Compositions and methods for treating hemoglobinopathies

Abstract

The present invention relates to compositions and methods for treating heme pathologies. The present invention features compositions and methods for editing deleterious mutations associated with heme pathologies such as sickle cell disease (SCD). In a specific embodiment, the present invention provides a method for correcting mutations in beta globin polynucleotides using a modified adenosine base editor called "ABE8" with unprecedented levels of efficiency (e.g., >60 to 70%).

Description

[0001] This invention is a divisional application of the PCT patent application entered into China with Chinese patent application number 202080028554.6, invention name “Compositions and methods for treating hemoglobinopathies”, and international application date February 13, 2020.

[0002] Cross-references to related applications

[0003] This application is an International PCT application, which claims priority to and the benefit of U.S. Provisional Application Nos. 62 / 805,271, filed February 13, 2019; 62 / 805,277, filed February 13, 2019; 62 / 852,224, filed May 23, 2019; 62 / 852,228, filed May 23, 2019; 62 / 931,722, filed November 6, 2019; 62 / 931,747, filed November 6, 2019; 62 / 941,569, filed November 27, 2019; and 62 / 966,526, filed January 27, 2020, all of which are incorporated herein by reference in their entirety.

[0004] Cited references

[0005] All publications, patents, and patent applications mentioned in this specification are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the absence of any other indication, the contents of the publications, patents, and patent applications mentioned in this specification are still incorporated by reference in their entirety. Technical Field

[0006] The present invention relates to a composition and method for treating hemoglobinopathies. Background Art

[0007] Sickle cell disease (SCD) is a disorder affecting hemoglobin, the molecule in red blood cells that carries oxygen to cells throughout the body. People with this condition have atypical hemoglobin molecules that deform their red blood cells into a sickle or crescent shape. The clinical symptoms of SCD are attributed to intermittent episodes of microvascular occlusion, leading to tissue ischemia / reperfusion injury and chronic hemolysis. Vaso-occlusive events are associated with ischemia / reperfusion injury to tissue, causing pain and acute or chronic damage affecting any organ system. Commonly affected are the bones / bone marrow, spleen, liver, brain, lungs, kidneys, and joints.

[0008] SCD is a genetic disorder characterized by the presence of at least one heme S allele (HbS; p.Glu6Val in HBB) and a second pathogenic variant in HBB, resulting in abnormal heme polymerization. HbS / S (homozygous p.Glu6Val in HBB) accounts for 60% to 70% of SCD cases in the United States. Life expectancy for men and women with SCD is only 42 and 48 years, respectively. Current treatments focus on managing disease symptoms. There is a great need for methods to edit the genetic mutations that cause SCD and other heme disorders. Summary of the Invention

[0009] As described below, the present invention features compositions and methods for editing deleterious mutations associated with sickle cell disease (SCD). In certain embodiments, the present invention provides for the use of a modified adenosine deaminase base editor, designated "ABE8," with unprecedented potency (e.g., >60 to 70%) to correct SCD mutations.

[0010] In one embodiment, the invention features a method for editing a beta globin polynucleotide comprising a sickle cell disease-associated single nucleotide polymorphism (SNP), the method comprising contacting the beta globin polynucleotide with one or more guide RNAs and a fusion protein comprising a polynucleotide-programmable DNA binding domain and at least one base editor domain, the base editor domain being an adenosine deaminase variant comprising a modification at amino acid position 82 and / or 166 of MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, wherein the guide RNA targets the base editor to modify the sickle cell disease-associated SNP.

[0011] In another embodiment, the invention features a method of editing a beta globin (HBB) polynucleotide comprising a sickle cell disease-associated single nucleotide polymorphism (SNP), the method comprising contacting the beta globin polynucleotide with one or more guide RNAs and a fusion protein comprising a polynucleotide-programmable DNA binding domain and at least one base editor domain comprising an adenosine deaminase variant, the polynucleotide-programmable DNA binding domain comprising the following sequence:

[0012]

[0013] The bold sequences indicate sequences derived from Cas9, the italic sequences represent linker sequences, and the underlined sequences represent bipartite nuclear localization sequences.

[0014] The adenosine deaminase variant comprises a modification at amino acid position 82 and / or 166 in MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD.

[0015] In another embodiment, the invention features a base editing system comprising a fusion protein of any of the foregoing embodiments or other embodiments described herein and a guide RNA comprising a nucleic acid sequence selected from the group consisting of CUUCUCCACAGGAGUCAGAU; ACUUCUCCACAGGAGUCAGAU; and GACUUCUCCACAGGAGUCAGAU. In one embodiment, the gRNA further comprises the nucleic acid sequence GUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACGAAACUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAA CACCCUGUCAUUUUAUGGCAGGGUG. In another embodiment, the gRNA comprises a nucleic acid sequence selected from the group consisting of

[0016] CUUCUCCACAGGAGUCAGAUGUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACGAAACUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG;

[0017] ACUUCUCCACAGGAGUCAGAUGUUUUUGUACUCUCAAGAUUUAAGUA ACUGUACAACGAAACUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAA GAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG; and

[0018] The nucleic acid sequence of GACUUCUCCACAGGAGUCAGAUGUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACGAAACUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG.

[0019] In another embodiment, the invention features a cell that is made by introducing into the cell or its progenitor: a base editor, a polynucleotide encoding a base editor into the cell, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain as described in any embodiment described herein; and one or more guide polynucleotides that target the base editor to perform the A·T to G·C modification of a sickle cell disease-associated SNP. In one embodiment, the cell made is a hematopoietic stem cell, a hematopoietic progenitor cell, a proerythrocyte, an erythroblast, a reticulocyte, or an erythrocyte. In another embodiment, the cell or its progenitor cell is a hematopoietic stem cell, a hematopoietic progenitor cell, a proerythrocyte, or an erythroblast. In another embodiment, the hematopoietic stem cell is a CD34 + In another embodiment, the cell is from an individual with sickle cell disease. In another embodiment, the cell is a mammalian cell or a human cell.

[0020] In another embodiment, the invention features a method of treating sickle cell disease in an individual, comprising administering to the individual a cell according to any prior embodiment or any other embodiment of the invention described in detail herein. In one embodiment, the cell is autologous to the individual. In another embodiment, the cell is allogeneic to the individual.

[0021] In another embodiment, the present invention provides an isolated cell or a cell population propagated or expanded from any preceding or any other embodiment of the invention detailed herein.

[0022] In another embodiment, the present invention provides a method for producing an erythrocyte or a progenitor thereof, which involves introducing a base editor or a polynucleotide encoding a base editor into an erythrocyte progenitor comprising a sickle cell disease-associated SNP, wherein the base editor comprises a polynucleotide programmable nucleotide binding domain and an adenosine deaminase variant domain as described in any of the preceding embodiments; and one or more guide polynucleotides, wherein the one or more guide polynucleotides target the base editor to perform the A·T to G·C modification of the sickle cell disease-associated SNP; and differentiating the erythrocyte progenitor into an erythrocyte. In one embodiment, the method involves differentiating the erythrocyte progenitor into one or more hematopoietic stem cells, hematopoietic progenitor cells, proerythroblasts, erythroblasts, reticulocytes, or erythrocytes. In one embodiment, the erythrocyte progenitor involved in the method is a CD34 + cells. In another embodiment, the erythroid progenitor cells are obtained from an individual with sickle cell disease. In another embodiment, the erythroid progenitor cells are mammalian or human cells. In another embodiment, the sickle cell disease-associated SNP is modified from A·T to G·C, changing valine to alanine in the HBB polypeptide. In another embodiment, the sickle cell disease-associated SNP results in the expression of an HBB polypeptide having valine at amino acid position 6. In another embodiment, the sickle cell disease-associated SNP replaces glutamic acid with valine. In another embodiment, cells are selected to modify the sickle cell disease-associated SNP from A·T to G·C. In another embodiment, the polynucleotide programmable DNA binding domain comprises a modified Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), a modified Streptococcus pyogenes Cas9 (SpCas9), or variants thereof.

[0023] In various embodiments of any or any other embodiment of the invention described herein, the adenosine deaminase variant comprises modifications at amino acid positions 82 and 166. In various embodiments of any or any other embodiment of the invention described herein, the adenosine deaminase variant comprises a V82S modification. In various embodiments of any or any other embodiment of the invention described herein, the adenosine deaminase variant comprises a T166R modification. In various embodiments of any or any other embodiment of the invention described herein, the adenosine deaminase variant comprises V82S and T166R modifications. In various embodiments of any or any other embodiment of the invention described herein, the adenosine deaminase variant further comprises one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, and Q154R. In various embodiments of any of the above or any other embodiments of the invention described herein, the adenosine deaminase variant comprises a member selected from the group consisting of Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y12 In the embodiments described above, the adenosine deaminase variant comprises Y147R+Q154R+Y123H. In the embodiments described above, the adenosine deaminase variant comprises Y147R+Q154R+Y123H. In the embodiments described above, the adenosine deaminase variant comprises Y147R+Q154R+I76Y. In the embodiments described above, the adenosine deaminase variant comprises Y147R+Q154R+T166R. In the embodiments described above, the adenosine deaminase variant comprises Y147T+Q154R. In the embodiments described above, the adenosine deaminase variant comprises Y147T+Q154S. In the embodiments described above, the adenosine deaminase variant comprises Y147R+Q154S. In the embodiments described above, the adenosine deaminase variant comprises V82S+Q154S. In the embodiments described above, the adenosine deaminase variant comprises V82S+Y147R. In the embodiments described above, the adenosine deaminase variant comprises V82S+Q154R. In the embodiments described above, the adenosine deaminase variant comprises V82S+Y123H.In the embodiments described above, the adenosine deaminase variant comprises I76Y + V82S. In the embodiments described above, the adenosine deaminase variant comprises V82S + Y123H + Y147T. In the embodiments described above, the adenosine deaminase variant comprises V82S + Y123H + Y147R. In the embodiments described above, the adenosine deaminase variant comprises V82S + Y123H + Q154R. In the embodiments described above, the adenosine deaminase variant comprises Y123H + Y147R + Q154R + I76Y. In the embodiments described above, the adenosine deaminase variant comprises V82S + Y123H + Y147R + Q154R. In the embodiments described above, the adenosine deaminase variant comprises I76Y + V82S + Y123H + Y147R + Q154R. In other embodiments of the above examples, the adenosine deaminase variant comprises a C-terminal deletion starting at a residue selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 157.

