or A, is an allyl at this position. SNPs underlie differences in disease susceptibility. The severity of the disease and the body's response to treatment are also an indication of genetic variation. SNPs can reside in the coding region of a gene, in the non-coding region of a gene, or in the intergenic region (the region between genes). In some embodiments, the SNPs within the coding sequence do not necessarily change the amino acid sequence of the produced protein due to degeneracy of the genetic code. There are two types of SNPs in the coding region: synonymous SNPs and non-synonymous SNPs. Synonymous SNPs have no effect on protein sequences, while non-synonymous SNPs change the amino acid sequence of a protein. There are two types of nonsynonymous SNPs: missense and nonsense. SNPs that are not in the protein-coding region can affect gene splicing, transcription factors binding, messenger RNA degradation, or sequences of non-coding RNAs. Gene expression affected by this type of SNP is called eSNP (expressed SNP) and can be upstream or downstream of the gene. Single-base variants (SNVs) are unlimited single-base variations and can occur in somatic cells. Somatic single base variations can also be called single base modifications.

[0164] "Specifically binding" is a nucleic acid molecule that recognizes and binds to the polypeptides and / or nucleic acid molecules of the present invention, but does not substantially recognize and bind to other molecules in a sample (eg, biological sample). , a polypeptide, or a complex thereof (eg, a nucleic acid-programmable DNA-binding domain and a guide nucleic acid), a compound, or a molecule.

[0165] 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 the endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the 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 the endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridize" means that a pair that forms a double-stranded molecule between or between complementary polynucleotide sequences (eg, genes described herein) under various stringency conditions. It means to make something. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0166] For example, stringent salt concentrations are typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, more preferably less than about 250 mM NaCl and 25 mM It is trisodium citrate. Low stringency hybridization can be obtained in the absence of an organic solvent, eg, formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Can do it. Stringent temperature conditions will 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, concentration of surfactants (eg, sodium dodecyl sulfate (SDS)), and the inclusion or elimination of carrier DNA are well known to those skilled in the art. Various levels of stringency can be achieved by combining these different conditions as necessary. In one embodiment, hybridization occurs at 30° C. in 750 mM NaCl, 75 mM trisodium citrate and 1% SDS. In another embodiment, hybridization occurs at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide and 100 μg / ml denatured salmon sperm DNA (ssDNA). In another embodiment, hybridization occurs at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide and 200 μg / ml ssDNA. Useful variations of these conditions will be readily apparent to those skilled in the art.

[0167] In most applications, the washing steps followed by hybridization also differ in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As noted above, washing stringency can be increased by lowering the salt concentration or increasing the temperature. For example, the stringent salt concentration for the washing step can be preferably less than about 30 mM NaCl and 3 mM trisodium citrate, most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the washing step typically include a temperature of at least about 25° C., more preferably at least about 42° C., even more preferably at least about 68° C. In one embodiment, the washing step is carried out at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step is carried out at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step is carried out at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations of these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art, such as 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: described in A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0168] "Split" means being split into two or more fragments.

[0169] "Splitted Cas9 protein" or "Splitted Cas9" refers to the 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 and C-terminal portions of the Cas9 protein can be spliced ​​to form a "reconstituted" Cas9 protein. In certain embodiments, the Cas9 protein is, for example, as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014, or Jiang et al. (2016) Science 351: 867-871. PDB file: As described in 5F9R (each incorporated by reference), it is divided into two fragments within an unordered region of the protein. In some embodiments, the protein is at any C, T, A, or S within the region between approximately amino acids A292-G364, F445-K483, or E565-T637 of SpCas9, or any other Cas9 , Cas9 variants (eg nCas9, dCas9), or at corresponding locations in other napDNAbp, are split into two fragments. In some embodiments, the protein is split into two fragments in 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.

[0170] In another embodiment, the N-terminal portion of the Cas9 protein comprises amino acids 1-573 or 1-637 of S. pyrogenes Cas9 wild type (SpCas9) (NCBI reference sequence: NC_002737.2, Uniprot reference sequence: Q99ZW2), The C-terminal portion of the Cas9 protein includes a portion of the SpCas9 wild-type amino acids 574-1368 or 638-1368, or a corresponding position thereof.

[0171] The C-terminal portion of the divided Cas9 can be linked to the N-terminal portion of the divided Cas9 to form the complete Cas9 protein. In some embodiments, the C-terminal portion of the Cas9 protein begins with the N-terminal portion of the Cas9 protein ending. Thus, in some embodiments, the C-terminal portion of the separated Cas9 comprises the portion of the amino acids (551-651) -1368 of spCas9. "(551-651) -1368" means starting with an amino acid between amino acids 551-651 (including both ends) and ending with amino acids 1368. For example, the C-terminal portion of the split Cas9 is the amino acids 551-1368, 552-1368, 553-1368, 554-1368, 555-1368, 555-1368, 556-1368, 557-1368, 558-1368, 559-1368, 560 of spCas9 -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 , 626-1368, 627- 1368, 628-1368, 629-1368, 630-1368, 631-1368, 632-1368, 633-1368, 633-1368, 634-1368, 635-1368, 636-1368, 637-1368, 638-1368, 639-1368, Whichever: 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 It may include one part. In some embodiments, the C-terminal portion of the divided Cas9 protein comprises the portion of amino acids 574-1368 or 638-1368 of SpCas9.

[0172] "Subject" means, but is not limited to, a mammal, including, but not limited to, humans, or non-human mammals such as cows, horses, dogs, sheep, or cats. The subject matter includes, but is not limited to, domestic animals, including cattle, goats, chickens, horses, pigs, rabbits, and sheep, that are raised to produce labor and provide food and other items; It can be mentioned.

[0173] "Substantially identical" means that a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or a nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein) ) is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity to ). In one embodiment, such sequences are at least 60%, 80% or 85%, 90%, 95%, or even 99% identical to the sequence used for comparison at amino acid levels or at nucleic acids.

[0174] Sequence identity is typically the case of sequence analysis software (e.g. the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705 Sequence Analysis Software Package, BLAST, BESTFIT, COBALT, EMBOSS Needle , GAP, or PILEUP / PRETTYBOX program) to measure. Such software matches identical or similar sequences by assigning a degree of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine. In an illustrative approach to determining the degree of identity, the BLAST program can be used, -3 And e -100 The probability scores between the two show closely related sequences. COBALT is used, for example, with the following parameters: a) Alignment parameters: Gap penalties-11,-1 and End-Gap penalties-5,-1, b) CDD parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved columns and Recompute on c) Query clustering parameters: Use query clusters on; Word Size 4; Max cluster distance 0.8; Alphabet Regular. EMBOSS Needle is used, for example, with the following parameters: a) Matrix: BLOSUM62; b) GAP OPEN: 10; c) GAP EXTEND: 0.5; d) OUTPUT FORMAT: pair; e) END GAP PENALTY: false; f) END GAP OPEN: 10; and g) END GAP EXTEND: 0.5.

[0175] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a nucleobase editor. In one embodiment, the target site is deamination by a deaminase or a fusion protein containing it (eg, adenine deaminase).

[0176] As used herein, the terms "treat", "treating", "treatment", and the like are to alleviate or ameliorate a disorder and / or related symptoms; Or refers to obtaining the desired pharmacological and / or physiological effect. It will be understood that treating a disorder or condition does not require the complete removal of the associated disorder, condition or condition (and not to rule out complete removal). In some embodiments, the effect is therapeutic, ie, not limited to, but the effect may partially or completely reduce, reduce, eliminate, relieve, reduce, or alleviate, the disease and / or the adverse symptoms resulting therefrom; Relax, decrease in intensity, or heal. In some embodiments, the effect is prophylactic, ie, the effect protects or prevents the occurrence or recurrence of a disease or condition. To this end, the methods of the present disclosure include administering a therapeutically effective amount of a composition as described herein.

[0177] "Uracil glycosylase inhibitor" or "UGI" means an agent that inhibits the uracil excision repair system. In one embodiment, the agent is a protein or fragment thereof that binds to the host uracil-DNA glycosylase and prevents removal of uracil residues from the DNA. In one embodiment, the UGI is a protein, fragment, or domain thereof capable of inhibiting uracil-DNA glycosylase base excision repair enzymes. In some embodiments, the UGI domain comprises a wild-type UGI or a modified version thereof. In some embodiments, the UGI domain comprises fragments of the exemplary amino acid sequences presented below. In some embodiments, the UGI fragment is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least of the exemplary UGI sequences provided below. Contains an amino acid sequence that includes 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, the UGI comprises an amino acid sequence homologous to an illustrative UGI amino acid sequence or a fragment thereof, as described below. In some embodiments, UGI or portions thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, relative to the wild-type UGI or UGI sequence or portions thereof, as described below. %, 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% identity. An exemplary UGI includes the following amino acid sequence: >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD APEYKPWALVIQDSNGENKIKML.

[0178] The term "vector" refers to the means for introducing a nucleic acid sequence into a cell and leading to a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, and episomes. An "expression vector" is a nucleic acid sequence that includes a nucleotide sequence that is expressed in a recipient cell. The expression vector may contain additional nucleic acid sequences to promote and / or facilitate expression of the introduced sequence, such as initiation, halt, enhancers, promoters, and secretory sequences.

[0179] Any compositions or methods provided herein can be combined with one or more of the other compositions and methods provided herein.

[0180] DNA editing has emerged as a viable means of altering pathogenic mutations by correcting at the genetic level. Until recently, all DNA editing platforms induced DNA double-strand breaks (DSBs) at designated genomic sites and determined product outcomes in a semi-stochastic manner, depending on the endogenous DNA repair pathway. It was functional and produced a complex population of gene products. Homologous recombination repair (HDR) pathways allow for accurate, user-defined repair results, but several challenges have been the high efficiency of using HDR in therapeutically relevant cell types. It has hindered the repair. In practice, this pathway is inefficient compared to competing, error-prone non-homologous terminal bonding pathways. Furthermore, HDR is severely restricted to G1 and S phases of the cell cycle, preventing the correct repair of DSBs in mitotic end cells. As a result, it has been found that modifying genomic sequences in high efficiency in user-defined programmable ways in these populations is difficult or impossible.

[0181] Features of the present disclosure are described in detail in the appended claims. A better understanding of the features and advantages of the present disclosure is made available to you by referring to the following detailed description which describes illustrative embodiments in which the principles of the present disclosure are utilized, and the accompanying drawings, in which the contents are as follows. You will get: [Brief description of the drawing]