[0024] In various embodiments of any or any other embodiment of the invention described herein, the cell is in vivo or ex vivo. In various embodiments of any or any other embodiment of the invention described herein, the A·T to G·C modification of the sickle cell disease-associated SNP changes a valine to an alanine in the HBB polypeptide. In various embodiments of any or any other embodiment of the invention described herein, the sickle cell disease-associated SNP results in the expression of an HBB polypeptide having a valine at amino acid position 6. In various embodiments of any or any other embodiment of the invention described herein, the sickle cell disease-associated SNP replaces a glutamic acid with a valine. In various embodiments of any or any other embodiment of the invention described herein, the A·T to G·C modification of the sickle cell disease-associated SNP results in the expression of an HBB polypeptide having an alanine at amino acid position 6. In various embodiments of any or any other embodiment of the invention described herein, the A·T to G·C modification of the sickle cell disease-associated SNP replaces a glutamic acid with an alanine.

[0025] In various embodiments of any or any other embodiment of the invention described herein, the polynucleotide programmable DNA binding domain is a modified Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), a modified Streptococcus pyogenes Cas9 (SpCas9), or a variant thereof. In various embodiments of any or any other embodiment of the invention described herein, the polynucleotide programmable DNA binding domain comprises a variant of SpCas9 having an altered protospacer adjacent motif (PAM) specificity or specificity for a non-G PAM. In various embodiments of any or any other embodiment of the invention described herein, the altered PAM has specificity for the nucleic acid sequence 5'-NGC-3'. In various embodiments of any or any other embodiment of the invention described herein, the modified SpCas9 comprises amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R, or their corresponding amino acid substitutions. In various embodiments of any or any other embodiment of the invention described herein, the polynucleotide programmable DNA binding domain is a nuclease-inactive or nickase variant. In various embodiments of any or any other embodiment of the invention described herein, the nickase variant comprises the amino acid substitution D10A, or its corresponding amino acid substitution. In various embodiments of any or any other embodiment of the invention described herein, the base editor further comprises a zinc finger domain. In various embodiments of any or any other embodiment of the invention described herein, the zinc finger domain comprises the recognition helix sequences RNEHLEV, QSTTLKR, and RTEHLAR or the recognition helix sequences RGEHLRQ, QSGTLKR, and RNDKLVP. In various embodiments of any of the above or any other embodiments of the invention described herein, the zinc finger domain is one or more zf1ra or zf1rb. In various embodiments of any of the above or any other embodiments of the invention described herein, the adenosine deaminase domain can deaminize adenine in deoxyribonucleic acid (DNA).In various embodiments of any or any other embodiment of the invention described herein, the one or more guide RNAs comprise a CRISPR RNA (crRNA) and a trans-encoded small RNA (tracrRNA), wherein the crRNA comprises a nucleic acid sequence complementary to an HBB nucleic acid sequence comprising a sickle cell disease-associated SNP. In various embodiments of any or any other embodiment of the invention described herein, the base editor is complexed with a single guide RNA (sgRNA), wherein the sgRNA comprises a nucleic acid sequence complementary to an HBB nucleic acid sequence comprising a sickle cell disease-associated SNP. In various embodiments of any or any other embodiment of the invention described herein, the A·T to G·C modification of the sickle cell disease-associated SNP changes a valine to an alanine in the HBB polypeptide. In another embodiment, the sickle cell disease-associated SNP results in expression of an HBB polypeptide having a valine at amino acid position 6. In another embodiment, the sickle cell disease-associated SNP replaces a glutamic acid with a valine. In another embodiment, the A·T to G·C modification of the sickle cell disease-associated SNP results in expression of an HBB polypeptide having an alanine at amino acid position 6. In another embodiment, the A·T to G·C modification of the sickle cell disease-associated SNP results in the substitution of alanine for glutamic acid. In another embodiment, cells are selected to have the A·T to G·C modification of the sickle cell disease-associated SNP. In another embodiment, the polynucleotide programmable DNA binding domain is a modified Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), a modified Streptococcus pyogenes Cas9 (SpCas9), or variants thereof.

[0026] In one embodiment, a method of treating sickle cell disease (SCD) in an individual is provided, wherein the method comprises administering to the individual a fusion protein comprising an adenosine deaminase variant inserted into a Cas9 or Cas12 polypeptide, or a polynucleotide encoding the fusion protein; and one or more guide polynucleotides that target the fusion protein to perform an A·T to G·C modification of an SCD-associated single nucleotide polymorphism (SNP), thereby treating SCD in the individual.

[0027] In another embodiment, a method of treating sickle cell disease (SCD) in an individual is provided, wherein the method comprises administering to the individual an adenosine base editor 8 (ABE8), or a polynucleotide encoding the base editor, wherein ABE8 comprises an adenosine deaminase variant inserted into a Cas9 or Cas12 polypeptide; and one or more guide polynucleotides that target ABE8 to perform an A·T to G·C modification of an SCD-associated SNP, thereby treating SCD in the individual.

[0028] In one embodiment of the method as described above, ABE8 is selected from the group consisting of: ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, ABE8.5-m, ABE8.6-m, ABE8.7-m, ABE8.8-m, ABE8.9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.13-m, ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8.23-m In one embodiment of the method described above, the adenosine deaminase variant comprises the following amino acid sequence:

[0029] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTA HAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGS LMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, and wherein the amino acid sequence comprises at least one modification. In one embodiment, the adenosine deaminase variant comprises a modification at amino acid position 82 and / or 166. In one embodiment, at least one modification comprises: V82S, T166R, Y147T, Y147R, Q154S, Y123H, and / or Q154R.

[0030] In one embodiment of the method described above, the adenosine deaminase variant comprises one of the following modifications: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H +Y147R;V82S+Y123H+Q154R;Y147R+Q154R+Y123H;Y147R+Q154R+I76Y;Y147R+Q154R+T166R;Y123H+Y147R+Q154R+I76Y;V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. In one embodiment of the method as described above, the adenosine deaminase variant is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24. In one embodiment, the adenosine deaminase variant comprises a C-terminal deletion starting at a residue selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 157. In one embodiment, the adenosine deaminase variant is an adenosine deaminase monomer comprising a TadA*8 adenosine deaminase variant domain. In one embodiment, the adenosine deaminase variant is an adenosine deaminase heterodimer comprising a wild-type adenosine deaminase domain and a TadA*8 adenosine deaminase variant domain. In one embodiment, the adenosine deaminase variant is an adenosine deaminase heterodimer comprising a TadA domain and a TadA*8 adenosine deaminase variant domain.

[0031] In one embodiment of the method described above, the SCD-associated SNP is located in the beta globin (HBB) gene. In one embodiment of the method described above, the SNP results in the expression of an HBB polypeptide having a valine at amino acid position 6. In one embodiment of the method described above, the SNP replaces glutamic acid with valine. In one embodiment of the method described above, the A·T to G·C modification of the SNP changes the valine in the HBB polypeptide to an alanine. In one embodiment of the method described above, the A·T to G·C modification of the SNP results in the expression of an HBB polypeptide having an alanine at amino acid position 6. In one embodiment of the method described above, the A·T to G·C modification of the SNP replaces glutamic acid with alanine.

[0032] In one embodiment of the method described above, the adenosine deaminase variant is inserted into a flexible loop, alpha-helical region, unstructured portion, or solvent-accessible portion of a Cas9 or Cas12 polypeptide. In one embodiment of the method described above, the adenosine deaminase variant is flanked by the N-terminal and C-terminal segments of a Cas9 or Cas12 polypeptide. In one embodiment of the method described above, the fusion protein or ABE8 comprises the formula NH2-[N-terminal segment of Cas9 or Cas12 polypeptide]-[adenosine deaminase variant]-[C-terminal segment of Cas9 or Cas12 polypeptide]-COOH, wherein "]-[" in each instance is an optional linker. In one embodiment, the C-terminus of the N-terminal segment or the N-terminus of the C-terminal segment comprises a portion of a flexible loop of a Cas9 or Cas12 polypeptide. In one embodiment, when the adenosine deaminase variant deaminates a target nucleobase, the flexible loop comprises amino acids adjacent to the target nucleobase.

[0033] In one embodiment of the method as described above, the method further comprises administering to the individual a guide nucleic acid sequence to deaminize a target nucleobase of a SNP associated with SCD. In one embodiment, deamination of the target nucleobase of the SNP results in replacement of the target nucleobase with a non-wild-type nucleobase, and wherein deamination of the target nucleobase alleviates a symptom of sickle cell disease. In one embodiment, deamination of the SNP associated with sickle cell disease results in substitution of alanine for glutamic acid.

[0034] In one embodiment of the method described above, the target nucleobase is 1-20 nucleobases away from the PAM sequence in the target polynucleotide sequence. In one embodiment, the target nucleobase is 2-12 nucleobases upstream of the PAM sequence. In one embodiment of the method described above, the N-terminal fragment or the C-terminal fragment of the Cas9 or Cas12 polypeptide binds to the target polynucleotide sequence. In certain embodiments, the N-terminal fragment or the C-terminal fragment comprises a RuvC domain; the N-terminal fragment or the C-terminal fragment comprises an HNH domain; neither the N-terminal fragment nor the C-terminal fragment comprises an HNH domain; or neither the N-terminal fragment nor the C-terminal fragment comprises a RuvC domain. In one embodiment, the Cas9 or Cas12 polypeptide comprises a partial or complete deletion of one or more structured domains, and wherein the deaminase is inserted at the location of the partial or complete deletion of the Cas9 or Cas12 polypeptide. In certain embodiments, the deletion is within the RuvC domain; the deletion is within the HNH domain; or the deletion bridges the RuvC domain and the C-terminal domain.

[0035] In one embodiment of the method described above, the fusion protein or ABE8 comprises a Cas9 polypeptide. In one embodiment, the Cas9 polypeptide is Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), or a variant thereof. In one embodiment, the Cas9 polypeptide comprises the following amino acid sequence (Cas9 reference sequence):

[0036] (Single underline: HNH domain; Double underline: RuvC domain; (Cas9 reference sequence), or a corresponding region thereof. In certain embodiments, the Cas9 polypeptide comprises a deletion of amino acids 1017-1069 (which are numbered according to the Cas9 polypeptide reference sequence), or amino acids corresponding thereto; the Cas9 polypeptide comprises a deletion of amino acids 792-872 (which are numbered according to the Cas9 polypeptide reference sequence), or amino acids corresponding thereto; or the Cas9 polypeptide comprises a deletion of amino acids 792-906 (which are numbered according to the Cas9 polypeptide reference sequence) , or their corresponding amino acids. In one embodiment of the method described above, the adenosine deaminase variant is inserted into a flexible loop of the Cas9 polypeptide. In one embodiment, the flexible loop comprises a region selected from the group consisting of amino acids at positions 530-537, 569-579, 686-691, 768-793, 943-947, 1002-1040, 1052-1077, 1232-1248, and 1298-1300 (numbered according to the Cas9 reference sequence), or their corresponding amino acid positions.

[0037] In one embodiment of the method described above, the deaminase variant is inserted between the following 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 (which are numbered according to the Cas9 reference sequence), or the corresponding amino acid positions. In one embodiment of the method described above, the deaminase variant is inserted between amino acid positions 768-769, 792-793, 1022-1023, 1026-1027, 1040-1041, 1068-1069, or 1247-1248 (numbered according to the Cas9 reference sequence), or corresponding amino acid positions thereof. In one embodiment of the method described above, the deaminase variant is inserted between amino acid positions 1016-1017, 1023-1024, 1029-1030, 1040-1041, 1069-1070, or 1247-1248 (numbered according to the Cas9 reference sequence), or corresponding amino acid positions thereof. In one embodiment of the method described above, the adenosine deaminase variant is inserted into a locus within a Cas9 polypeptide indicated in Table 14A. In one embodiment, the N-terminal fragment comprises amino acid residues 1-529, 538-568, 580-685, 692-942, 948-1001, 1026-1051, 1078-1231, and / or 1248-1297 of the Cas9 reference sequence, or residues corresponding thereto. In one embodiment, the C-terminal fragment comprises amino acid residues 1301-1368, 1248-1297, 1078-1231, 1026-1051, 948-1001, 692-942, 580-685, and / or 538-568 of the Cas9 reference sequence, or residues corresponding thereto.

[0038] In one embodiment of the method as described above, the Cas9 polypeptide is a modified Cas9 and is specific for an altered PAM or a non-G PAM. In one embodiment of the method as described above, the Cas9 polypeptide is a nickase or wherein the Cas9 polypeptide is nuclease-inactivated. In one embodiment of the method as described above, the Cas9 polypeptide is a modified SpCas9 polypeptide. In one embodiment, the modified SpCas9 polypeptide comprises amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER), and is specific for an altered PAM 5'-NGC-3'.

[0039] In another embodiment of the method as described above, the fusion protein or ABE8 comprises a Cas12 polypeptide. In one embodiment, the adenosine deaminase variant is inserted into the Cas12 polypeptide. In one embodiment, the Cas12 polypeptide is Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, or Cas12i. In one embodiment, the adenosine deaminase variant is inserted between the following amino acid positions: a) 153-154, 255-256, 306-307, 980-981, 1019-1020, 534-535, 604-605, or 344-345 of BhCas12b, or the corresponding amino acid residues of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, or Cas12i; b) 147 and 148, 248 and 249, 299 and 300 of BvCas12b. 0, 991 and 992, or 1031 and 1032, or between the corresponding amino acid residues of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, or Cas12i; or c) 157 and 158, 258 and 259, 310 and 311, 1008 and 1009, or 1044 and 1045 of AaCas12b, or between the corresponding amino acid residues of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, or Cas12i. In one embodiment, the adenosine deaminase variant is a locus inserted into the Cas12 polypeptide indicated in Table 14B. 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.

[0040] In one embodiment of the method described above, the guide RNA comprises CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). In one embodiment of the method described above, the individual is a mammal or a human.

[0041] In another embodiment, a pharmaceutical composition is provided, comprising a base editing system comprising a fusion protein according to any of the methods, embodiments, and implementation schemes described above, and a pharmaceutically acceptable carrier, solvent, or excipient. In one embodiment, the pharmaceutical composition further comprises a guide RNA comprising a nucleic acid sequence selected from the group consisting of CUUCUCCACAGGAGUCAGAU; ACUUCUCCACAGGAGUCAGAU; and GACUUCUCCACAGGAGUCAGAU. In one embodiment, the gRNA further comprises a nucleic acid sequence selected from the group consisting of the nucleic acid sequence GUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACGAAACUUACACAGUUACUUAAAUC UUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCA GGGUG. In one embodiment, the gRNA comprises a moiety selected from the group consisting of CUUCUCCACAGGAGUCAGAUGUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACGAAA CUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG;

[0042] ACUUCUCCACAGGAGUCAGAUGUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAA CGAAACUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG; and

[0043] The nucleic acid sequence of GACUUCUCCACAGGAGUCAGAUGUUUUUGUACUCUCAAGAUUUAAGUAACUGUACA ACGAAACUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG.

[0044] In one embodiment, a pharmaceutical composition is provided, comprising a base editor or a polynucleotide encoding a base editor, wherein the base editor comprises any one of the polynucleotide programmable DNA binding domains and adenosine deaminase domains described in the methods, examples, and embodiments described above; and one or more guide polynucleotides that target the base editor to perform A·T to G·C modification of a sickle cell disease-associated SNP; and a pharmaceutically acceptable carrier, solvent, or excipient.

[0045] In another embodiment, a pharmaceutical composition is provided, comprising the cells according to the above embodiments and implementation schemes, and a pharmaceutically acceptable carrier, solvent, or excipient.

[0046] In another embodiment, a kit is provided, comprising a base editing system comprising a fusion protein according to any of the methods, embodiments, and implementations described above. In one embodiment, the kit further comprises a guide RNA comprising a nucleic acid sequence selected from the group consisting of CUUCUCCACAGGAGUCAGAU; ACUUCUCCACAGGAGUCAGAU; and GACUUCUCCACAGGAGUCAGAU.

[0047] In another embodiment, a kit is provided, comprising a base editor or a polynucleotide encoding a base editor, wherein the base editor comprises any one of the polynucleotide programmable DNA binding domains and adenosine deaminase domains described in the methods, examples, and embodiments described above; and one or more guide polynucleotides that target the base editor to perform A·T to G·C modification of a sickle cell disease-associated SNP.

[0048] In another embodiment, a kit is provided, comprising any one of the cells of the above embodiments and embodiments. In one embodiment of the kit, the kit further comprises a package insert with instructions for use.

[0049] In one embodiment, the present invention provides a base editor system comprising a polynucleotide programmable DNA binding domain and at least one base editor domain comprising an adenosine deaminase variant, wherein the variant comprises a modification at amino acid position 82 or 166 of the following sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD; and a guide RNA, wherein the guide RNA targets the base editor to modify a SNP associated with alpha-1 antitrypsin deficiency. In some embodiments, the adenosine deaminase variant comprises a V82S modification 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 a truncated TadA8 that loses 1, 2, 3, 4, 5, 66, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to full-length TadA8. In some embodiments, the adenosine deaminase variant is a truncated TadA8 that loses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to full-length TadA8. In some embodiments, the polynucleotide programmable DNA binding domain is a modified Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), a modified Streptococcus pyogenes Cas9 (SpCas9), or a variant thereof. In some embodiments, the polynucleotide programmable DNA binding domain is a SpCas9 variant with a modified protospacer adjacent motif (PAM) specificity or specificity for a non-G PAM. In some embodiments, the polynucleotide programmable DNA binding domain is a nuclease-inactivated Cas9. In some embodiments, the polynucleotide programmable DNA binding domain is a Cas9 nickase.

[0050] In one embodiment, a base editor system is provided herein that comprises one or more guide RNAs and a fusion protein comprising a polynucleotide-programmable DNA-binding domain comprising the following sequence:

[0051]

[0052] In one embodiment, a cell is provided, comprising any of the base editor systems described above. In some embodiments, the cell is a human cell or a mammalian cell. In some embodiments, the cell is in vitro, in vivo, or in vitro.

[0053] The description and examples herein are illustrative of embodiments of the present invention. It is generally understood that the present invention is not limited to the specific embodiments described herein and that variations are possible. Those skilled in the art will generally appreciate that many variations and modifications of the present invention are within the scope of the present invention.

[0054] The present invention provides compositions and methods for editing mutations associated with sickle cell disease (SCD). The isolation or manufacture of the compositions and articles defined herein is described in connection with the examples provided below. Additional features and advantages of the present invention will become apparent from the detailed description and claims. Unless otherwise indicated, the practice of certain embodiments disclosed herein employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art. See, for example: Sambrook and Green, 4th edition, Molecular Cloning: A Laboratory Manual (2012); a series of Current Protocols in Molecular Biology (FM Ausubel et al., eds.); a series of Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th edition (RI Freshney, ed. (2010)).

[0055] The section headings provided herein are for organizational purposes only and are not intended to limit the subject matter described.

[0056] Although various features of the present invention may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the invention is described herein in the context of separate embodiments for purposes of illustration, the invention may also be implemented in a single embodiment. The section headings used herein are for organizational purposes only and are not intended to be limiting of the subject matter described.

[0057] The features of the present invention are particularly described in the appended claims. The features and advantages of the present invention will be more clearly understood by referring to the following detailed description of the embodiments of the present invention and the accompanying drawings.