[0182] [Figure 1] FIG. 1 shows the G6PC nucleotide target sequence and the corresponding amino acid sequences, indicating the bystander and on-target A>G bases for correction for the GSD1a Q347X mutation. [Figure 2] FIG. 2 shows accurate base correction and bystander editing. Figure 2A shows the positions of the target nucleobase and bystander nucleobase. Figure 2B shows the percentage of accurate on-target and bystander correction for the GSD1a G6PC Q347X mutation in HEK293T cells using the ABE8 variant. [Figure 3] Figures 3A and 3B show editor optimization of correction for GSD1a G6PC Q347X mutations in HEK293T cells. Figure 3A shows the G6PC nucleotide target sequence and the corresponding amino acid sequence, showing the bystander and on-target A>G bases and GGA PAM sequences for correction for the GSD1a Q347X mutation. FIG. 3B is a graph showing the percentage correction for the GSD1a G6PC Q347X mutation using ABE8 monomer and heterodimeric variants. [Figure 4] Figure 4 is a graph showing the percentage correction for the GSD1a G6PC Q347X mutation in HEK293T cells using the ABE8 double mutant variant, with A>G with bystander (A2) and on-target (A6) Compare bases. [Figure 5] Figure 5 is a graph showing the percentage of corrected GSD1a Q347X mutations in patient-derived B lymphocytes using the ABE8 variant. [Figure 6] Figures 6A and 6B show accurate corrections for the GSD1a G6PC Q347X mutation in iPS-derived hepatocytes of complex heterozygous (Q347X, G222R) patients. Figure 6A shows the G6PC nucleotide target sequence, the corresponding amino acid sequence, and the GGA PAM sequence, indicating the bystander and on-target A>G bases for correction for the GSD1a Q347X mutation. Figure 6B is a graph showing the A>G base editing efficiency of the GSD1a Q347X mutation using the ABE8 variant, comparing the on-target and bystander corrections. [Figure 7] Figures 7A and 7B show editor optimization of correction for GSD1a Q347X mutations in patient iPS-derived hepatocytes. Figure 7A shows the NGA PAM sequence and the corresponding target sequence of GSD1a, showing the bystander and on-target A>G bases. Figure 7B is a graph showing the base editing efficiency of the GSD1a Q347X mutation using the ABE8 variant. [Figure 8]Figures 8A and 8B provide an in vitro transduction schedule for the GSD1a Q347X mutation in primary hepatocyte co-culture systems. Figure 8A provides an in vitro transduction schedule timeline in either hepatocyte monolayers or hepatocyte cocultures and shows representative time points. Figure 8B shows images of donor-derived transduced primary hepatocytes used in a co-culture system for the GSD1a Q347X mutation. [Figure 9] Figure 9 shows day 6 (D6) in primary hepatocyte cocultures transduced at 30, 100, and 300 lentivirus multiplicities of infection (MOI) using a lentiviral vector containing the GSD1a Q347X mutation. ) shows images of GFP expression (GFP, brightfield, merge). [Figure 10] Figures 10A, 10B, and 10C show corrections for the GSD1a Q347X mutation in lentivirus-transduced primary hepatocyte co-culture systems. Figure 10A shows images of GFP expression in primary hepatocyte cocultures (donor RSE) transduced at MOI of 500 using a lentiviral vector containing the GSD1a Q347X mutation. Figure 10B is a graph showing the A>G base editing efficiency of correction for on-target GSD1a Q347X mutations and indels in transduced primary hepatocyte cocultures. The dashed line represents A>G base editing efficiency for therapeutic benefits. Figure 10C in transduced primary hepatocyte co-cultures treated or remained untreated with collagenases type III, type IV and hyaluronic acid in media with or without polyethylene glycol 8000 (PEG8K). 1 is a graph showing the A>G base editing efficiency of the GSD1a Q347X mutation. [Figure 11] FIG. 11 shows the G6PC nucleotide target sequence and the corresponding amino acid sequences, indicating the bystander and on-target A>G bases for correction for the GSD1a R83C mutation. [Figure 12] Figures 12A and 12B show accurate corrections for the GSD1a G6PC R83C mutation in HEK293T cells. FIG. 12A shows the G6PC nucleotide target sequence and corresponding amino acid sequences indicating bystander, synonymous, and on-target A>G bases for correction for the GSD1a R83C mutation. Figure 12B is a graph showing the A>G base editing efficiency of GSD1a R83C mutations using the ABE8 variant, comparing the on-target and bystander corrections. [Figure 13] Figures 13A and 13B show base editing of G6PC R83C mutations by plasmid transfection in HEK293T lench model cells. Figure 13A shows the GAGAAT PAM sequence of GSD1a gRNA#820 and the AGA PAM sequence of GSD1a gRNA#1121 and the corresponding target sequence, which provides the bystander of the target sequence and the on-target A>G base. show. FIG. 13B is a graph showing percentage of on-target and bystander correction for GSD1a R83C mutations using an ABE base editor with gRNA1121 or gRNA820. [Figure 14] FIG. 14 is a graph showing the A>G base editing efficiency of GSD1a R83C mutations using the saABE8 variant. [Figure 15] FIG. 15 is a graph showing the A>G base editing efficiency of GSD1a R83C mutations using the saABE8 double mutant variant. [Figure 16] FIG. 16 is a graph showing the A>G base editing efficiency of on-target, bystander, and synonymous bystander correction of GSD1a R83C mutations in HEK293T cells using the ABE8 variant. [Figure 17] Figures 17A and 17B show corrections for the GSD1a Q347X mutation in primary mouse hepatocytes isolated from a transgenic mouse model of GSD1a. Figure 17A shows images of primary mouse hepatocytes isolated from the ASC transgenic mouse model, huG6PC, R83C (V166L). Figure 17B is a graph showing the base editing efficiencies of positions A12G, A10G, A6G and Indel for correction of GSD1a R83C mutations using the ABE8 variant in primary mouse hepatocytes isolated from the GSD1a transgenic mouse model. be. [Figure 18] Figure 18 shows 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 RNA of various lengths as shown. It is a graph. Data were obtained in HEK293T cells. Details of the target site and other editing are also provided. [Figure 19] Figure 19 shows levels (percentages) of A>G base editing at on-target (12A) and off-target (6A) sites using ABE8 (TadA*8 variant-SaCas9) in combination with 20nt and 21nt guide RNA. ) is a graph showing edited. Data were obtained in HEK293T cells. [Figure 20] Figure 20 shows the level of A>G base editing at on-target (12A) and off-target (6A) sites using an ABE base editor (TadA variant-SaCas9) in combination with a 20nt or 21nt guide RNA (R83C). 1 is a graph showing % correction of ). Data were obtained in HEK293T cells. [Figure 21] Figure 21 shows A>G(%) base editing at on-target (12A) and off-target (6A) sites using an ABE base editor (TadA variant-SaCas9) in combination with a 20nt or 21nt guide RNA. 1 is a graph showing the levels. Data were obtained in a primary human hepatocyte lentiviral model of GSD1a R83C. [Figure 22] Figure 22 shows the level of A>G base editing at on-target (12A) and off-target (6A) sites using an ABE base editor (TadA variant-SaCas9) in combination with a 20-nt or 21-nt guide RNA (R83C). 1 is a graph showing % correction of ). Data were obtained in a primary human hepatocyte lentiviral model of GSD1a R83C. [Figure 23] Figure 23 is a graph showing the level of A>G(%) accurate base editing at on-target and off-target sites in heterozygous transgenic GSD1a R83C mice. [Figure 24] FIG. 24 is a table showing the Cas9 variants for accessing all possible PAMs of an NRNN PAM. Only Cas9 variants that require recognition of up to three defined nucleotides in that PAM are listed. Non-G PAM variants include SpCas9-NRRH, SpCas9-NRTH, and SpCas9-NRCH. [Form for implementing the invention]

[0183] The present invention uses a composition containing a novel adenosine base editor (eg, ABE8) with increased efficiency, and a base editor containing an adenosine deaminase variant to modify mutations associated with glycogen disease type 1a (GSD1a). Provide a method.

[0184] The present invention provides a base editor, at least in part, characterized by an adenosine deaminase variant (ie, ABE8), to accurately administer single nucleotide polymorphisms (eg, R83C, Q347X) in the endogenous glucose-6-phosphatase (G6PC) gene. Based on the discovery of correcting.

[0185] The GSD1a mutations, R83C and Q347X, are cytidine-to-thymidine (C→T), leading to base pair substitutions of C·G-to-T. These substitutions can be returned to wild-type non-pathogenic genomic sequences using an adenosine base editor (ABE) that catalyzes the A·T to G·C substitution. In broad interpretation, mutations that cause GSD1a are potentially reverted to wild-type sequences using ABE without the risk of inducing overexpression of the G6PC gene, which can occur when gene therapy is used. It is a target. Thus, DNA base editing from A.T to G.C accurately corrects one or more of the most common GSD1a-induced mutations in the G6PC gene.

[0186] [Nucleobase Editor] A base or nucleobase editor is disclosed herein for editing, modifying or modifying the target nucleotide sequence of a polynucleotide. Description of this specification is a nucleobase or base editor that includes a polynucleotide-programmable nucleotide-binding domain and a nucleobase editing domain (eg, adenosine deaminase). Polynucleotide programmable nucleotide binding domains, when combined with a bound guide polynucleotide (eg, gRNA), are used to combine (complementary base pairs between the base of the bound guide nucleic acid and the base of the target polynucleotide sequence. (via formation) it is possible to specifically bind to the target polynucleotide sequence, thereby localizing the base editor to the target nucleic acid sequence that is desired to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded 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.

[0187] [Polynucleotide Programmable Nucleotide Binding Domain] It is to be understood that the polynucleotide programmable nucleotide binding domain can also include a nucleic acid programmable protein that binds to RNA. For example, a polynucleotide programmable nucleotide binding domain may be linked to a nucleic acid that guides the polynucleotide programmable nucleotide binding domain to the RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, but they are not specifically listed in this disclosure.

[0188] The polynucleotide programmable nucleotide binding domain of the base editor can itself contain one or more domains. For example, a polynucleotide programmable nucleotide binding domain can include one or more nuclease domains. In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain can comprise an endonuclease or an exonuclease. As used herein, the term "exonuclease" refers to a protein or polypeptide capable of digesting a nucleic acid (eg, RNA or DNA) from its free end, and the term "endonuclease" refers to a nucleic acid (eg, DNA or RNA) Refers to a protein or polypeptide capable of catalyzing (eg, cleaving) the internal region. In some embodiments, the endonuclease is capable of cleaving a single strand of double-stranded nucleic acid. In some embodiments, the endonuclease is capable of cleaving both strands of the double-stranded nucleic acid molecule. In some embodiments, the polynucleotide programmable nucleotide binding domain can be a deoxyribonuclease. In some embodiments, the polynucleotide programmable nucleotide binding domain can be a ribonuclease.

[0189] In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain is capable of cutting zero, one or two strands of the target polynucleotide. In some embodiments, the polynucleotide programmable nucleotide binding domain can include a nickase domain. As used herein, the term "niccase" refers to a polynucleotide-programmable nucleotide binding domain that includes a nuclease domain that is capable of cleaving only one strand of the double stranded nucleic acid molecule (eg, DNA). In some embodiments, the nickase is derived from a fully catalytically active (eg, naturally occurring) form of the polynucleotide programmable nucleotide binding domain by introducing one or more mutations into the active polynucleotide programmable nucleotide binding domain. It can be done. For example, if 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 a histidine at position 840. In such an embodiment, residue H 840 retains catalytic activity, thereby allowing the single strand of the nucleic acid double strand to be cleaved. In another example, the Cas9-derived nickase domain can include an H840A mutation, while the amino acid residue at position 10 remains at D. In some embodiments, the nickases are derived from a fully catalytically active (eg, naturally occurring) form of the polynucleotide programmable nucleotide binding domain by removing all or part of the nuclease domain that is not required for nickase activity. It can be done. For example, if the polynucleotide-programmable nucleotide-binding domain includes a nickase domain derived from Cas9, the nickase domain derived from Cas9 can include deletions of all or part of the RuvC domain or the HNH domain.

[0190] The amino acid sequence of an exemplary catalytic active Cas9 is as follows: MDKKYSIGLDIGTNS VGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEEN PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQ LPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSE QEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK LPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD.

[0191] Thus, a base editor containing a polynucleotide programmable nucleotide binding domain containing a nickase domain may be used to cleavage single-stranded DNA cleavage (nick) at a particular polynucleotide target sequence (eg, determined by the complementary sequence of the bound guide nucleic acid). can be generated. In some embodiments, the strand of the nucleic acid double-stranded target polynucleotide sequence that is cleaved by a base editor containing a nickase domain (eg, a nickase domain from Cas9) is a strand that is not edited by the base editor (ie, by the base editor). The strand to be cleaved is the opposite strand to the strand that contains the base to be edited). In other embodiments, a base editor containing a nickase domain (eg, a nickase domain from Cas9) is capable of cutting the strands of the targeted DNA molecule for editing. In such an embodiment, the non-target strand is not cleaved.

[0192] Also provided herein are base editors containing catalytically dead (ie, unable to cleave the target polynucleotide sequence) polynucleotide programmable nucleotide binding domains. As used herein, the terms "catalytically dead" and "nuclease inactivation" are polynucleotide programmable with one or more mutations and / or deletions that result in inability to cleave the strands of the nucleic acid. It is used interchangeably to refer to a nucleotide binding domain. In some embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain base editor can lack nuclease activity as a result of a particular point mutation in one or more nuclease domains. For example, in the case of a base editor that contains the Cas9 domain, Cas9 can contain both the D10A and H840A mutations. Such mutations inactivate both nuclease domains, resulting in loss of nuclease activity. In other embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain can include one or more deletions in all or part of the catalytic domain (eg, RuvC1 and / or HNH domains). In a further embodiment, the catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (eg, D10A or H840A) and deletion of all or part of the nuclease domain.