[0058] definition

[0059] The following definitions are provided to supplement those in the related art and are relevant to the present application and are not intended to imply any related or unrelated cases, such as any commonly owned patents or applications. Although any methods and materials similar or equivalent to those described herein can be used to perform the experiments of the present invention, preferred materials and methods are described herein. Therefore, the terminology used herein is for description of specific embodiments only and is not intended to be limiting.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide general definitions of many of the terms used herein by those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th ed., R. Rieger et al. (eds.), Springer-Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0061] Throughout this application, the singular includes the plural, unless expressly stated otherwise. It should be noted that the singular forms "a," "an," and "the" used throughout this specification include the plural unless expressly stated otherwise. Throughout this application, the use of "or" means "and / or," unless otherwise stated, and is generally understood to be inclusive. Furthermore, the term "including," as well as other forms such as "include," "includes," and "included," are used without limitation.

[0062] As used in this specification and claims, the terms "comprising" (and any comprising forms, such as "comprise" and "comprises"), "having" (and any having forms, such as "have" and "has"), "including" (and any including forms, such as "includes" and "include"), or "containing" (and any containing forms, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Any embodiment discussed in this specification contemplates implementation using any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0063] The terms "about" or "approximately" mean that a particular value is within an acceptable error range as determined by one skilled in the art, part of which will depend on the manner in which the value is measured or determined, i.e., on the limitations of the measurement system. For example, depending on the operation of the relevant art, "about" can mean within 1 or more standard deviations. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of the specified value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, such as within 5 times or within 2 times the specified value. If the application and claims specify a specific value, it should be assumed that the term "about" means within an acceptable error range for the specific value unless otherwise specified.

[0064] 47, 48, 49, or 50.

[0065] References in this specification to "some embodiments," "one embodiment," "an embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least some embodiments of the invention, but not necessarily in all embodiments of the invention.

[0066] "Adenosine deaminase" refers to a polypeptide or fragment thereof that can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to form inosine or the formation of deoxyinosine from deoxyadenosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine from deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) may be derived from any organism, such as bacteria. In some embodiments, the adenosine deaminase comprises a modification in the following sequence:

[0067] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

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

[0069] In some embodiments, TadA*7.10 comprises at least one modification. In some embodiments, TadA*7.10 comprises modifications at amino acids 82 and / or 166. In certain embodiments, variants of the sequences mentioned above comprise one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, variants of the TadA7.10 sequence comprise a sequence selected from the group consisting of Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V Modified combinations of 82S+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.

[0070] In other embodiments, the present invention provides adenosine deaminase variants comprising deletions, for example, TadA*8, comprising a C-terminal deletion beginning at residues 149, 150, 151, 152, 153, 154, 155, 156, or 157. In other embodiments, the adenosine deaminase variant is a TadA (e.g., TadA*8) monomer comprising one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant comprises a member selected from the group consisting of Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+ Monomers of modified combinations of Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.

[0071] In yet other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each having one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In yet other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each comprising a domain selected from the group consisting of Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y1 Modified combinations of the group consisting of 23H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.

[0072] In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type TadA adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type TadA adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising a moiety selected from the group consisting of Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y1 47T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and modified combinations of I76Y+V82S+Y123H+Y147R+Q154R.

[0073] In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising the following modifications: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123 H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q1 54R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; or I76Y+V82S+Y123H+Y147R+Q154R.

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

[0075] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

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

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

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

[0079] MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN

[0080] Bacillus subtilis (B.subtilis) TadA:

[0081] MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE

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

[0083] MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV

[0084] Shewanella putrefaciens (S. putrefaciens) TadA:

[0085] MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE

[0086] Haemophilus influenzae F3031 (H. influenzae) TadA:

[0087] MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTΑΗAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK

[0088] Caulobacter crescentus (C. crescentus) TadA:

[0089] MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI

[0090] Geobacter sulfurreducens (G.sulfurreducens) TadA:

[0091] MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP

[0092] TadA*7.10

[0093] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0094] By "adenosine deaminase base editor 8 (ABE8) polypeptide" is meant a base editor (BE) as defined and / or described herein, comprising an adenosine deaminase variant comprising a modification at amino acid position 82 and / or 166 of the following reference sequence:

[0095] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTA HAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGS LMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD. In some embodiments, ABE8 comprises other modifications relative to the reference sequence.

[0096] "Adenosine deaminase base editor 8 (ABE8) polynucleotide" means a polynucleotide (polynucleotide sequence) encoding an ABE8 polypeptide.

[0097] "Administering" herein refers to providing one or more compositions described herein to a patient or individual. For example, and not limited to, administration of an injectable composition may be by intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more of these routes may be used. Parenteral administration may be, for example, by bolus injection or by prolonged intravenous infusion. Alternatively or concurrently, oral administration may be used.

[0098] "Agent" means any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or a fragment thereof.

[0099] "Alteration" means changing (e.g., increasing or decreasing) the structure, expression level, or activity of a gene or polypeptide, as detected by standard art-known methods, such as those described herein. As used herein, alteration includes changing the polynucleotide or polypeptide sequence or changing the expression level, such as by 25%, 40%, 50%, or more.

[0100] "Alteration" means to reduce, suppress, attenuate, eliminate, arrest, or stabilize the development or progression of a disease.

[0101] "Analog" refers to molecules that are not identical but possess similar functional or structural features. For example, polynucleotide or polypeptide analogs retain the biological activity of the corresponding naturally occurring polynucleotide or polypeptide but have modifications that enhance the analog's function relative to the naturally occurring polynucleotide or polypeptide. These modifications may increase the analog's affinity for DNA, potency, specificity, protease or nuclease resistance, membrane permeability, and / or half-life without altering, for example, ligand binding. Analogs may include non-natural nucleotides or amino acids.

[0102] "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 comprises a nucleobase modifying polypeptide (e.g., a deaminase) and a nucleic acid programmable nucleotide binding domain, and is linked to a guide polynucleotide (e.g., a guide RNA). In various embodiments, the agent is a biomolecular complex comprising a protein domain with base editing activity, that is, a domain that can modify bases (e.g., A, T, C, G, or U) in a nucleic acid molecule (e.g., DNA). In some embodiments, the polynucleotide programmable DNA binding domain is fused or linked to a deaminase domain. In one embodiment, the agent is a fusion protein comprising a domain with base editing activity. In another embodiment, the protein domain with base editing activity is linked to a guide RNA (e.g., via an RNA binding motif on the guide RNA and an RNA binding domain fused to a deaminase). In some embodiments, the domain with base editing activity can deaminize bases in nucleic acid molecules. In some embodiments, a base editor can deaminize one or more bases within a DNA molecule. In some embodiments, a base editor can deaminize adenosine (A) within DNA. In some embodiments, a base editor is an adenosine base editor (ABE).

[0103] In some embodiments, base editors (e.g., ABE8) are generated by selecting an adenosine deaminase variant (e.g., TadA*8) into a backbone comprising a circular permutation of Cas9 (e.g., spCAS9 or saCAS9) and a bipartite nuclear localization sequence. Circular permutations of Cas9 are known in the art and are described, for example, in Oakes et al., Cell 176, 254–267, 2019. In the following circular permutation examples, bold sequences indicate sequences derived from Cas9, italic sequences represent linker sequences, and underlined sequences represent bipartite nuclear localization sequences.

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

[0105]

[0106]

[0107] In some embodiments, ABE8 is selected from the group consisting of: base editors from Tables 6 to 9, 13, or 14 below. In some embodiments, ABE8 comprises an adenosine deaminase variant evolved from TadA. In some embodiments, the adenosine deaminase variant of ABE8 is a TadA*8 variant described in Tables 7, 9, 13, or 14 below. In some embodiments, the adenosine deaminase variant is a TadA*7.10 variant (e.g., TadA*8) comprising one or more modifications selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In various embodiments, ABE8 comprises a TadA*7.10 variant (e.g., TadA*8) and a residue selected from Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y In some embodiments, ABE8 is a monomeric construct. In some embodiments, ABE8 is a heterodimeric construct. In some embodiments, ABE8 comprises the sequence:

[0108] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0109] In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-related (e.g., Cas or Cpf1) enzyme. In some embodiments, the base editor is a catalytically inactivated Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. The details of the base editor are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), the contents of which have been incorporated herein by reference in their entirety. See also Komor, AC et al. "Programmable base editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, NM et al. "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage" Nature 551,464-471(2017); "Improved base excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A baseeditors with higher efficiency and product purity" Science Advances 3:eaao4774(2017) by Komor, AC et al., and "Base editing: precision chemistry on the genome and transcriptome of living" by Rees, HA et al. cells." Nat Rev Genet. 2018 Dec; 19(12): 770-788. doi: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0110] For example, the adenine base editor (ABE) used in the base editing compositions, systems, and methods described herein has a nucleic acid sequence (8877 base pairs) as 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.). Polynucleotide sequences having at least 95% or greater identity to the ABE nucleic acid sequence are also encompassed.

[0111] ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT

[0112] GACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG

[0113] TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCATTG

[0114] ACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCC

[0115] ATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGT

[0116] CAGATCCGCTAGAGATCCGCGGCCGCTAATACGACTCACTATAGGGAGAGCCGCCACCATGAAACGGACA

[0117] GCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTCTCTGAAGTCGAGTTTAGCCACGAGT

[0118] ATTGGATGAGGCACGCACTGACCCTGGCAAAGCGAGCATGGGATGAAAGAGAAGTCCCCGTGGGCGCCGT

[0119] GCTGGTGCACAACAATAGAGTGATCGGAGAGGGATGGAACAGGCCAATCGGCCGCCACGACCCTACCGCA

[0120] CACGCAGAGATCATGGCACTGAGGCAGGGAGGCCTGGTCATGCAGAATTACCGCCTGATCGATGCCACCC

[0121] TGTATGTGACACTGGAGCCATGCGTGATGTGCGCAGGAGCAATGATCCACAGCAGGATCGGAAGAGTGGT

[0122] GTTCGGAGCACGGGACGCCAAGACCGGCGCAGCAGGCTCCCTGATGGATGTGCTGCACCACCCCGGCATG

[0123] AACCACCGGGTGGAGATCACAGAGGGAATCCTGGCAGACGAGTGCGCCGCCCTGCTGAGCGATTTCTTTA

[0124] GAATGCGGAGACAGGAGATCAAGGCCCAGAAGAAGGCACAGAGCTCCACCGACTCTGGAGGATCTAGCGG

[0125] AGGATCCTCTGGAAGCGAGACACCAGGCACAAGCGAGTCCGCCACACCAGAGAGCTCCGGCGGCTCCTCC

[0126] GGAGGATCCTCTGAGGTGGAGTTTTCCCACGAGTACTGGATGAGACATGCCCTGACCCTGGCCAAGAGGG

[0127] CACGCGATGAGAGGGAGGTGCCTGTGGGAGCCGTGCTGGTGCTGAACAATAGAGTGATCGGCGAGGGCTG

[0128] GAACAGAGCCATCGGCCTGCACGACCCAACAGCCCATGCCGAAATTATGGCCCTGAGACAGGGCGGCCTG

[0129] GTCATGCAGAACTACAGACTGATTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTGATGTGCGCCG

[0130] GCGCCATGATCCACTCTAGGATCGGCCGCGTGGTGTTTGGCGTGAGGAACGCAAAAACCGGCGCCGCAGG

[0131] CTCCCTGATGGACGTGCTGCACTACCCCGGCATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCA

[0132] GATGAATGTGCCGCCCTGCTGTGCTATTTCTTTCGGATGCCTAGACAGGTGTTCAATGCTCAGAAGAAGG

[0133] CCCAGAGCTCCACCGACTCCGGAGGATCTAGCGGAGGCTCCTCTGGCTCTGAGACACCTGGCACAAGCGA

[0134] GAGCGCAACACCTGAAAGCAGCGGGGGCAGCAGCGGGGGGTCAGACAAGAAGTACAGCATCGGCCTGGCC

[0135] ATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGG

[0136] TGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGA

[0137] AACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGC

[0138] TATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGT

[0139] CCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGC

[0140] CTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGAC

[0141] CTGCGGCTGATCTATCTGGCGCTGGCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACC

[0142] TGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGA

[0143] GGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGA

[0144] CGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGGAGAAAGAATGGCCTGTTCGGAAACCTGATTGCCC

[0145] TGAGCCTGGGCCTGACCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAG

[0146] CAAGGACACCTACGACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGGCCGACCTGTTTT

[0147] CTGGCCGCCAAGAACCTGCTCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCA

[0148] AGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGC

[0149] TCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCC

[0150] GGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGG

[0151] ACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAA

[0152] CGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTAC

[0153] CCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCC

[0154] CTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAA

[0155] CTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAG

[0156] AACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGC

[0157] TGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGC

[0158] CATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAG

[0159] AAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACAT

[0160] ACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGA

[0161] AGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCC

[0162] CACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCC

[0163] GGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGG

[0164] CTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAA

[0165] GCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTA

[0166] AGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA

[0167] GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGA

[0168] ATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACA

[0169] CCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGA

[0170] ACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGAC

[0171] TCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAG

[0172] AGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTT

[0173] CGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAG

[0174] CTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACG

[0175] ACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCG

[0176] GAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAAC

[0177] GCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACA

[0178] AGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTT

[0179] CTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGG

[0180] CCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGC

[0181] GGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAA

[0182] AGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAG

[0183] TACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGT

[0184] CCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAA

[0185] TCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAG

[0186] TACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAA

[0187] ACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGG

[0188] CTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATC

[0189] GAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCT

[0190] ACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAA

[0191] TCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAA

[0192] GAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTC

[0193] AGCTGGGAGGTGACTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAG

[0194] GAAAGTCTAACCGGTCATCATCACCATCACCATTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTT

[0195] CTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC

[0196] TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGT

[0197] GGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCT

[0198] CTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGATACCGTCGACCTCTAGCTAGAGCTTGGCGTA

[0199] ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA

[0200] AGCATAAAGTGTAAAGCCTAGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGC

[0201] CCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGG

[0202] TTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGA

[0203] GCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA

[0204] TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCT

[0205] CCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA

[0206] AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGAT

[0207] ACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC

[0208] GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA

[0209] TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTA

[0210] ACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTA

[0211] CACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC

[0212] TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA

[0213] GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACACTCAGTGGAACGAAAACTC

[0214] ACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA

[0215] AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG

[0216] CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTAC

[0217] GATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA

[0218] GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCT

[0219] CCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGT

[0220] TGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCC

[0221] CAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGA

[0222] TCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTAC

[0223] TGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGT

[0224] ATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAA

[0225] AAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG

[0226] TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGA

[0227] GCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATAC

[0228] TCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATG

[0229] TATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGA

[0230] TCGGGAGATCGATCTCCCGATCCCCTAGGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAA

[0231] GCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAAC

[0232] AAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGAT

[0233] GTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCAT

[0234] TAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC

[0235] CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCAT

[0236] TGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC

[0237] "Base editing activity" means changing the base within a polynucleotide by chemical action. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is adenosine or adenine deaminase activity, for example: converting A·T to G·C. In some embodiments, base editing activity is analyzed using editing efficacy. Base editing efficacy can be measured in any suitable manner, for example: using Sanger sequencing or next-generation sequencing. In some embodiments, base editing efficacy is a measure of the percentage of total sequenced reads with nuclear base conversions performed by base editors, for example: the percentage of total sequenced reads with target AT base pairs converted to GC base pairs. In some embodiments, when base editing is performed in a group of cells, base editing efficacy is a measure of the percentage of total cells with nuclear base conversions performed by base editors.

[0238] The term "base editor system" refers to a system for editing the nucleobases of a target nucleotide sequence. In various embodiments, the base editor system comprises (1) a polynucleotide programmable nucleotide binding domain (e.g., Cas9); (2) a deaminase domain (e.g., adenosine deaminase) that deaminates the nucleobase; and (3) one or more guide polynucleotides (e.g., guide RNA). In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor system is ABE8.

[0239] In some embodiments, a base editor system may include more than one base editing component. For example, a base editor system may include more than one deaminase. In some embodiments, a base editor system may include one or more adenosine deaminases. In some embodiments, a single guide polynucleotide may be used to target different deaminases to a target nucleic acid sequence. In some embodiments, a pair of guide polynucleotides may be used to target different deaminases to a target nucleic acid sequence.

[0240] The deaminase domain and the polynucleotide-programmable nucleotide-binding component of a base editor system can be associated with each other using any combination of covalent or non-covalent methods, or by association. For example, in some embodiments, the deaminase domain can be targeted to a target nucleotide sequence using 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 can target the deaminase domain to a target nucleotide sequence by non-covalently interacting with the deaminase domain or by associating with the deaminase domain. For example, in some embodiments, the deaminase domain can include an additional heterologous moiety or domain that can interact, associate, or form a complex with the additional heterologous moiety or domain that is part of the polynucleotide-programmable nucleotide-binding domain. In some embodiments, the additional heterologous moiety can bind, interact, associate, or form a complex with a polypeptide. In some embodiments, the additional heterologous moiety can bind, interact, associate, or form a complex with a polynucleotide. In some embodiments, the additional heterologous moiety can be associated with a guide polynucleotide. In some embodiments, the additional heterologous moiety can be associated with a polypeptide linker. In some embodiments, the additional heterologous moiety can be associated with a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0241] The base editor system may further comprise a guide polynucleotide component. It should be understood that the components of the base editor system may be associated with each other through covalent bonds, non-covalent interactions, or any combination of their association and interaction. In some embodiments, the deaminase domain can be targeted to the target nucleotide sequence through the guide polynucleotide. For example, in some embodiments, the deaminase domain may comprise an additional heterologous portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) that can interact, associate, or form a complex with a portion or segment of the guide polynucleotide (e.g., a polynucleotide motif). In some embodiments, the additional heterologous portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) can be fused or linked to the deaminase domain. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous moiety may be conjugated to a guide polynucleotide. In some embodiments, the additional heterologous moiety may be conjugated to a polypeptide linker. In some embodiments, the additional heterologous moiety may be conjugated to a polynucleotide linker. The additional heterologous moiety may be a protein domain. In some embodiments, the additional heterologous moiety may be a Kappa homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0242] In some embodiments, the base editor system may further comprise an inhibitor of a base excision repair (BER) component. It should be understood that the components of the base editor system may be associated with each other through covalent bonds, non-covalent interactions, or any combination of their associations and interactions. The inhibitor of the BER component may comprise a BER inhibitor. In some embodiments, the inhibitor of BER may be a uracil DNA glycosidase inhibitor (UGI). In some embodiments, the inhibitor of BER may be an inosine BER inhibitor. In some embodiments, the inhibitor of BER may be targeted to a target nucleotide sequence via a polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain may be fused or linked to an inhibitor of BER. In some embodiments, the polynucleotide programmable nucleotide binding domain may be fused or linked to a deaminase domain and an inhibitor of BER. In some embodiments, the polynucleotide programmable nucleotide binding domain may target the inhibitor of BER to a target nucleotide sequence by non-covalent interaction or association with the inhibitor of BER. For example, in some embodiments, the BER inhibitor component can include an additional heterologous moiety or domain that can interact with, associate with, or form a complex with an additional heterologous moiety or domain that is part of the programmable nucleotide binding domain of the polynucleotide.