[0193] Also contemplated herein are mutations capable of generating catalytically dead polynucleotide programmable nucleotide binding domains from previously functional versions of the polynucleotide programmable nucleotide binding domain. For example, in the case of catalytically dead Cas9 ("dCas9"), a variant is provided that has mutations other than D10A and H840A resulting in nuclease-inactivated Cas9. Such mutations include, for example, other amino acid substitutions in D10 and H840, or other substitutions within the nuclease domain of Cas9 (eg, substitutions in the HNH nuclease subdomain and / or RuvC1 subdomain). Additional suitable nuclease-inactive dCas9 domains may become apparent to those skilled in the art based on this disclosure and knowledge in the art, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains. (See, for example, 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 hereby incorporated by reference. is incorporated into)).

[0194] Non-limiting examples of polynucleotide programmable nucleotide binding domains that can be incorporated into the base editor include CRISPR protein-derived domains, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). . In some embodiments, the base editor may be a naturally-occurring or modified protein that is capable of binding to a nucleic acid sequence via a binding guide nucleic acid upon CRISPR (ie, Clustered Regularly Interspaced Short Palindromic Repeats)-mediated modification of the nucleic acid, or its Includes a portion-containing polynucleotide programmable nucleotide binding domain. Such proteins are referred to herein as "CRISPR proteins." Therefore, disclosed herein is a base editor that includes a polynucleotide-programmable nucleotide binding domain that includes all or part of the CRISPR protein (ie, a base editor that includes all or part of the CRISPR protein as a domain ( It is also known as the "CRISPR protein-derived domain" in the base editor)). The CRISPR protein-derived domains incorporated in the base editor can be modified in comparison to wild-type or native CRISPR proteins. For example, as described below, a CRISPR protein derived domain may include one or more mutations, insertions, deletions, rearrangements and / or recombination compared to a wild-type or native CRISPR protein.

[0195] CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, metastatic factors, conjugation plasmids). The CRISPR cluster includes a spacer, a sequence complementary to the preceding mobile element, and a target invasion nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In the type II CRISPR system, the correct processing of pre-crRNA requires the transcoding small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. tracrRNA serves as a guide to the processing of pre-crRNA by ribonuclease 3. Cas9 / crRNA / tracrRNA then cleaves a linear or circular dsDNA target complementary to the spacer with an endonuclease. Target strands that are not complementary to crRNA are first endonucleaseically cleaved and then exonucleaseically trimmed 3'-5'. In nature, both proteins and RNA are required for DNA binding and cleavage. However, a single guide RNA ("sgRNA" or simply "gRNA") can be created to incorporate both crRNA and tracrRNA aspects 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 by reference herein. . Cas9 recognizes short motifs (PAM or protospacer flanking motifs) in CRISPR repeats to help distinguish between "self" and "non-self."

[0196] In some embodiments, the methods described herein may 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 approximately 20 base spacers defining the genomic target to be modified. Therefore, one skilled in the art can alter Cas protein specificity genomic targets, which may be partially dependent on how specific the gRNA targeting sequence is relative to the genomic target compared to other parts of the genome. is decided to

[0197] In some embodiments, the gRNA scaffold sequence is: GUUUAGAGC UAGAAAUAGC AAGUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU.

[0198] In some embodiments, the CRISPR protein-derived domain incorporated in the base editor is an endonuclease (eg, a deoxyribonuclease or ribonuclease) that is capable of binding to a target polynucleotide when combined with a binding guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated in the base editor is a nickase that is capable of binding to a target polynucleotide when combined with a binding guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated in the base editor is a catalytically dead domain that is capable of binding to a target polynucleotide when combined with a binding guide nucleic acid. In some embodiments, the target polynucleotide that binds to the CRISPR protein-derived domain of the base editor is DNA, and in some embodiments, the target polynucleotide that binds to the CRISPR protein-derived domain of the base editor is RNA.

[0199] CAs proteins that can be used herein include class 1 and class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Cssx16, Cx16, Cx, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Css1, Cssd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpf1, Examples of these include Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i, CARF, DinG, homologues thereof, or variants thereof. Unmodified CRISPR enzymes, like Cas9, can have DNA cleavage activity with two functional endonuclease regions, RuvC and HNH. The CRISPR enzyme is capable of inducing cleavage of one or both strands in a target sequence, such as within the target sequence and / or in the complementary strand of the target sequence. For example, the CRISPR enzyme is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 bases from the first or last nucleotide of the target sequence. Breaks of one or both strands in pairs or more can be induced.

[0200] Vectors encoding CRISPR enzymes mutated against the corresponding wild-type enzyme can be used to deny the ability to cleave one or both strands of the target polynucleotide containing the target sequence. Cas9 is a wild-type exemplary Cas9 polypeptide (eg, Cas9 from S. pyogenes) and at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93 It can refer to a polypeptide having %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology. Cas9 is at most or approximately 50%, 60%, 70%, 80%, 90%, 91 for example wild-type Cas9 polypeptides (eg, those from S. pyogenes). It can refer to a polypeptide having %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology. Cas9 can refer to a wild type or a modified form of a Cas9 protein that may contain amino acid changes such as a deletion, insertion, substitution, variant, mutation, fusion, chimeras, or any combination thereof.

[0201] In one embodiment, the CRISPR protein-derived domain of the base editor is Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Refs: NC_021284 .1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (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); Neisseria meningitidis (NCBI Ref: YP_002342100.1), Streptococcus pyogenes, or Cas9 from Staphylococcus aureus can be included in all or part.

[0202] [Cas9 domain of the nucleobase editor] The sequence and structure of the Cas9 nuclease are well known to those skilled in the art (eg, "Complete genome sequence of an Ml strain of Streptococcus pyogenes." Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G. , Lyon K., Primeaux C, Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J. , Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 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., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunology.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337 See:816-821 (2012), the entire contents of which are incorporated by reference herein). Cas9 orthologs have been described in a variety of species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will become apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences are described in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunocy systems (2013) RNA Biology 10:5, 726-737 Cas9 sequences derived from organisms and loci. The entire contents of which are incorporated by reference herein.

[0203] In some embodiments, the 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, a Cas9 domain can be a Cas9 domain that cleaves both strands of a double-stranded nucleic acid (eg, both strands of a double-stranded DNA molecule). In some embodiments, the Cas9 domain comprises any one of the amino acid sequences described herein. In some embodiments, the Cas9 domain is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, for any one of the amino acid sequences described herein. Includes an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity. In some embodiments, the Cas9 domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, compared to any one of the amino acid sequences described herein. 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, Includes amino acid sequences having 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations. In some embodiments, the Cas9 domain is at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, compared to any one of the amino acid sequences described herein. 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 includes an amino acid sequence having at least 1200 identical continuous amino acid residues.

[0204] In some embodiments, a protein comprising a fragment of Cas9 is provided. For example, in some embodiments, the protein comprises one of the following two Cas9 domains: (1) the gRNA-binding domain of Cas9; (2) the DNA cleavage domain of Cas9. In some embodiments, a protein containing Cas9 or a fragment thereof is referred to as a "Cas9 variant." The Cas9 variant shares homology with Cas9 or a fragment thereof. For example, the 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% The same, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the Cas9 mutant is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, compared to wild-type Cas9. 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, It can have amino acid changes of 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (eg, a gRNA-binding domain or DNA-cleaving domain), the fragments being at least about 70% identical to the corresponding fragment of wild-type Cas9, and 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 and is at least about 99.5% identical, or at least about 99.9% identical. 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 of the amino acid length of the corresponding wild-type Cas9. %, 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%. In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, It is 1150, 1200, 1250, or at least 1300 amino acids in length.

[0205] In some embodiments, the Cas9 fusion protein provided herein comprises the full-length amino acid sequence of the Cas9 protein, eg, one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not include the full-length Cas9 sequence and only include one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and further suitable sequences of the Cas9 domains and fragments will be apparent to those skilled in the art.

[0206] A Cas9 protein can bind to a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to that guide RNA. In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a Cas9 domain, such as a nuclease active Cas9, Cas9 nickase (nCas9), or nuclease-inactive Cas9 (dCas9). Examples of nucleic acid programmable DNA binding proteins include, but are not limited to, Cas9 (eg, dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b / C2cl, and Cas12c / C2c3.

[0207] 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). ATGGATAAGAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGGGGGTGATCACTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTAT CAAAAAAATCTTATAGGGCTCTTTATTTGGCAGTGGAGAGAGAGCGGAAGCGACTCGTCCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTGTTATCTACAGGAGATTT TTTCAAATGAGATGGCGAAAGTAGATGATAGTTCTTCATCATCGACTTGAAGAGTCATTTTTGGTGGAAGAGAAGAAGCATGAACGTCATCCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTAT CATGAGAAATATCCAACTATCATCATCTGCGAAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATGCGCATATGATTAAGTTTCGTGGTCATTTTTGAG GGAGATTTAAATCCTGATAATAGTGTGGACAAACTATTATCCAGTTGGTACAAATCTACAATCAATCAATTATTGAAGAAACCCTATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTGC ACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAGAAATGGCTTGTTGGGAATCTCATTGCTTTGTCATGTCATGGGATTGACCCCTAATTTTAAATCAAATTTG ATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTATACGATGATGATTTAGATAATTGGCGCAAATTGGAGATCAATATGCTGATTTGGCAGCTAAGAATTTATCAGATGCT ATTTTACTTCAGATATCCTCAGAGAGTAAATAGTGAAATAACTAAGGCTCCCCCTATCAGCTCAATGATTAAGCGCTACGATGAACATCAAGACTTAGCTTTTAAAAGCTTTTAGTTCGACAACAA CTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGG TACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTGACAACGGCTCTATTCCCATCAAATTCACACTTGGGTGAGCTGCATGCTATTTGGAGAAGAAGAAG ACTTTTATCCATTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAA GAAACAATTACCCCATGGAATTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAAATTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAAACATAGTTG CTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAATGCGAAAACCAGCATTTCTTCAGGTGAACAGAGAAAGCCATTGTTGATTTACTCTCAAAACAAAA TCGAAAAGTAACCGTTAAGCAATTAAAGAGAATTATTCAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGCGCCTACCATGATTTGCTAA AAATTAAAGATAAAGATTTTTTGGATAATGAGAAAATGAAATGAAGATATTGTTTTTAACATGATTGACCCTTATTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATGCTCAC CTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGGACGTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTGAAA TCAGATGGTTTTGCCAATCGCAATTTATGCAGCTGATCCATGATGATAGTTGACATTTAAGAGATATTCAAAGAAGCAGGTGTCTGGACAAGGCCATAGTTACATGAACAGATTGCTAACTT AGCTGGCAGTCCTGCTATTAAAAAGGTATTTACAGACTGTAAAAATTGTTGATGAACTGGTCAAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGAACTC AAAAGGCCAGAAAATTCGCGAGAGAGCGTATGAAACGAATCGAAGAGGTATCAAGAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCCAATTGCAAAATGAAAGCTCTATC TATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATGTTTAAGTGATTATGTCGATCACAAAGTTTCATACAAGTTTCATACAAGTTTCATCAATAGACAATAAGGTA CTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAAGATGAAAACTATTGGAGACAACTTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTTGATAA TTTAACGAAAGCTGAACGTGGAGTTTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTGGATAGTCGCATGAATACTA AATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATTAGTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATGCC CATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAACTTGAATCGGAGTTGTGTGATTATAAAGTTGTTGGTGATTATAAAGTTGTTGTAAAATGATTGCTAAGTCTGAGCAA GAAATAGGCAAAGCAACCGCAAAATATTTCTTTATACATGAACTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGG AGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGCCCCAAGTCAAGTCAATATTGTCAAGAAAACAGAGAGAGAGGCGGATTCTCCAAGGAGTCAATTTTTACCAA AAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTGGTTGCTAAGGTGGAAAAGGGAAATCGAAG AAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAGTTCCTTGAAAAAATCCGATTGACTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAGACTTAATCATTAAACTA CCTAAATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTTTATAGCTAGTCA TTATGAAAGTTGAAGGTAGTCCAGAGAAGAAGAAAAACAATTGTTGTGGAGCATAAGCATAAGCATTTAGATGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAG ATGCCAATTTAGATAAAGTTCTTAGTGCATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAAATATATAT TTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTAGATGCCACTCATCAATCCACTGGTCTTATATGAAACACGCATTGATGAGGTGACTGA TIFF0007631209000009.tif170162 (Single underline: HNH domain, double underline: RuvC domain)