[0243] In some embodiments, a BER inhibitor can be targeted to a target nucleotide sequence via a guide polynucleotide. For example, in some embodiments, a BER inhibitor can comprise an additional heterologous moiety or domain (e.g., a polynucleotide binding domain, such as an RNA- or DNA-binding protein) that can interact, associate, or form a complex with a portion or segment of a guide polynucleotide (e.g., a polynucleotide motif). In some embodiments, the additional heterologous moiety or domain (e.g., a polynucleotide binding domain, such as an RNA- or DNA-binding protein) of a guide polynucleotide can be fused or linked to the BER inhibitor. In some embodiments, the additional heterologous moiety can bind to, interact with, associate with, or form a complex with the polynucleotide. In some embodiments, the additional heterologous moiety can bind to the guide polynucleotide. In some embodiments, the additional heterologous moiety can bind to a polypeptide linker. In some embodiments, the additional heterologous moiety can bind to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous portion can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0244] "β-globin (HBB) protein" means a polypeptide or fragment thereof having at least about 95% amino acid sequence identity to NCBI Accession No. NP_000509. In certain embodiments, the β-globin protein comprises one or more modifications relative to the following reference sequence. In a specific embodiment, the sickle cell disease-associated β-globin protein comprises an E6V (also known as an E7V) mutation. Examples of β-globin amino acid sequences are provided below.

[0245]

[0246] "HBB polynucleotide" means a nucleic acid molecule encoding a β-globin protein or a fragment thereof. The sequence of an example HBB polynucleotide can be obtained from NCBI accession number NM_000518 and is provided below:

[0247]

[0248] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease that comprises a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and / or a gRNA binding domain of Cas9). Cas9 nucleases are sometimes also referred to as Casn1 nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)-related nucleases. CRISPR is an acquired immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). The CRISPR cluster comprises a spacer, a sequence complementary to the aforementioned mobile elements, and a target invasive nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, the corrective processing of pre-crRNA requires trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. TracrRNA is a guide that assists pre-crRNA processing as ribonuclease 3. Subsequently, Cas9 / crRNA / tracrRNA cleaves linear or circular dsDNA targets complementary to the spacer in an endonucleolytic manner. The target chain that is not complementary to the crRNA is first cut by an endonucleolytic manner. It is then trimmed 3′-5′ in an exonucleolytic manner. DNA-binding and cleavage in nature usually require proteins and two RNAs. However, a single guide RNA ("sgRNA" or "gRNA" for short) can be engineered to incorporate both crRNA and tracrRNA into a single RNA species. See, for example: Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821 (2012), the entire contents of which have been incorporated herein by reference. Cas9 recognizes a short motif (PAM or protospacer adjacent motif) within the CRISPR repeat sequence to help distinguish self from non-self.The Cas9 nuclease sequence and structure are known to those skilled in the art (see, for example, “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., JJ, McShan W.M., Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski J., et al., “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” (See, for example, "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," by Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E., Science 337:816-821 (2012), each of which is hereby incorporated by reference in its entirety.) Cas9 orthologs have been described in various species, including, but not limited to, Streptococcus pyogenes and Streptococcus thermophilus.Other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on the present disclosure, and include Cas9 sequences from organisms and loci disclosed in "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737 by Chylinski, Rhun, and Charpentier; the entire contents of which are incorporated herein by reference.

[0249] One example of Cas9 is Streptococcus pyogenes Cas9 (spCas9), whose amino acid sequence is provided below:

[0250]

[0251]

[0252] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0253] Nuclease-inactivated Cas9 proteins can be interchangeably referred to as "dCas9" proteins (nuclease-"inactivated" Cas9) or catalytically inactivated Cas9. Methods for producing Cas9 proteins (or fragments thereof) with inactivated DNA cleavage domains are known (see, for example, Jinek et al., Science. 337: 816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152(5): 1173-83, the entire contents of which are incorporated herein by reference). For example, it is known that the DNA cleavage domain of Cas9 includes two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337:816-821 (2012); Qi et al., Cell. 28;152(5):1173-83 (2013)). In some embodiments, the Cas9 nuclease has an inactivated (e.g., inactive) DNA cleavage domain, i.e., Cas9 is a nickase, referred to as a "nCas9" protein (for "nickase" Cas9).

[0254] In some embodiments, a protein comprising a fragment of Cas9 is provided. For example, in some embodiments, the protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, a protein comprising Cas9 or a fragment thereof is referred to as a "Cas9 variant." A Cas9 variant shares common homology with Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 variant can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the corresponding fragment of wild-type Cas9.

[0255] In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length. In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, nucleotide and amino acid sequences are as follows):

[0256] ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGAT GATTATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAAT CTTATAGGGGGCTCTTTTATTTGGCAGTGGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGT AGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAA GTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGT CATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTG CGAAAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATG ATTAAGTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTA TTTATCCAGTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTAGAGTAGAT GCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCC GGTGAGAAGAGAAATGGCTTGTTTGGGAATCTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAA TCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAGATACTTACGATGATGATTTAGAT AATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCT ATTTTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATT AAGCGCTACGATGAACATCATCAAGACTTGACTCTTTTAAGCTTTAGTTCGACAACAACTTCCAGAA AAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGC CAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTG AAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATT CACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGT GAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGT CGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGAT AAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAA GTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATAT GTTACTGAGGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTC TTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTT GATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGCGCCTACCATGATTTGCTA AAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTA ACATTGACCTTATTTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGAT GATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATT AATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAAT CGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGATATTCAAAAAGCACAGGTG TCTGGACAAGGCCATAGTTTACATGAACAGATTGCTAACTTAGCTGGCAGTCCTGCTATTAAAAAAGGT ATTTTACAGACTGTAAAAATTGTTGATGAACTGGTCAAAGTAATGGGGCATAAGCCAGAAAATATCGTT ATTGAAATGGCACGTGAAAATCAGACAACTCAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGA ATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTG CAAAATGAAAAGCTCTATCTCTATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGAT ATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCAATA GACAATAAGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTA GTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGAT AATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTG GTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGAT GAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGA AAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAAT GCCGTTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTAT AAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAAATAGGCAAAGCAACCGCAAAATAT TTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAA CGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACA GTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTC TCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCA AAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAA GGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTT GAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAA CTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTA CAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAA AAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTA GATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAA GTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTA TTTACGTTGACGAATCTTGGAGCTCCCGCTTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGA TATACGTCTACAAAAGAAGTTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACA CGCATTGATTTGAGTCAGCTAGGAGGTGACTGA

[0257]

[0258] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0259] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence:

[0260] ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGAT GAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAAT CTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGG AGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAA GTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGG CACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTC AGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATG ATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTG TTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGAT GCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAACCTGATCGCACAATTACCC GGAGAGAAAAAAATGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTTAAG TCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGAC AATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTCCAAAAACCTTAGCGATGCA ATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCCGCTTCAATGATC AAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAG AAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGGAGT CAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTA AAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGACTTTCGACAACGGTAGCATTCCACATCAAATC CACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGT GAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCT CGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGAT AAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAA GTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTAT GTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTA TTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTC GATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTA AAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTG ACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGAC GATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATC AACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAAT AGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTT TCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGC ATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATT GTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAG AGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAA TTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTG GACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCA ATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAA GTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTC GATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAG CTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAGATTCCCGAATGAATACGAAATAC GACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTC AGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTT AATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGAT TACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAA TACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGC AAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCG ACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGG TTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGAC CCGAAAGGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAG AAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCT TTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATT AACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAG CTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTAC GAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTAT CTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGAC AAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCAT TTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAA CGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCATCCATCACGGGATTATATGAA ACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGAC TACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCA GGA

[0261]

[0262]

[0263] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0264] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI reference sequence: NC_002737.2 (nucleotide sequence below); and Uniprot reference sequence: Q99ZW2 (amino acid sequence below):

[0265] ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGAT GAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAAT CTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGT AGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAA GTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGT CATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTG CGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATG ATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTA TTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGAT GCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCC GGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAA TCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGAT AATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCT ATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATT AAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAA AAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGC CAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTG AAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATT CACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGT GAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGT CGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGAT AAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAA GTACTACCAAAAACATAGTTTGCTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATAT GTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTC TTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTT GATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTA AAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTA ACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGAT GATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATT AATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAAT CGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTG TCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGT ATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATC GTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAA CGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAA TTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTA GATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCA ATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAA GTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTT GATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAA TTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATAC GATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTC CGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTA AATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGAT TATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAA TATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGC AAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCC ACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGA TTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGAT CCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAA AAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCC TTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATT AAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAA TTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTAT GAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTAT TTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGAT AAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCAT TTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAA CGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAA ACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGA

[0266] (SEQ ID NO:1. Single underline: HNH domain; double underline: RuvC domain).

[0267] In some embodiments, Cas9 is Cas9 from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma yrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense, China (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021847.1); Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis I (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1), or Neisseria meningitidis (NCBI Ref: YP_002342100.1), or Cas9 from any other organism.

[0268] In some embodiments, Cas9 is Neisseria meningitidis Cas9 (NmeCas9) or a variant thereof. In some embodiments, NmeCas9 is specific for NNNNGAYW PAM, wherein Y is C or T and W is A or T. In some embodiments, NmeCas9 is specific for NNNNGYTT PAM, wherein Y is C or T. In some embodiments, NmeCas9 is specific for NNNNGTCT PAM. In some embodiments, NmeCas9 is Nme1 Cas9. In some embodiments, NmeCas9 is specific for NNNNGATT PAM, NNNNCCTA PAM, NNNNCCTC PAM, NNNNCCTT PAM, NNNNCCTG PAM, NNNNCCGT PAM, NNNCCGGPAM, NNNNCCCA PAM, NNNNCCCT PAM, NNNNCCCC PAM, NNNNCCAT PAM, NNNNCCAG PAM, NNNNCCAT PAM, or NNNGATT PAM. In some embodiments, Nme1Cas9 is specific for NNNGATT PAM, NNNNCCT A PAM, NNNNCTC PAM, NNNCCTT PAM, or NNNNCCTGPAM. In some embodiments, NmeCas9 is specific for CAA PAM, CAAA PAM, or CCA PAM. In some embodiments, NmeCas9 is Nme2 Cas9. In some embodiments, NmeCas9 is specific for NNNNCC (N4CC) PAM, where N is any one of A, G, C, or T. In some embodiments, NmeCas9 is specific for NNNNCCGT PAM, NNNNCCGGPAM, NNNNCCCA PAM, NNNNCCCT PAM, NNNNCCCC PAM, NNNNCCAT PAM, NNNNCCAG PAM, NNNNCCAT PAM, or NNNGATT PAM. In some embodiments, NmeCas9 is Nme3Cas9. In some embodiments, NmeCas9 is specific for NNNNCAAA PAM, NNNNCC PAM, or NNNNCNNN PAM. In some embodiments, the PAM-interaction domain for Nme1, Nme2, or Nme3 is N4GAT, N4CC, and N4CAAA, respectively.Other NmeCas9 features and PAM sequences are described in Edraki et al., A Compact, High-Accuracy Cas9 with aDinucleotide PAM for In Vivo Genome Editing, Mol. Cell. (2019) 73(4): 714-726, the contents of which are incorporated herein by reference in their entirety.

[0269] An example of a Neisseria meningitidis Cas9 protein, Nme1Cas9, (NCBI reference: WP_002235162.1; type II CRISPR RNA-guide endonuclease Cas9) has the following amino acid sequence:

[0270]

[0271]

[0272] Another example of a Neisseria meningitidis Cas9 protein, Nme2Cas9, (NCBI reference: WP_002230835; type II CRISPR RNA-guide endonuclease Cas9) has the following amino acid sequence:

[0273]

[0274] In some embodiments, dCas9 corresponds to or comprises a portion or all of a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, a dCas9 domain comprises D10A and H840A mutations or corresponding mutations in another Cas9. In some embodiments, dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A):

[0275]

[0276]

[0277] (Single underline: HNH domain; double underline: RuvC domain).

[0278] In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequence provided above, or at the corresponding position in any of the amino acid sequences provided herein.

[0279] In other embodiments, dCas9 variants are provided that have mutations other than D10A and H840A, for example, resulting in nuclease-inactive Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs of dCas9 are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants of dCas9 are provided having shorter or longer amino acid sequences that differ by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, or by about 100 or more amino acids.

[0280] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of a Cas9 protein, such as one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but rather comprise only one or more fragments thereof. Examples of amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and those skilled in the art will recognize additional suitable sequences of Cas9 domains and fragments.

[0281] It is understood that additional Cas9 proteins (e.g., nuclease-inactivated Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including variants and homologs thereof, are within the scope of the present invention. Examples of Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is a nuclease-inactivated Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease-active Cas9.

[0282] Catalytically inactive Cas9 (dCas9) example:

[0283] DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL KIIKDKDFLDNEENEDILEDIVLTLTLFEDRAMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0284] Examples of catalytic Cas9 nickase (nCas9):

[0285]

[0286] Examples of catalytically active Cas9:

[0287]

[0288] In some embodiments, Cas9 refers to Cas9 from an archaeon (e.g., nanoarchaea), which belongs to the domain and kingdom Unicellular Prokaryotes. In some embodiments, Cas9 refers to CasX or CasY, which are 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, the entire contents of which are incorporated herein by reference. Using genome-resolved metagenomics, numerous CRISPR-Cas systems have been identified, including the first Cas9 reported in the archaeal domain of life. This diverse Cas9 protein has been found in the little-studied Nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, have been discovered, which are the most robust systems discovered to date. In some embodiments, Cas9 refers to CasX, or a variant of CasX. In some embodiments, Cas9 refers to CasY, or a variant of CasY. It should be understood that other RNA-guide DNA-binding proteins can be used as nucleic acid-programmable DNA-binding proteins (napDNAbp) and are within the scope of the present invention.

[0289] In some embodiments, Cas9 is a Cas9 variant that is specific for an altered PAM sequence. In some embodiments, additional Cas9 variants and PAM sequences are described in Miller et al., Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat Biotechnol (2020), doi.org / 10.1038 / s41587-020-0412-8, the entire contents of which are incorporated herein by reference. In some embodiments, Cas9 variants do not require a specific PAM. In some embodiments, Cas9 variants, for example, SpCas9 variants, are specific for NRNH PAMs, where R is A or G and H is A, C, or T. In some embodiments, SpCas9 variants are specific for the PAM sequences AAA, TAA, CAA, GAA, TAT, GAT, or CAC. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1218, 1219, 1221, 1249, 1256, 1264, 1290, 1318, 1317, 1320, 1321, 1323, 1332, 1333, 1335, 1337, or 1339 (numbered relative to the above reference sequence), or a corresponding position thereof.

[0290] (Single underline: HNH domain; double underline: RuvC domain).

[0291] In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1135, 1218, 1219, 1221, 1249, 1320, 1321, 1323, 1332, 1333, 1335, or 1337 (numbered relative to the reference sequence above), or a corresponding position thereof. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1219, 1221, 1256, 1264, 1290, 1318, 1317, 1320, 1323, 1333 (numbered relative to the reference sequence above), or a corresponding position thereof. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1131, 1135, 1150, 1156, 1180, 1191, 1218, 1219, 1221, 1227, 1249, 1253, 1286, 1293, 1320, 1321, 1332, 1335, 1339 (numbered relative to the reference sequence above), or a corresponding position thereof. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1127, 1135, 1180, 1207, 1219, 1234, 1286, 1301, 1332, 1335, 1337, 1338, 1349 (numbered relative to the reference sequence above). Examples of amino acid substitutions and PAM specificities of SpCas9 variants are shown in Tables AD and Figure 49 .

[0292] Table A

[0293]

[0294] Table B

[0295]

[0296]

[0297] Table C

[0298]

[0299] Table D

[0300]

[0301]

[0302] In certain embodiments, napDNAbp suitable for use in the methods of the present invention include circular arrangements known in the art and described, for example, in Oakes et al., Cell 176, 254–267, 2019. An example of a circular arrangement is as follows, where bold sequences indicate sequences derived from Cas9, italic sequences represent linker sequences, and underlined sequences represent bipartite nuclear localization sequences.

[0303] CP5 (with MSP "NGC = Pam variant, with mutation pattern Cas9 such as: NGG" PID = protein interaction domain and "D10A" nickase):

[0304]

[0305]

[0306] Non-limiting examples of polynucleotide programmable nucleotide-binding domains that can be incorporated into base editors include CRISPR protein-derived domains, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs).

[0307] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein may be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a native CasX or CasY protein. In some embodiments, the napDNAbp is a native CasX or CasY protein. In some embodiments, the amino acid sequence comprised by napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any CasX or CasY protein described herein. It should be understood that Cas12b / C2c1, CasX, and CasY from other bacterial species can also be used according to the present invention.

[0308] Cas12b / C2c1(uniprot.org / uniprot / T0D7A2#2)

[0309] sp|T0D7A2|C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acidoterrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB13137 / GD3B) GN=c2c1 PE=1 SV=1

[0310]

[0311] CasX(uniprot.org / uniprot / F0NN87;uniprot.org / uniprot / F0NH53)

[0312] >tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx protein OS=Sulfolobus islandicus (strain HVE10 / 4) GN=SiH_0402 PE=4 SV=1

[0313] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAAGWVLTRLGKAK VSEGDYVGVNVFTTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0314] >tr|F0NH53|F0NH53_SULIR CRISPR-associated protein, Casx OS=Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4 SV=1

[0315] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTSNIILPLSGNDKNPWTETLKCYNFPTTTVALSEVFKNFSQVKECEEVSAPSFVKPFEYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0316] δ- proteobacteria (Deltaproteobacteria) CasX

[0317] MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQK WYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0318] CasY(ncbi.nlm.nih.gov / protein / APG80656.1)

[0319] >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria bacteria]

[0320]

[0321] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid that has common properties. One functional approach to defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins from homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Based on such analyses, groups of amino acids can be defined that are preferentially exchanged with each other and therefore have the most similar effects on the overall protein structure (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include amino acid substitutions, such as: lysine for arginine and vice versa, thereby maintaining a positive charge; glutamic acid for aspartic acid and vice versa, thereby maintaining a negative charge; serine for threonine, thereby maintaining a free -OH group; and glutamine for aspartic acid, thereby maintaining a free -NH2 group.

[0322] The terms "coding sequence" or "protein coding sequence" are used interchangeably herein to refer to a segment of a polynucleotide that encodes a protein. The region or sequence is bounded near the 5' end by a start codon and at the 3' end by a stop codon. A coding sequence may also be referred to as an open reading frame.

[0323] As used herein, the term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine to form hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine or adenine (A) to form inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine or deoxyadenosine to form 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 (e.g., engineered adenosine deaminases, evolved adenosine deaminases) can be from any organism, such as bacteria. In some embodiments, the adenosine deaminase is from bacteria, such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Caulobacter crescentus.

[0324] In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is a TadA variant. In some embodiments, the TadA variant is TadA*8. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain 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 to a naturally occurring deaminase. For example, deaminase domains are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), the contents of each of which are incorporated herein by reference in their entirety.See also Komor, AC et al. "Programmable base editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, NM et al. "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage" Nature 551,464-471(2017); "Improved base excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A baseeditors with higher efficiency and product purity" Science Advances 3:eaao4774(2017)) by Komor, AC et al.; and "Base editing: precision chemistry on the genome and transcriptome of living" by Rees, HA et al. cells." Nat Rev Genet. 2018 Dec; 19(12): 770-788. doi: 10.1038 / s41576-018-0059-1, the entire contents of which have been incorporated herein by reference.

[0325] "Detection" refers to determining the presence, absence, or amount of an analyte to be detected. In one embodiment, detection involves detecting sequence modifications in a polynucleotide or polypeptide. In another embodiment, detection involves detecting the presence of insertions / deletions (indels).

[0326] "Detectably labeled" means that the composition, when linked to a molecule of interest, can be detected by microscopic, photochemical, biochemical, immunochemical, or chemical means. Examples of suitable labels include radioactive isotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin, or haptens.

[0327] "Disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In one embodiment, the disease is SCD. In one embodiment, the disease is beta-thalassemia.