[0208] In some embodiments, wild-type Cas9 corresponds to or includes the following nucleotide and / or amino acid sequences: ATGGATAAAAAGTATTCTATGGTTTAGACATCGGCACTAATTCCGTTGGGCTGTCATAACCGATGAATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAA AAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACAAGAAATTTTTTAGCAA TGAGATGGCCAAAGTTGACGATTCTTTCTCACCGTTGGAAGAGTCCTTCTTGGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAGT ACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTGGCTCTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATC CGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATCTTAGCGCCCGCCTCTCTAAATCC CGACGGCTAGAAAACCTGATCGCACAATTACCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTGACACCAAATTTTAAGTCGAACTTCGAAGATGCCAAA TTGCAGCTTAGTAAGGACACGTACGATCTCGACAATCTACTGGCACAAATTGGAGAATCAGTATGCGGACTTATTTTGGCTGCCAAAAACCTTAGCGATGCAATCCCTATCTGACATACTGAGA GTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAGGTACATGAACATCAAGACTTGACACCTCAAGGCCTAGTCCGTCAGCAACTGCCTGAGAATATAAGGAAATATTC TTTGATCAGTCGAAAAACGGGTACGGTTATGACGGGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCCATATTGAGAAGATGGATGGGGACGGAAGAGTTGCTTGTAAAACTCAATCG CGAAGATCTACTGCGAAAGCAGCGGACTTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATATGAAGGCAGGAGGATTTTTTATCCGTTCCAAAGACAATCGTGAAAA GATTGAGAAAATCCTAACCTTTTCGCATACCTTATGTGGGACCCTGCCGAGGGAACTCTCGGTTCGCATGGACAAGAAAGTCCGAAGAAACGATTACTCCCATGGAATTTGGAAGTTGTCG ATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGAGGATGACCAACTTTGACAAGAATTTGAACGAAAAAGTATTGCCTAAGCAGTTTACTATTACGAGAGTGTACAATGAACTCACGAAAG TTAAGTATGTCACTGAGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAGAAGAAGCAATAGTAGATCTGTTATCAAGACCGCAAAGTGACAGTTAAGCAATTGAAGAGGACTATTTAAG AAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCCTCCTAAAGATAATTAAGATAAGATAAGGACTTCCTGGATAACGAGAGAATGAAGAT ATCTTAGAAGATATAGTGTTGACTCTTACCCTTTGAAGATCGGGAAATGATTGAGGAAAAACATACGCTCACCTGTTCGGAACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGC TGGGGACGATTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTATGCAGCTGATCCATGATGACTCTTTA ACCTTCAAAGAGGATATACAAAGGCACAGTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTGCTGGTTCGCCAGCCATCAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGA GCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGATAGAGAGGTATTAAAGA ACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCCAATTGCAGAACGAGAAACTTGCAAAATGGAAGGGACATGTATGTTGATCAGGATAAACCGTTTAT CTGATTACGACGTCGATCACATTGTACCCCCAATCCCTTTTTGAAGGACGAATTCGAATAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGA AAATGAAGAACTATTGGCGCAGCTCCTAAATGCGAAACTGATAACGCAAGAAGTTCGATAACTTAACTAAGCTGAGAGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTC GTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGAGATAGATTCCCGAATGAATACGAAATACGAGAGAGAGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAATTGGTGTCGGAC TTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTAATGCCGTCGTAGGGACCGCACTCATTAAGAATACCCCGAAGCTAGAAAGTGAGTTTGTG TATGGTGATTACAAAGTTATATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCCAAATACTTCTTTATATGAATTTCTTAAGACGGAAATCACTCTGGCA AACGGAGAGATACGCAAACGACCTTTAATGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCAAGTCAACATAGTAAAGAA AACTGAGGTGCAGACCGGAGGTTTTCAAAGGAATCGATTCTCCAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGGACCGAAAAAGTACGGTGGCTTCGATAGCCTACAGTTGCCTAA TTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAACTGAAGTCAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCGTCTTTTGAAAAGAACCCCATCGACTTCCTGGGCGA AAGGTTACAAGGAAGTAAAAGGATCTCATAATTAAACTACCAAGTATAGTCTGTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGAGCTTCAAAGGGGAACGAACTCGCACTAC CGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAGAGAAGCAACTTTTGTTGAGCAGCAAAACATTATCTCGACGAAATCATAGAGCAAA ATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGCCAATCTGGACAAAGTATTAAGCGCATACAAGCAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCC AACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACAACGATAGATCGCAAACGATAGATCGCAAACGATACACTTCCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCACGGGATTATATGAAACTCGGATA GATTTGTCACAGCTTGGGGTGACGGATCCCCCAAGAGAGAAGAGGAAAGTCTCGAGCGACTACAAGACCATGACGGTGATTATAAAGATCATGACAAGGATGACGATGACAAGGCTGCAGGA TIFF0007631209000010.tif169164 (single underlined: HNH domain, double underlined: RuvC domain).

[0209] In some embodiments, wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI reference sequence: NC_002737.2 (the nucleotide sequence is as follows) and Uniprot reference sequence: Q99ZW2 (the amino acid sequence is as follows) It's the street)): ATGGATAAGAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGGGGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTAT CAAAAAAATCTTATAGGGCTCTTTATTATTGACAGTGGAGAGAGAGCGGAAGCGACTCGTCCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTGTTATCTACAGGAGATTT TTTCAAATGAGATGGCGAAAGTAGATGATAGTTCTTCATCATCGACTTGAAGAGTCATTTTTGGTGGAAGAGAAGAAGCATGAACGTCATCCTATTTTGGAAATATAGTAGATGAAGTTGCTTAT CATGAGAAATATCCAACTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATGCGCATATGATTAAGTTTCGTGGTCATTTTTGAG GGAGATTTAAATCCTGATAATAGTGTGGACAAACTATTATCCAGTTGGTACAAACCTACAATCAATCAATTATTGAAGAAAACCCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGC ACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTGGGAATCTCATTGCTTTGTCATGGGTTGACCCCCTAATTTTAAATCAAATTTG ATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTATACGATGATGATTTAGATAATTGGCGCAAATTGGAGATCAATATGCTGATTTGGCAGCTAAGAATTTATCAGATGCT ATTTTACTTCAGATATCCTCAGAGAGTAAATACTGAAATAACTAAGGCTCCCCCTATCAATGATGAACGCTACGATGAACATCAAGACTTAGCTTTTAAAAGCTTTTAGTTCGACAACAA CTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGG TACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTGACAACGGCTCTATTCCCATCAAATTCACACTTGGGTGAGCTGCATGCTATTTGGAGAAGAAGAAG ACTTTTATCCATTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAA GAAACAATTACCCCATGGAATTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAAATTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAAACATAGTTG CTTTATGAGTATTTTACGGTTTATAACGAATTAGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTCAGGTGAACAGAGAAAGCCATTGTTGATTTACTCTCAAAACAAAA TCGAAAAGTAACCGTTAAGCAATTAAAGAGAATTATTCAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATAGATTTAATGCTTCATTAGGTACCATGATTTGCTAAAA AATTATTAAAGATAAAGATTTTTTGGATAATGAGAGAAAATGAAATGATATTGTTTTTAACATGACC TCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGGACGTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTGAAAT CAGATGGTTTTGCCAATCGCAATTTATGCAGCTGATCCATGATGATAGTTGACATTTAAGAGAGAGATTCAAAAGCAAGTGTCTGGACAAGGCGATAGTTACATGAACATATGCAAATTTA GCTGGTAGCCCTGCTATTAAAAAGGTATTTACAGACTGTAAAAGTTGTTGATGAATGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGAAC TCAAAAGGCCAGAAAATTCGCGAGAGAGCGTATGAAACGAATCGAAGAGGTATCAAGAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTC TCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATTAATCGTTTAAGTGATTATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGAATAAGG TCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAAGATGAAAACTATTGGAGACAACTTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTGAT AATTTAACGAAAGCTGAACGTGGAGTTTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTGGATAGTCGCATGAATACT AAATACGATGAAAATGATAAACTTATTCGAGAGAGGTTAAAGTGATTACCTTAAAATTAGTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATGC CCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCCAAAACTTGAATCGGAGTTGTGTGATTATAAAGTTGTTGTGATTATAAAGTTGTTGTAAAATGATTGCTAAGTCTGAGCA AGAAATAGGCAAAGCAACCGCAAAATATTTCTTTATACATGAACTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTG GAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGCCCCAAGTCAAGTCAAGTCAATATTGTCAAGAAACAGAGAGAGGCGGATTCTCCAAGGAGTCAATTTTACCA AAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAA GAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAGTTCCTTGAAAAAATCCGATTGACTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAGACTTAATCATTAAACT ACCTAAATATAGTCTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTTTAGCTAGTC ATTATGAAAGTTGAAGGTAGTCCAGAGAAGAAGAAAAACAATTGTTGTGGAGCATAAGCATAAGCATTATTAGATGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTAGCAG ATGCCAATTTAGATAAAGTTCTTAGTGCATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAAATATATAT TTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTAGATGCCACTCATCAATCCACTGGTCTTATATGAAACACGCATTGATGAGGTGACTGA TIFF0007631209000011.tif169163 (Single underline: HNH domain, double underline: RuvC domain)

[0210] In some embodiments, Cas9 includes Corynebacterium ulcerans (NCBI reference sequence: NC_015683.1, NC_017317.1), Corynebacterium diphtheria (NCBI reference sequence: NC_016782.1, NC_016786.1), Spiroplasma syrphidicola (NCBI reference sequence: NC_021284 .1), Prevotella intermedia (NCBI reference sequence: NC_017861.1), Spiroplasma taiwanense (NCBI reference sequence: NC_021846.1), Streptococcus iniae (NCBI reference sequence: NC_021314.1), Belliella baltica (NCBI reference sequence: NC_018010.1), Psychroflexus torquisI (NCBI reference sequence: NC_018721.1), Streptococcus thermophilus (NCBI reference sequence: YP_820832.1), Listeria innocua (NCBI reference sequence: NP_472073.1), Campylobacter jejuni ( Refers to Cas9 from NCBI reference sequence: YP_002344900.1), or from Neisseria meningitidis (NCBI reference sequence: YP_002342100.1), or Cas9 from any other organism.

[0211] It is to be understood that additional Cas9 proteins, including their variants and homologues (eg, nuclease-inactive Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), are within the scope of this disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is the nuclease-inactive form of Cas9 (dCas9). In some embodiments, the Cas9 protein is Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is nuclease-active form Cas9.

[0212] 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 without cleaving any strand of the double-stranded nucleic acid molecule (eg, via a gRNA molecule). In some embodiments, the nuclease-inactive dCas9 domain corresponds in any of the D10X and H840X mutations of the amino acid sequences described herein, or in any of the amino acid sequences provided herein. Includes mutations, X is any amino acid change. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10A and H840A mutation of the amino acid sequences described herein, or a corresponding mutation in any of the amino acid sequences described herein. . By way of example, the nuclease-inactive Cas9 domain comprises the following amino acid sequences provided in the cloning vector pPlatTET-gRNA 2 (access number BAV54124):

[0213] The amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows: MDKKYSIGLAIGTNS VGWAVIT DEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEEN PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQ LPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSE QEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK LPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (See, e.g. Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.” Cell. 2013; 152(5):1173-83, the entire contents of which are hereby incorporated by reference. is incorporated into).

[0214] Additional suitable nuclease-inactive dCas9 domains will become apparent to those skilled in the art based on their knowledge in the present disclosure and within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, mutant domains of D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A ( 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 by reference herein.) .

[0215] In some embodiments, the Cas9 nuclease has an inactive (eg, inactivated) DNA cleavage domain, ie, Cas9 is a nickase referred to as the "nCas9" protein (meaning "nickase" Cas9). . Nuclease-inactivated Cas9 proteins may be referred to as compatible as "dCas9" proteins (meaning nuclease "dead" Cas9) or catalytically inactive Cas9. Methods for creating Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains are known (e.g., Jinek et al., Science. 337:816-821 (2012), Qi et al., “Repurposing See CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28;152(5):1173-83, the entire contents of each of which are incorporated by reference herein. . For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to gRNA, while the RuvC1 subdomain cleaves the strand non-complementary. Mutations within these subdomains may silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of S. pyrogenes Cas9 (Jinek et al., Science. 337:816-821 (2012), Qi et al., Cell. 28;152 ( 5):1173-83 (2013)).

[0216] In some embodiments, the dCas9 domain is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, for any of the dCas9 domains provided herein. Contains an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity. In some embodiments, the Cas9 domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, compared to any of the amino acid sequences described herein. 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, Includes amino acid sequences having 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations. In some embodiments, the Cas9 domain is at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, compared to any of the amino acid sequences described herein. , 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 It includes an amino acid sequence having at least 1200 identical contiguous amino acid residues.

[0217] In some embodiments, dCas9 corresponds to, or includes, or in part, or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises a D10A and H840A mutation or a corresponding mutation in another Cas9.

[0218] In some embodiments, dCas9 comprises the amino acid sequences of dCas9 (D10A and H840A): TIFF0007631209000012.tif168162 (single underlined: HNH domain; double underlined: RuvC domain).

[0219] In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 in the amino acid sequence provided above, or at the corresponding position in any of the amino acid sequences provided herein. The group remains histidine.

[0220] In other embodiments, there is provided a dCas9 variant with mutations other than D10A and H840A, leading to, for example, nuclease-inactivated Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions in D10 and H840, or other substitutions within the nuclease domain of Cas9 (eg, substitutions in the HNH nuclease subdomain and / or RuvC1 subdomain). In some embodiments, a variant or homolog of dCas9 is 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 Those having about 99.5% identical, or at least about 99.9% identity are provided. In certain embodiments, short or long, or 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 A variant of dCas9 having an amino acid sequence is provided.

[0221] In some embodiments, the Cas9 domain is Cas9 nickase. Cas9 nickases can be Cas9 proteins that are capable of cleaving only one strand of a double-stranded nucleic acid molecule (eg, double-stranded DNA molecule). In some embodiments, the Cas9 nickase cleaves the target strand of a double-stranded nucleic acid molecule, which forms a base pair with the gRNA (eg, sgRNA) that the Cas9 nickase is bound to Cas9 ( It means to cut the strands (complementary). In some embodiments, the Cas9 nickase comprises a D10A mutation and has histidine at position 840. In some embodiments, Cas9 nickase cleaves the non-target, non-base editing strand of double-stranded nucleic acid molecules, which causes the Cas9 nickase to base pair with a gRNA (eg, sgRNA) bound to Cas9. It means to cut off unformed chains. In some embodiments, the Cas9 nickase contains an H840A mutation and has an aspartate residue at position 10, or a corresponding mutation. In some embodiments, the Cas9 nickase is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, with any of the Cas9 nickases provided herein. , comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity. Further suitable Cas9 nickases will be apparent to those skilled in the art based on this disclosure and knowledge in the art, and are within the scope of this disclosure.

[0222] The amino acid sequence of an exemplary catalytic Cas9 nickase (nCas9) is as follows: MDKKYSIGLAIGTNS VGWAVIT DEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEEN PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQ LPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSE QEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK LPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0223] In some embodiments, Cas9 refers to Cas9 from archaeals (eg, nanoarchia) that make up the domains and territories of single-cell prokaryotic microorganisms. In some embodiments, the nucleic acid programmable DNA binding protein is described, for example, in Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21 It is designated as CasX or CasY, the entire contents of which are incorporated by reference herein. Using genomic degradation metagenomics, many CRISPR-Cas systems have been identified, including Cas9, which was first reported in the archaeal domain of life. This branched Cas9 protein was discovered in very little-studied nanoarchia as part of the active CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, have been discovered, which are among the most compact systems ever discovered. In some embodiments, in the base editor systems described herein, Cas9 is replaced by a CasX or a variant of CasX. In some embodiments, in the base editor systems described herein, Cas9 is replaced by a CasY or a CasY variant. It is to be understood that other RNA-induced DNA-binding proteins may also be used as nucleic acid programmable DNA-binding proteins (napDNAbp) and are within the scope of this disclosure.

[0224] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein can be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, napDNAbp is a CasY protein. In some embodiments, 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, relative to the naturally occurring CasX or CasY protein. Contains an amino acid sequence having 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity. In some embodiments, the programmable nucleotide binding protein is a naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide binding protein is at least 85%, at least 90%, at least 91%, at least 92%, at least 93, relative to any of the CasX or CasY proteins described herein. Includes an amino acid sequence having an identity of %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%. It is to be understood that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.

[0225] Exemplary CasX ((uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53) tr|F0NN87|F0NN87_SULIHCRISPR-associatedCasx protein OS = Sulfolobus islandicus (strain HVE10 / 4) GN = SiH_0402 PE=4 SV=1) The amino acid sequence of: MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRRVKLE VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0226] The amino acid sequence for an example CasX (>tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS = Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4 SV=1) is as follows: MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRRVKLEVEPHYLIMAAAGWVLTRLGKAKV SEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0227] Deltaproteobacteria CasX MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTT AGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDI IIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGV TAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQ KWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLL SLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASK SRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEY QITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0228] The amino acid sequence of an example CasY ((ncbi.nlm.nih.gov / protein / APG80656.1) >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacteria]) is as follows: MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPREIVSAINDDYVGLYGLSNFDDLYNAEKRNEEKVYSVLDFWYDCVQYGAVFSYTAPGLLKNVAEVRGGSYELTKTLKGSHLYDELQIDKVIKFLNKKEISRAN GSLDKLKKDIID CFKAEYRERHKDQCNKLADDIKNAKKDAGASLGERQKKLFRDFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNRNRGEVLFNKLKEYAQKLDKNEGSLEMWEYIGIGNSGTAFSNFLGEGFLGRLRENKITELKKA MMDITDAWRGQEQEEELEKRLRILAALTIKLREPKFDNHWGYRSDINGKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEMINRFGESDTKEEAVVSSLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDVQEALI KERLEAEKKKPKRKKSDAEDEKETIDFKELFPHLAKPLKLVPNFYGDSKRELYKKYKNAAIYTDALWKAVEKIYKSAFSSSLKNSFFDTDFDKDFFIKRLQKIFSVYRRFNTDKWKPIVKNSFAPYCDIVSLAENEVLYKPKQSRSRKS AAIDKNRVRLPSTENIAKAGIALARELSVAGFDWKDLLKKEEHEEYIDLIELHKTALALLLAVTETQLDISALDFVENGTVKDFMKTRDGNLVLEGRFLEMFSQSIVFSELRGLAGLMSRKEFITRSAIQTMNGKQAELLYIPHEFQSAKI TTPKEMSRAFLDLAPAEFATSLEPESLSEKSLLKLKQMRYYPHYFGYELTRTGQGIDGGVAENALRLEKSPVKKREIKCKQYKTLGRGQNKIVLYVRSSYYQTQFLEWFLHRPKNVQTDVAVSGSFLIDEKKVKTRWNYDALTVALEPVSGS ERVFVSQPFTIFPEKSAEEEGQRYLGIDIGEYGIAYTALEITGDSAKILDQNFISDPQLKTLREEVKGLKLDQRRGTFAMPSTKIARIRESLVHSLRNRIHHLALKHKAKIVYELEVSRFEEGKQKIKKVYATLKKADVYSEIDADKNLQT TVWGKLAVASEISASYTSQFCGACKKLWRAEMQVDETITTQELIGTVRVIKGGTLIDAIKDFMRPPIFDENDTPFPKYRDFCDKHHISKKMRGNSCLFICPFCRANADADIQASQTIALLRYVKEEKKVEDYFERFRKLKNIKVLGQMKKI.

[0229] The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Cas9 undergoes conformational changes upon target binding, which causes the nuclease domain to be placed so as to cleave the reverse strand of the target DNA. The final result of Cas9-mediated DNA cleavage is a double-stranded break (DSB) within the target DNA (upstream of approximately 3-4 nucleotide PAM sequences). The resulting DSB is then repaired by one of two general repair pathways: (1) the efficient but often false non-homologous terminal join (NHEJ) pathway, or (2) the less efficient is a high fidelity homologous recombination repair (HDR) pathway.

[0230] The "efficiency" of non-homologous terminal joining (NHEJ) and / or homologous recombination repair (HDR) can be calculated by any convenient method. For example, in some embodiments, efficiency can be expressed in terms of percentage of successful HDR. For example, cleavage products can be generated using a test nuclease assay, and the product-to-substrate ratio can be used to calculate the percentage. For example, a test nuclease enzyme can be used that directly cleaves DNA containing the newly incorporated restriction sequence as a result of successful HDR. More cleaved substrates exhibit a higher percentage HDR (higher HDR efficiency). As an illustrative example, the percentage of HDR can be calculated using the following equation [(cleavage product) / (substrate+cleavage product)] (e.g. (b+c) / (a) +b+c), where "a" is the band strength of the DNA substrate, and "b" and "c" are cleavage products).

[0231] In some embodiments, efficiency can be expressed in terms of percentage of successful NHEJs. For example, the T7 endonuclease I assay can be used to generate cleavage products, and the product-to-substrate ratio can be used to calculate the percentage of NHEJ. T7 endonuclease I cleaves mismatched heteroduplex DNA resulting from hybridization of wild type with mutant DNA strands (NHEJ results in small random insertions or deletions (indels) at the original cleavage site. ). More cuttings show a higher percentage of NHEJ (higher NHEJ efficiency). As an illustrative example, the 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 strength of the DNA substrate, and "b" and "c" are 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).

[0232] The NHEJ repair pathway is the most active repair mechanism, frequently causing small nucleotide insertions or deletions (indels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical implications, as cell populations expressing Cas9 and gRNA or guide polynucleotides can result in a diverse array of mutations. In some embodiments, the NHEJ produces small indels in the target DNA, resulting in deletion, insertion, or frameshift of an amino acid that results in an immature stop codon within the open reading frame (ORF) of the target gene. Mutations are brought about. The ideal end result is a loss-of-function mutation within the target gene.

[0233] While NHEJ-mediated DSB repair often disrupts the open reading frame of genes, homologous recombination repair (HDR) is a specific nucleotide range from single nucleotide changes to large insertions such as fluorophores or tag additions. It can be used to generate changes. To utilize HDR for gene editing, a DNA repair template containing the desired sequence can be delivered in the cell type of interest along with gRNA and Cas9 or Cas9 nickase. The repair template can contain the desired edits and additional homologous sequences immediately upstream and downstream of the target (referred to as the left and right homologous arms). The length of each homologous arm may depend on the magnitude of the change introduced, with larger inserts requiring longer homologous arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. The efficiency of HDR is generally low (<10% of modified alleles) even in cells expressing Cas9, gRNA, and exogenous repair templates. Because HDR occurs during the S and G2 phases of the cell cycle, the efficiency of HDR can be enhanced by synchronizing the cells. Chemical or genetically inhibiting genes involved in NHEJ can also increase the frequency of HDR.

[0234] In some embodiments, Cas9 is a modified Cas9. A given gRNA targeting sequence can have additional sites throughout the genome where partial homology is present. These sites are called off-targets and need to be considered when designing gRNA. In addition to optimizing gRNA design, CRISPR specificity can also be increased by modification to Cas9. Cas9 results in double-stranded breaks (DSBs) due to the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase, a D10A mutant of SpCas9, carries a single nuclease domain and produces a DNA nick rather than a DSB. The nickase system can also be combined with HDR-mediated gene editing for specific gene editing.

[0235] In some embodiments, Cas9 is a variant Cas9 protein. Variant Cas9 polypeptides have an amino acid sequence that differs (eg, having one deletion, insertion, substitution, fusion) when compared to the amino acid sequence of the wild-type Cas9 protein. In some instances, the variant Cas9 polypeptide has an amino acid change (eg, a deletion, insertion, or substitution) that reduces the nuclease activity of the Cas9 polypeptide. For example, in some examples, the variant Cas9 polypeptide is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or 1 of the nuclease activity of the corresponding wild-type Cas9 protein Has less than %. In some embodiments, the variant Cas9 protein does not have substantial nuclease activity. If the Cas9 protein of interest is a variant Cas9 protein that does not have substantial nuclease activity, this can be referred to as "dCas9."

[0236] In some embodiments, the variant Cas9 protein has reduced nuclease activity. For example, the variant Cas9 protein is less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or about 0.1 of the endonuclease activity of a wild-type Cas9 protein, such as a wild-type Cas9 protein indicates less than %.

[0237] In some embodiments, the variant Cas9 protein is capable of cleaving the complementary strand of the guide target sequence, but has a reduced ability to cleave the non-complementary strand of the double-stranded guide target sequence. For example, the variant Cas9 protein can have mutations (amino acid substitutions) that reduce the function of the RuvC domain. By way of non-limiting example, in some embodiments, the variant Cas9 protein has D10A (aspartic acid to alanine at amino acid position 10) and thus may cleave the complementary strand of the double-stranded guide target sequence. Although it is possible, its ability to cleave non-complementary strands of double-stranded guide target sequences is reduced (and therefore, when variant Cas9 protein cleaves double-stranded target nucleic acids, instead of double-stranded breaks (DSB). Single-strand breaks (SSBs) occur) (see, e.g. Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21).

[0238] In some embodiments, the variant Cas9 protein is capable of cleaving the non-complementary strand of a double-stranded guide target sequence, but has a reduced ability to cleave the complementary strand of a guide target sequence. For example, the variant Cas9 protein can have mutations (amino acid substitutions) that reduce the function of the HNH domain (RuvC / HNH / RuvC domain motif). By way of non-limiting example, in some embodiments, the variant Cas9 protein has a H840A (histidine to alanine at amino acid position 840) mutation, and thus can cleave the non-complementary strand of the guide target sequence. , the ability to cleave the complementary strand of the guide target sequence is reduced (and therefore, SSBs occur in place of the DSB when the variant Cas9 protein cleaves the double-stranded guide target sequence). Such Cas9 proteins have reduced ability to cleave guide target sequences (eg, single-stranded guide target sequences), but retain the ability to bind to guide target sequences (eg, single-stranded guide target sequences). There is.

[0239] In some embodiments, the variant Cas9 protein has a reduced ability to cleave both complementary and non-complementary strands of double-stranded target DNA. By way of non-limiting example, in some embodiments, the variant Cas9 protein carries both D10A and H840A mutations, so that the polypeptide is a complementary and non-complementary strand of the double-stranded target DNA. The ability to cut both is reduced. Such Cas9 proteins have a reduced ability to cleave target DNA (eg, single-stranded target DNA), but retain the ability to bind to target DNA (eg, single-stranded target DNA).

[0240] As another non-limiting example, in some embodiments, the variant Cas9 protein carries mutations in W476A and W1126A, resulting in a reduced ability of the polypeptide to cleave target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (eg, single-stranded target DNA), but retain the ability to bind to target DNA (eg, single-stranded target DNA).

[0241] As another non-limiting example, in some embodiments, the variant Cas9 protein carries mutations in P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, so that the polypeptide is capable of forming a target DNA. The ability to cut is reduced. Such Cas9 proteins have a reduced ability to cleave target DNA (eg, single-stranded target DNA), but retain the ability to bind to target DNA (eg, single-stranded target DNA).

[0242] As another non-limiting example, in some embodiments, the variant Cas9 protein carries mutations in H840A, W476A, and W1126A, resulting in a reduced ability of the polypeptide to cleave target DNA. It's. Such Cas9 proteins have a reduced ability to cleave target DNA (eg, single-stranded target DNA), but retain the ability to bind to target DNA (eg, single-stranded target DNA). As another non-limiting example, in some embodiments, the variant Cas9 protein carries mutations in H840A, D10A, W476A, and W1126A, so that the polypeptide is capable of cleaving target DNA. It's falling. Such Cas9 proteins have a reduced ability to cleave target DNA (eg, single-stranded target DNA), but retain the ability to bind to target DNA (eg, single-stranded target DNA). In some embodiments, the variant Cas9 has a catalytic His residue repaired at position 840 of the Cas9 HNH domain (A840H).

[0243] As another non-limiting example, in some embodiments, the variant Cas9 protein carries mutations in H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, so that the polypeptide is a targeted target The ability to cleave DNA is reduced. Such Cas9 proteins have a reduced ability to cleave target DNA (eg, single-stranded target DNA), but retain the ability to bind to target DNA (eg, single-stranded target DNA). As another non-limiting example, in some embodiments, the variant Cas9 protein carries mutations in D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, resulting in the polypeptide being , its ability to cleave target DNA is reduced. Such Cas9 proteins have a reduced ability to cleave target DNA (eg, single-stranded target DNA), but retain the ability to bind to target DNA (eg, single-stranded target DNA). In some embodiments, if the variant Cas9 protein carries mutations in W476A and W1126A, or if the variant Cas9 protein carries mutations in P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A does not efficiently bind to PAM sequences. Thus, in some such embodiments, when such variant Cas9 protein is used in methods of binding, the method does not require a PAM sequence. In other words, in some embodiments, when such variant Cas9 protein is used in the method of binding, the method can include guide RNA, but this method cannot be performed in the absence of a PAM sequence. (hence, binding specificity is provided by the targeting segment of the guide RNA). Other residues can be mutated to achieve the above effects (ie, inactivate one or the other nuclease moieties). By way of non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 can be modified (ie, replaced). Also, mutations other than alanine substitution are suitable.

[0244] In some embodiments, a variant Cas9 protein with reduced catalytic activity (eg, Cas9 proteins are suddenly in a 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), are variants as long as they retain the ability to interact with the guide RNA. Cas9 proteins are still able to bind to target DNA in a site-specific manner (as they are still induced to target DNA sequences by guide RNA).

[0245] 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.

[0246] Some embodiments include amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER), and have specificity for the modified PAM5'-NGC-3' Modified SpCas9 was used.

[0247] An alternative to S. pyrogenes Cas9 can include RNA-induced endonucleases from the Cpf1 family that exhibit cleavage activity in mammalian cells. CRISPR (CRISPR / Cpf1) from Prevotella and Francisella 1 is a DNA editing technique similar to the CRISPR / Cas9 system. Cpf1 is an RNA-induced endonuclease of the class II CRISPR / Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses guide RNA to search for and cleave viral DNA. Cpf1 is a smaller, simple endonuclease than Cas9, and overcomes some of the constraints of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, the result of Cpf1-mediated DNA cleavage is a double-stranded cleavage with a short 3' overhang. The staggered cleavage pattern of Cpf1 can open up the possibility of directional gene transfer similar to conventional restriction enzyme cloning, which can increase the efficiency of gene editing. Similar to the Cas9 variants and homologous species mentioned above, Cpf1 expands the number of sites that can be targeted by CRISPR to an AT-rich region or AT-rich genome that lacks the NGG PAM site that SpCas9 prefers. It can also be done. The Cpf1 locus contains a mixed alpha / beta domain, RuvC-I followed by a helical region, RuvC-II, and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, Cpf1 does not have an HNH endonuclease domain and the N-terminus of Cpf1 does not have an alpha-helix recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain structure indicates that Cpf1 is functionally unique and is classified as a class 2, V-type CRISPR system. The Cpf1 locus encodes Cas1, Cas2, and Cas4 proteins that are more similar to Type I and III than the Type II system. Functional Cpf1 does not require transactivated CRISPR RNA (tracrRNA), and therefore only CRISPR (crRNA). This is beneficial for genome editing, as Cpf1 is not only smaller than Cas9, but also has smaller sgRNA molecules (about half the number of nucleotides in Cas9). In contrast to the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex is targeted by the identification of protospacers flanking the motif 5'-YTN-3' or 5'-TTN-3'. Or cleave the RNA. After identification of PAM, Cpf1 introduces an adhesive end-like DNA double-stranded break with overhangs of 4 or 5 nucleotides.

[0248] In some embodiments, Cas9 is a Cas9 variant that has specificity for the modified PAM sequence. In some embodiments, additional Cas9 variants and PAM sequences are described in Miller, S.M., et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs, Nat. Biotechnol. (2020), which is The present invention is incorporated by reference in its entirety. In some embodiments, the Cas9 variant does not have a PAM requirement for specificity. In some embodiments, the Cas9 variant, eg, the SpCas9 variant, has specificity for NRNH PAM, where R is A or G and H is A, C, or T. In some embodiments, the SpCas9 variant has specificity for the PAM sequence AAA, TAA, CAA, GAA, TAT, GAT, or CAC. In some embodiments, the SpCas9 variant is positioned at numbering in SEQ ID NO: 1, 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 at the corresponding position thereof, contains amino acid substitutions. In some embodiments, the SpCas9 variant is numbered in SEQ ID NO: 1, or positions 1114, 1135, 1218, 1219, 1221, 1249, 1320, 1321, 1323, 1332, 1333, 1335, or 1337, or corresponding to positions In positions contain amino acid substitutions. In some embodiments, the SpCas9 variant is positioned at numbering in SEQ ID NO: 1, 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1219, 1221, 1256, 1264, 1290, 1318, 1317 , 1320, 1323, 1333, or at the corresponding position thereof, contains amino acid substitutions. In some embodiments, the SpCas9 variant is positioned at numbering in SEQ ID NO: 1, 1114, 1131, 1135, 1150, 1156, 1180, 1191, 1218, 1219, 1221, 1227, 1249, 1253, 1286, 1293, 1320 , 1321, 1332, 1335, 1339, or at the corresponding position thereof, contains amino acid substitutions. In some embodiments, the SpCas9 variant is positioned at numbering in SEQ ID NO: 1, 1114, 1127, 1135, 1180, 1207, 1219, 1234, 1286, 1301, 1332, 1335, 1337, 1338, 1349, or its correspondence The amino acid substitution is included in the position. Exemplary amino acid substitutions and PAM specificity of the SpCas9 variant are shown in Tables 1A-1D.

[0249] Table 1A [Table 1-1]

[0250] Table 1B [Table 1-2]

[0251] Table 1C [Table 1-3]

[0252] Table 1D [Table 1-4]

[0253] In some embodiments, Cas9 is Neisseria menigitidis Cas9 (NmeCas9) or a variant thereof. In some embodiments, NmeCas9 has specificity for NNNNGAYW PAM, where Y is C or T and W is A or T. In some embodiments, NmeCas9 has specificity for NNNNGYTT PAM, where Y is C or T. In some embodiments, NmeCas9 has specificity for NNNNGTCT PAM. In some embodiments, NmeCas9 is Nme1 Cas9. In some embodiments, NmeCas9 is NNNNGATT PAM, NNNCCTA PAM, NNNCCTC PAM, NNNNCCTT PAM, NNNNCCTG PAM, NNNNCCTG PAM, NNNNCCGT PAM, NNNNCGGPAM, NNNNCCA PAM, NNNNCCCCT PAM, NNNNCCAG PAM, NNNNCAT PAM, or NNNGATT It has specificity for PAM. In some embodiments, Nme1Cas9 has specificity for NNNNGATT PAM, NNNNCCTA PAM, NNNNCCTC PAM, NNNNCCTC PAM, NNNNCCTT PAM, or NNNNCCTG PAM. In some embodiments, NmeCas9 has specificity for CAA PAM, CAAA PAM, or CCA PAM. In some embodiments, NmeCas9 is Nme2 Cas9. In some embodiments, NmeCas9 has specificity for NNNNCC(N4CC)PAM, where N is any one of A, G, C, or T. In some embodiments, NmeCas9 has specificity for NNNNCCGT PAM, NNNNCCGGPAM, NNNNCCCA PAM, NNNNCCCCT PAM, NNNNCCC PAM, NNNNCCAT PAM, NNNNCCAG PAM, NNNNCCAT PAM, or NNNGATT PAM. In some embodiments, NmeCas9 is Nme3Cas9. In some embodiments, NmeCas9 has specificity for NNNNCAAA PAM, NNNNCC PAM, or NNNNCNN PAM. Additional NmeCas9 features and PAM sequences described in Edraki et al. Mol. Cell. (2019) 73(4): 714-726 are incorporated by reference in their entirety.

[0254] An exemplary amino acid sequence for Nme1Cas9 is provided below: Type II CRISPR RNA-induced endonuclease Cas9 [Neisseria meningitidis]WP_002235162.1 1 maafkpnpin yilgldigia svgwamveid edenpiclid lgvrvferae vpktgdslam 61 arrlarsvrr ltrrrahrll rarrllkreg vlqaadfden glikslpntp wqlraaaldr 121 kltplewsav llhlikhrgy lsqrkneget adkelgallk gvadnahalq tgdfrtpael 181 alnkfekesg hirnqrgdys htfsrkdlqa elillefekqk efgnphvsgg lkegietllm 241 tqrpalsgda vqkmlghctf epaepkaakn tytaerfiwl tklnnlrile qgserpltdt 301 Eatlmdepy rkskltyaqa rkllgledta ffkglrygkd naeastlmem kayhaisral 361 ekeglkdkks plnlspelqd eigtafslfk tdeditgrlk driqpeilea llkhisfdkf 421 vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee kiylppipad eirnpvvlra 481 lsqarkving vvrrygspar ihietarevg ksfkdrkeie krqeenrkdr ekaaakfrey 541 fpnfvgepks kdilklrlye qqhgkclysg keinlgrlne kgyveidhal pfsrtwddsf 601 nnkvlvlgse nqnkgnqtpy ​​eyfngkdnsr ewqefkarve tsrfprskkq rillqkfded 661 gfkernlndt ryvnrflcqf vadrmrltgk gkkrvfasng qitnllrgfw glrkvraend 721 rhhaldavvv acstvamqqk itrfvrykem nafdgktidk etgevlhqkt hfpqpweffa 781 qevmirvfgk pdgkpefeea dtpeklrtll aeklssrpea vheyvtplfv srapnrkmsg 841 qghmetvksa krldegvsvl rvpltqlklk dlekmvnrer epklyealka rleahkddpa 901 kafaepfyky dkagnrtqqv kavrveqvqk tgvwvrnhng iadnatmvrv dvfekgdkyy 961 lvpiyswqva kgilpdravv qgkdeedwql iddsfnfkfs lhpndlvevi tkkarmfgyf 1021 aschrgtgni nirhdldhk igkngilegi gvktalsfqk yqidelgkei rpcrlkkrp 1081 vr

[0255] An exemplary amino acid sequence for Nme2Cas9 is provided below: Type II CRISPR RNA-induced endonuclease Cas9 [Neisseria meningitidis]WP_002230835.1 1 maafkpnpin yilgldigia svgwamveid eeenpirlid lgvrvferae vpktgdslam 61 arrlarsvrr ltrrrahrll rarrllkreg vlqaadfden glikslpntp wqlraaaldr 121 kltplewsav llhlikhrgy lsqrkneget adkelgallk gvannahalq tgdfrtpael 181 alnkfekesg hirnqrgdys htfsrkdlqa elillefekqk efgnphvsgg lkegietllm 241 tqrpalsgda vqkmlghctf epaepkaakn tytaerfiwl tklnnlrile qgserpltdt 301 Eatlmdepy rkskltyaqa rkllgledta ffkglrygkd naeastlmem kayhaisral 361 ekeglkdkks plnlsselqd eigtafslfk tdeditgrlk drvqpeilea llkhisfdkf 421 vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee kiylppipad eirnpvvlra 481 lsqarkving vvrrygspar ihietarevg ksfkdrkeie krqeenrkdr ekaaakfrey 541 fpnfvgepks kdilklrlye qqhgkclysg keinlvrlne kgyveidhal pfsrtwddsf 601 nnkvlvlgse nqnkgnqtpy ​​eyfngkdnsr ewqefkarve tsrfprskkq rillqkfded 661 gfkecnlndt ryvnrflcqf vadhilltgk gkrrvfasng qitnllrgfw glrkvraend 721 rhhaldavvv acstvamqqk itrfvrykem nafdgktidk etgkvlhqkt hfpqpweffa 781 qevmirvfgk pdgkpefeea dtpeklrtll aeklssrpea vheyvtplfv srapnrkmsg 841 ahkdtlrsak rfvkhnekis vkrvwlteik ladlenmvny kngreielye alkarleayg 901 gnakqafdpk dnpfykkggq lvkavrvekt qesgvllnkk naytiadngd mvrvdvfckv 961 dkkgknqyfi vpiyawqvae nilpdidckg yriddsytfc fslhkydlia fqkdekskve 1021 fayyincdss ngrfylawhd kgskeqqfri stqnlvliqk yqvnelgkei rpcrlkkrp 1081 vr

[0256] [Cas12 domain of the nucleobase editor] Typically, the microbial CRISPR-Cas system is divided into class 1 and class 2 systems. The class 1 system has a multi-subunit effector complex, while the class 2 system has a single protein effector. For example, Cas9 and Cpf1 are class 2 effects but are of different types (type II and V, respectively). In addition to Cpf1, class 2, V-type CRISPR-Cas systems also include Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. 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,” See Science, 2019 Jan. 4;363:88-91, the entire contents of each of which are incorporated by reference herein. The V-type Cas protein contains a RuvC (or RuvC-like) endonuclease domain. While the production of mature CRISPR RNA (crRNA) is generally tracrRNA-independent, for example Cas12b / C2c1 requires tracrRNA for the production of crRNA. Cas12b / C2c1 depends on both crRNA and tracrRNA for DNA cleavage.

[0257] Nucleic acid programmable DNA binding proteins contemplated in the present invention include Cas proteins classified as Class 2, V type (Cas12 protein). Non-limiting examples of Cas class 2, V-type proteins include Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i, their homologues, or modified versions thereof. can be mentioned. As used herein, the Cas12 protein can also be referred to as a Cas12 nuclease, a Cas12 domain, or a Cas12 protein domain. In some embodiments, the Cas12 proteins of the invention comprise an amino acid sequence interrupted by an internally fused protein domain, such as a deaminase domain.

[0258] In some embodiments, the Cas12 domain is a nuclease-inactive Cas12 domain or Cas12 nickase. In some embodiments, the Cas12 domain is a nuclease-active domain. For example, a Cas12 domain can be a Cas12 domain that nicks one strand of a double-stranded nucleic acid (eg, a double-stranded DNA molecule). In some embodiments, the Cas12 domain comprises any one of the amino acid sequences described herein. In certain embodiments, the Cas12 domain is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least Contains an amino acid sequence that is 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical. In some embodiments, the Cas12 domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 compared to any one of the amino acid sequences described herein. ,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 , includes amino acid sequences having mutations of 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more. In some embodiments, the Cas12 domain is at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least compared to any one of the amino acid sequences described herein. 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, It includes an amino acid sequence having at least 1100, or at least 1200 identical contiguous amino acid residues.

[0259] In some embodiments, a protein comprising a fragment of Cas12 is provided. For example, in some embodiments, the protein comprises one of two Cas12 domains: (1) a gRNA-binding domain of Cas12 or (2) a DNA cleavage domain of Cas12. In some embodiments, proteins containing Cas12 or fragments thereof are referred to as "Cas12 variants." The Cas12 variant shares homology with Cas12 or a fragment thereof. For example, the Cas12 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% The same, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the Cas12 variant is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, compared to wild-type Cas12. 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, It can have 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes. In some embodiments, the Cas12 variant comprises a fragment of Cas12 (eg, a gRNA-binding domain or DNA cleavage domain), the fragment being at least about 70% identical to the corresponding fragment of wild-type Cas12, and 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 be. 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% of the amino acid length of the corresponding wild-type Cas12. , 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%. In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 , 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0260] In some embodiments, Cas12 includes, or part or in whole, a Cas12 amino acid sequence having one or more mutations that modify Cas12 nuclease activity. By way of example, such mutations include amino acid substitutions within the RuvC nuclease domain of Cas12. In some embodiments, 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 Variants or homologs of Cas12 are provided that are %identical or at least about 99.9%identical. In some embodiments, 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 Provided are variants of Cas12 having a short or long amino acid sequence by 50 amino acids, about 75 amino acids, about 100 amino acids, or more amino acids.

[0261] In some embodiments, the Cas12 fusion protein provided herein includes a Cas12 protein full-length amino acid sequence, eg, one of the Cas12 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not include the full-length Cas12 sequence and contain only one or more fragments thereof. Exemplary amino acid sequences for suitable Cas12 domains are provided herein, and additional suitable sequences for the Cas12 domain and fragments will be apparent to those skilled in the art.

[0262] Generally, class 2, V-type Cas proteins have a single functional RuvC endonuclease domain (eg, Chen et al., “CRISPR-Cas12a target binding unleased indiscriminate single-stranded DNase activity,” Science 360:436-439 (See 2018). In some cases, the Cas12 protein is the variant Cas12b protein. (See Strecker et al., Nature Communications, 2019, 10(1): Art. No.: 212). In one embodiment, the variant Cas12 polypeptide differs in 1, 2, 3, 4, 5, or more amino acids when compared to the amino acid sequence of the wild-type Cas12 protein (eg, deletion, insertion, It has an amino acid sequence with substitutions, fusions). In some instances, the variant Cas12 polypeptide has an amino acid change (eg, a deletion, insertion, or substitution) that reduces the activity of the Cas12 polypeptide. For example, in some examples, variant Cas12 is 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. is a Cas12b polypeptide having In some cases, the variant Cas12b protein does not have substantial nickase activity.

[0263] 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.

[0264] In some embodiments, the Cas12 protein includes RNA-induced endonucleases from the Cas12a / Cpf1 family that exhibit activity in mammalian cells. CRISPR (CRISPR / Cpf1) from Prevotella and Francisella 1 is a DNA editing technique similar to the CRISPR / Cas9 system. Cpf1 is an RNA-induced endonuclease of the class II CRISPR / Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses guide RNA to search for and cleave viral DNA. Cpf1 is a smaller, simple endonuclease than Cas9, and overcomes some of the constraints of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, the result of Cpf1-mediated DNA cleavage is a double-stranded cleavage with a short 3' overhang. The staggered cleavage pattern of Cpf1 can open up the possibility of directional gene transfer similar to conventional restriction enzyme cloning, which can increase the efficiency of gene editing. Similar to the Cas9 variants and homologous species mentioned above, Cpf1 expands the number of sites that can be targeted by CRISPR to an AT-rich region or AT-rich genome that lacks the NGG PAM site that SpCas9 prefers. It can also be done. The Cpf1 locus contains a mixed alpha / beta domain, RuvC-I followed by a helical region, RuvC-II, and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, unlike Cas9, Cpf1 does not have an HNH endonuclease domain and the N-terminus of Cpf1 does not have an alpha-helix recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain structure indicates that Cpf1 is functionally unique and is classified as a class 2, V-type CRISPR system. The Cpf1 locus encodes Cas1, Cas2, and Cas4 proteins that are more similar to Type I and III than the Type II system. Functional Cpf1 does not require transactivated CRISPR RNA (tracrRNA), and therefore only CRISPR (crRNA). This is beneficial for genome editing, as Cpf1 is not only smaller than Cas9, but also has smaller sgRNA molecules (about half the number of nucleotides in Cas9). In contrast to the G-rich PAM targeted by Cas9, t...

Claims

1. 化合物分合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物� or contacting a base editor complexed with multiple guide polynucleotides, the base editor comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain is: TadA*7.10 amino acid sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD or comprises an amino acid sequence having at least 90% sequence identity to a fragment thereof lacking only N-terminal methionine, the adenosine deaminase domain being numbered V82S, Y147T, Y147R, and 合方法分合物分合物分合物合物合物合物合物合物合物合物。 154S, including at least one modification selected from the group consisting of Q154S, One or more of the guide polynucleotides target the base editor to result in an A.T to G.C modification of the SNP associated with GSD1a, where the SNP associated with GSD1a is the amino acid position It terminates early at 347 or leads to expression of a G6PC polypeptide containing a cysteine ​​at position 83; method.

2. whether the polynucleotide programmable DNA binding domain is Streptococcus pyogenes Cas9 (SpCas9); Does the polynucleotide programmable DNA binding domain include modified SpCas9 having modified protospacer flanking motif (PAM) specificity or having specificity for non-G PAM; Is the polynucleotide programmable DNA binding domain Staphylococcus aureus Cas9 (SaCas9)? whether the polynucleotide programmable DNA binding domain is nuclease-inactive or nickase; or The polynucleotide programmable DNA binding domain is a nickase containing an amino acid substitution D10A or a corresponding amino acid substitution; 2. The method of claim 1.

3. 3. The method of claim 1 or 2, wherein the adenosine deaminase domain comprises a V82S modification with numbering based on the TadA*7.10 amino acid sequence.

4. 4. The method of claim 3, wherein the adenosine deaminase domain further comprises a Y123H modification with numbering based on the TadA*7.10 amino acid sequence.

5. The adenosine deaminase domain is numbered based on the TadA*7.10 amino acid sequence; Y147T and Q154R; Y147T and Q154S; Y147R and Q154S; V82S and Q154S; V82S and Y147R; V82S and Q154R; V82S and Y123H; I76Y and V82S; V82S, Y123H, and Y147T; V82S, Y123H, and Y147R; V82S, Y123H, and Q154R; Y147R, Q154R, and Y123H; Y147R, Q154R, and I76Y; Y147R, Q154R, and T166R; Y123H, Y147R, Q154R, and I76Y; V82S, Y123H, Y147R, and Q154R; and I76Y, V82S, Y123H, Y147R, and Q154R 3. The method of claim 1 or 2, comprising a combination of modifications selected from the group consisting of:

6. 6. The method of any one of claims 1 to 5, wherein the base editor further comprises a wild-type adenosine deaminase domain.

7. Polynucleotide programmable DNA-binding domain and the following TadA*7.10 amino acid sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD or a base editor comprising an adenosine deaminase domain containing an amino acid sequence having at least 90% sequence identity to a fragment thereof lacking only the N-terminal methionine, wherein the adenosine deaminase domain is said to be described as TadA*7.10 amino acid sequence a base editor, or a polynucleotide encoding the base editor, comprising at least one modification selected from the group consisting of V82S, Y147T, Y147R, and Q154S, by numbering according to: One or more guide polynucleotides that target the base editor to result in the A.T to G.C modification of the SNP associated with GSD1a, the SNP associated with GSD1a is early at amino acid position 347. and a guide polynucleotide that terminates or leads to expression of a G6PC polypeptide containing a cysteine ​​at position 83. Includes in vitro or ex vivo cells.

8. 8. The cell of claim 7, which is a mammalian hepatocyte or a hepatocyte precursor.

9. 9. The cell of claim 8, wherein the hepatocyte is an iPSc-derived hepatocyte.

10. The guide polynucleotide is a) GACCUAGGCGAGGCAGUAGG; b) CCAGUAUGGACACUGUCCAAA; c) CAGUAUGGACACUGUCCAAA; and d) AGUAUGGACACUGUCCAAAG 9. The cell of claim 7 or 8, comprising a nucleic acid sequence selected from the group consisting of:

11. A base editor containing a polynucleotide programmable DNA-binding domain and an adenosine deaminase domain, said adenosine deaminase domain having the following TadA*7.10 amino acid sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD or comprises an amino acid sequence having at least 90% sequence identity to a fragment thereof lacking only the N-terminal methionine, where the adenosine deaminase domain is numbered based on the TadA*7.10 amino acid sequence; and a base editor, or a polynucleotide encoding the base editor, comprising at least one modification selected from the group consisting of: One or more guide polynucleotides targeting the base editor to result in the A.T to G.C modification of the SNP associated with GSD1a, the SNP associated with GSD1a is at amino acid position 347 One or more guide polynucleotides and that lead to expression of glucose-6-phosphatase (G6PC) polypeptides that terminate early or contain cysteine ​​at position 83. a pharmaceutical composition for the treatment of GSD1a in a subject, comprising:

12. (a) For induced pluripotent stem cells or hepatocyte precursors containing SNPs associated with glycogen disease type 1a (GSD1a), A base editor containing a polynucleotide programmable nucleotide binding domain and an adenosine deaminase domain, said adenosine deaminase domain having the following TadA*7.10 amino acid sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD or comprises an amino acid sequence having at least 90% sequence identity to a fragment thereof lacking only the N-terminal methionine, where the adenosine deaminase domain is numbered based on the TadA*7.10 amino acid sequence; and a base editor, or a polynucleotide encoding the base editor, comprising at least one modification selected from the group consisting of: One or more guide polynucleotides that target the base editor to result in an A.T to G.C modification of the SNP associated with GSD1a, the SNP associated with GSD1a, wherein the SNP associated with GSD1a is at amino acid position 347 and guide polynucleotides that terminate early or lead to expression of a glucose-6-phosphatase (G6PC) polypeptide containing a cysteine ​​at position 83. to introduce and (b) Differentiating said induced pluripotent stem cells into hepatocytes or precursors thereof; 合物合物合物合物合物。 合物合物合物合物合物。 合物合物合物合物合物合物。 合物合物合物合物

13. 13. The method of claim 12, wherein the hepatocyte precursor is obtained from a subject having GSD1a.

14. 14. The method of claim 12 or 13, wherein the adenosine deaminase domain comprises a V82S modification with numbering based on the TadA*7.10 amino acid sequence.

15. 15. The method of claim 14, wherein the adenosine deaminase domain further comprises a Y123H modification with numbering based on the TadA*7.10 amino acid sequence.

16. The adenosine deaminase domain is numbered based on the TadA*7.10 amino acid sequence Y147T and Q154R; Y147T and Q154S; Y147R and Q154S; V82S and Q154S; V82S and Y147R; V82S and Q154R; V82S and Y123H; I76Y and V82S; V82S, Y123H, and Y147T; V82S, Y123H, and Y147R; V82S, Y123H, and Q154R; Y147R, Q154R, and Y123H; Y147R, Q154R, and I76Y; Y147R, Q154R, and T166R; Y123H, Y147R, Q154R, and I76Y; V82S, Y123H, Y147R, and Q154R; and I76Y, V82S, Y123H, Y147R, and Q154R 14. The method of claim 12 or claim 13, comprising a combination of modifications selected from the group consisting of:

17. 17. The method of any one of claims 12 to 16, wherein the base editor further comprises a wild-type adenosine deaminase domain.

18. 化合物分合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物合物� or contacting a base editor complexed with multiple guide polynucleotides; The base editor includes an adenosine deaminase domain inserted into a Cas9 or Cas12 polypeptide, the adenosine deaminase domain having the following TadA*7.10 amino acid sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD or comprises an amino acid sequence having at least 90% sequence identity to a fragment that lacks only N-terminal methionine, where the adenosine deaminase domain is numbered based on the TadA*7.10 amino acid sequence; and at least one modification selected from the group consisting of: Q154S; One or more of the guide polynucleotides target the base editor to result in an A.T to G.C modification of the SNP associated with GSD1a, where the SNP associated with GSD1a is amino acid It terminates early at position 347 or leads to expression of a G6PC polypeptide containing a cysteine ​​at position 83; method.

19. A fusion protein containing an adenosine deaminase domain inserted within a Cas9 or Cas12 polypeptide, said adenosine deaminase domain having the following TadA*7.10 amino acid sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD or comprises an amino acid sequence having at least 90% sequence identity to a fragment that lacks only N-terminal methionine, where the adenosine deaminase domain is numbered based on the TadA*7.10 amino acid sequence; and a fusion protein, or a polynucleotide encoding the fusion protein, comprising at least one modification selected from the group consisting of: Q154S; One or more guide polynucleotides that target the fusion protein to result in the A.T to G.C modification of a single nucleotide polymorphism (SNP) associated with GSD1a, the SNP associated with GSD1a is , and a guide polynucleotide that terminates early at amino acid position 347 or leads to expression of a G6PC polypeptide containing a cysteine ​​at position 83. a pharmaceutical composition for the treatment of glycogenic disease type 1a (GSD1a) in a subject, comprising:

20. 20. The pharmaceutical composition of claim 19, wherein the adenosine deaminase domain comprises a V82S modification with numbering based on the TadA*7.10 amino acid sequence.

21. 21. The pharmaceutical composition of claim 20, wherein the amino acid sequence further comprises a Y123H modification with numbering based on the TadA*7.10 amino acid sequence.

22. a) A base editor containing a polynucleotide programmable DNA-binding domain and an adenosine deaminase domain, said adenosine deaminase domain having the following TadA*7.10 amino acid sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD or comprises an amino acid sequence having at least 90% sequence identity to a fragment that lacks only N-terminal methionine, where the adenosine deaminase domain is numbered based on the TadA*7.10 amino acid sequence; and a base editor comprising at least one modification selected from the group consisting of: b) one or more guide polynucleotides that target the base editor to result in the A.T to G.C modification of the SNP associated with GSD1a, wherein the SNP associated with GSD1a is amino acid position 347 and guide polynucleotides that terminate early in or lead to expression of a G6PC polypeptide containing cysteine ​​at position 83. A pharmaceutical composition for the treatment of glycogen disease type 1a (GSD1a) containing an effective amount of:

23. 11. A pharmaceutical composition for the treatment of glycogenic disease type 1a (GSD1a), comprising an effective amount of a cell according to any one of claims 7 to 10.

24. A kit for the treatment of glycogen disease type 1a (GSD1a), a) A base editor containing a polynucleotide programmable DNA-binding domain and an adenosine deaminase domain, said adenosine deaminase domain having the following TadA*7.10 amino acid sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD or comprises an amino acid sequence having at least 90% sequence identity to a fragment that lacks only N-terminal methionine, where the adenosine deaminase domain is numbered based on the TadA*7.10 amino acid sequence; and a base editor comprising at least one modification selected from the group consisting of: b) one or more guide polynucleotides capable of targeting the base editor to result in the A, T to G, C modification of the SNP associated with GSD1a, the SNP associated with GSD1a, A guide polynucleotide that terminates early at amino acid position 347 or leads to expression of a G6PC polypeptide containing a cysteine ​​at position 83. kit including.

25. 11. A kit for the treatment of glycogenic disease type 1a (GSD1a), comprising the cells according to any one of claims 7 to 10.

26. A base editor system comprising a base editor complexed with one or more guide polynucleotides, the base editor comprising a polynucleotide programmable DNA binding domain and the following TadA*7.10 amino acid sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD or an adenosine deaminase domain containing an amino acid sequence having at least 90% sequence identity to a fragment thereof lacking only the N-terminal methionine, wherein the adenosine deaminase domain is numbered based on the TadA*7.10 amino acid sequence 合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合合� Providing a modification from to G.C, where the SNP associated with GSD1a is one that terminates early at amino acid position 347 or results in expression of a G6PC polypeptide containing a cysteine ​​at position 83; Base editor system.

27. 30. The base editor system of claim 26, wherein the adenosine deaminase domain further comprises a Y123H modification with numbering based on the TadA*7.10 amino acid sequence.

28. The polynucleotide programmable DNA binding domain is a modified Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), or a modified Streptococcus pyogenes Cas9 (SpCas9); and / or The polynucleotide programmable DNA binding domain is SpCas9 with modified protospacer adjacent motif (PAM) specificity or specificity for non-G PAM; and / or The polynucleotide programmable DNA binding domain is either nuclease-inactive Cas9 or Cas9 nickase; 28. The base editor system of claim 26 or claim 27.

29. One or more guide RNAs, and The following array: EIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSL FELENGRKRMLASAKFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSGG SGGSGGSGGMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGH FLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEN PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLP EKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLI HDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSE EVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEGADKRTADGSEFESPKKKRKV* a polynucleotide programmable DNA binding domain containing The following TadA*7.10 amino acid sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD or an adenosine deaminase domain containing an amino acid sequence having at least 90% sequence identity to a fragment thereof lacking only the N-terminal methionine, and with a numbering based on the TadA*7.10 amino acid sequence; A fusion protein comprising at least one base editor domain containing an adenosine deaminase domain, comprising at least one modification selected from the group consisting of Q154S 合方法合物合物合物合物合物合物合物。 The base editor system comprising one or more of the guide RNAs target the base editor to result in an alteration from A, T to G, C of the SNP associated with GSD1a, where: The SNPs associated with are those that terminate early at amino acid position 347 or lead to expression of a G6PC polypeptide containing a cysteine ​​at position 83; Base editor system.

30. 30. In vitro or ex vivo cells comprising the base editor system according to any of claims 26-29.

31. The cell is a mammalian cell and / or The cells are ex vivo or in vitro; 31. The cell of claim 30.

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