[0328] As used herein, the term "effective amount" refers to an amount of a bioactive agent sufficient to elicit a desired biological response. The effective amount of the active compound(s) used in the treatment of a disease using the present invention will vary depending on the route of administration, the age, weight, and general health of the individual. The appropriate dosage and course of treatment will ultimately be determined by the attending physician or veterinarian. Such amounts are referred to as "effective" amounts. In certain embodiments, an effective amount is an amount in which the base editor system of the present invention (e.g., a fusion protein comprising a programmable DNA-binding protein, a nucleobase editor, and a gRNA) is sufficient to modify an SCD mutation in a cell to achieve a therapeutic effect (e.g., reducing or controlling an individual's SCD or its symptoms or signs). Such therapeutic effects do not necessarily require fully modifying the SCD of all cells in a tissue or organ, but only about 1%, 5%, 10%, 25%, 50%, 75%, or more cells in the tissue or organ. In one embodiment, the effective amount is sufficient to alleviate one or more SCD symptoms, including anemia and ischemia.

[0329] "Fragment" means a portion of a polypeptide or nucleic acid molecule. This portion comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of a reference nucleic acid molecule or polypeptide. A fragment can comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0330] "Guide RNA" or "gRNA" refers to a polynucleotide that is specific for a target sequence and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpf1). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). The gRNA can be a complex of two or more RNAs, or a single RNA molecule. A gRNA that is a single RNA molecule can be referred to as a single guide RNA (sgRNA), but "gRNA" can be used interchangeably with a guide RNA that is a single molecule or a complex of two or more molecules. Typically, a gRNA that is a single RNA species comprises two domains: (1) a domain that shares common homology with the target nucleic acid (e.g., and directs the Cas9 complex and target binding); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA provided by Jinek et al., Science 337:816-821 (2012), the entire disclosure of which is incorporated herein by reference. Other examples of gRNAs (e.g., those that include domain 2) can be found in U.S. Provisional Patent Application No. USSN 61 / 874,682, filed September 6, 2013, entitled "Switchable Cas9 Nucleases and Uses Thereof"; and U.S. Provisional Patent Application No. USSN 61 / 874,746, filed September 6, 2013, entitled "Delivery System For Functional Nucleases," the entire disclosures of which are each incorporated herein by reference. In some embodiments, a gRNA comprises two or more domains (1) and (2) and can be referred to as an "extended gRNA." The extended gRNA will bind to two or more Cas9 proteins and bind to the target nucleic acid in two or more independent regions described herein. The gRNA comprises a nucleotide sequence that complements the target site and mediates the binding of the nuclease / RNA complex to the target site, providing sequence specificity to the nuclease:RNA complex. Those skilled in the art generally understand that RNA polynucleotide sequences, such as gRNA sequences, include the nucleobase uracil (U), a pyrimidine derivative, rather than the nucleobase thymine (T) found in DNA polynucleotide sequences. In RNA, uracil base pairs with adenine and replaces thymine during DNA transcription.

[0331] "Hb G-Makassar" or "Makassar" refers to a human beta-heme variant, human hemoglobin (Hb) G-Makassar variant or mutation (HB Makassar variant), which is an asymptomatic naturally occurring variant (E6A) hemoglobin. Hb G-Makassar was first discovered in Indonesia (Mohamad, AS et al., 2018, Hematol. Rep., 10(3):7210 (doi:10.4081 / hr.2018.7210). Hb G-Makassar has a slow mobility during electrophoresis. Makassar β-heme variants have anatomical abnormalities at the β-6 or A3 positions, where the glutamyl residue is usually replaced by an alanyl residue. The substitution of a single amino acid β-6 glutamyl residue to valine in the gene encoding the β-globin subunit causes sickle cell disease. Routine procedures such as isoelectric focusing, heme electrophoresis using cation exchange, high performance liquid chromatography (HPLC), and cellulose acetate electrophoresis are unable to separate Hb G-Makassar and HbS globin types because they have been found to have the same properties when analyzed using these methods. The results cannot correctly distinguish Hb G-Makassar and HbS are confused with each other by those skilled in the art, thus leading to the misdiagnosis of sickle cell disease (SCD).

[0332] "Hybridization" refers to hydrogen bonding, which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through hydrogen bonding.

[0333] The term "inhibitor of base repair" or "IBR" refers to a protein that can inhibit the activity of a nucleic acid repair enzyme, such as a base excision repair (BER) enzyme. In some embodiments, the IBR is an inhibitor of inosine base excision repair. Examples of such base repair inhibitors include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGG1, hNEIL1, T7Endol, T4PDG, UDG, hSMUG1, and hAAG. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is catalytically inactive EndoV or catalytically inactive hAAG. In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, the base repair inhibitor is catalytically inactive EndoV or catalytically inactive hAAG.

[0334] In some embodiments, the base repair inhibitor is a uracil glycosidase inhibitor (UGI). UGI refers to a protein that can inhibit the uracil-DNA glycosidase base excision repair enzyme. In some embodiments, the UGI domain comprises wild-type UGI or a fragment of wild-type UGI. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to UGI or UGI fragments. 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 "inactivated inosine-specific nuclease." Without being bound by any particular theory, catalytically inactive inosine glycosidases (e.g., alkyladenine glycosidase (AAG)) can bind to inosine but do not create an abasic site or exclude inosine, thereby sterically blocking the newly formed inosine moiety from the DNA damage / repair machinery. In some embodiments, catalytically inactive inosine-specific nucleases can bind to inosine in nucleic acids but do not cleave the nucleic acids. Non-limiting examples of catalytically inactive inosine-specific nucleases include catalytically inactive alkyl adenosine glycosidase (AAG nuclease), e.g., from humans, and catalytically inactive endonuclease V (EndoV nuclease), e.g., from Escherichia coli. In some embodiments, the catalytically inactive AAG nuclease comprises an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0335] "Increase" means a positive change of at least 10%, 25%, 50%, 75%, or 100%.

[0336] An "intein" is a fragment of a protein that can self-excise and join other fragments (exteins) via peptide bonds during a process called protein splicing. Inteins are also called "introns." The process of self-excision and joining of an intein to other protein parts is referred to herein as "protein splicing" or "intein-mediated protein splicing." In some embodiments, the precursor intein (the protein containing the intein before intein-mediated protein splicing) is derived from two genes. These inteins are referred to herein as split inteins (e.g., split intein-N and split intein-C). For example, in cyanobacteria, DnaE (the catalytic subunit of DNA polymerase III) is encoded by two separate genes, dnaE-n and dnaE-c. The intein encoded by the dnaE-n gene is referred to herein as "intein-N." The intein encoded by the dnaE-c gene is referred to herein as "intein-C."

[0337] Other intein systems may also be employed. For example, synthetic intein pairs based on the dnaE intein: Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C) have been described (e.g., Stevens et al., J Am Chem Soc. 2016 Feb. 24;138(7):2162-5, incorporated herein by reference). Non-limiting examples of intein pairs that may be used in accordance with the present invention include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, and Cne Prp8 intein (e.g., as described in U.S. Patent No. 8,394,604, incorporated herein by reference).

[0338] Examples of nucleotide and amino acid sequences of inteins are provided.

[0339] DnaE intein-N DNA:

[0340] TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTTGCCAATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCGATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGGGGA GAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAGGGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTTCCTAAT

[0341] DnaE intein-N protein:

[0342] CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN

[0343] DnaE intein-C DNA:

[0344] ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGATATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAGCTTCTAAT

[0345] Intein-C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN

[0346] Cfa-N DNA:

[0347] TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCCTATTGGAAAGATTGTCGAAGAGAGAATTGAATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGGCACAATCGCGGCGAACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACGAGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAATAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTGCCA

[0348] Cfa-N protein:

[0349] CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP

[0350] Cfa-C DNA:

[0351] ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAGGAAAGTAAAGATAATATCTCGAAAAAGTCTTGGTACCCAAAATGTCTATGATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTAGCCAGCAAC

[0352] Cfa-C protein:

[0353] MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKN GLVASN

[0354] Intein-N and intein-C may be fused to the N-terminal portion of split Cas9 and the C-terminal portion of split Cas9, respectively, to facilitate the splicing of the N-terminal portion of split Cas9 and the C-terminal portion of split Cas9. For example, in some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of split Cas9, i.e., forming the structural formula N-[N-terminal portion of split Cas9]-[intein-N]--C. In some embodiments, intein-C is fused to the N-terminus of the C-terminal portion of split Cas9, i.e., forming the structural formula N-[intein-C]--[C-terminal portion of split Cas9]-C. The mechanism of intein-mediated protein splicing of the protein to which the intein is fused (e.g., split Cas9) is known in the art, for example, as described in Shah et al., Chem Sci. 2014; 5(1):446-461, which is incorporated herein by reference. Methods for designing and using inteins are known in the art and are described in, for example, WO2014004336, WO2017132580, US20150344549, and US20180127780, the contents of each of which are incorporated herein by reference in their entirety.

[0355] The terms "isolated," "purified," or "biologically pure" refer to compositions that are free to varying degrees of various materials normally associated with them in nature. "Isolated" refers to a degree of separation from the original source or environment. "Purified" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other undesirable consequences. In other words, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or if it is free of chemical precursors or other chemicals when chemically synthesized. Purity and homology are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" may refer to a nucleic acid or protein that produces essentially a single band on an electrophoretic gel. Proteins that have been modified, for example, by phosphorylation or glycosylation, with different modifications resulting in different isolated proteins, can be isolated and purified.

[0356] "Isolated polynucleotide" means a nucleic acid (e.g., DNA) flanking a gene that is excluded from the native genome of the organism from which the nucleic acid molecule of the invention is derived. Thus, the term includes, for example, recombinant DNA that has been incorporated into a vector; incorporated into an autonomously replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryotic or eukaryotic organism; or such recombinant DNA that is present as a separate molecule independent of other sequences (e.g., a cDNA or genome or cDNA fragment generated by PCR or restriction endonuclease digestion). Furthermore, the term includes RNA molecules transcribed from a DNA molecule, as well as recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0357] An "isolated polypeptide" refers to a polypeptide of the present invention that has been separated from naturally associated components. Generally, a polypeptide is isolated when it has been freed from at least 60% by weight of the proteins and organic molecules with which it is naturally associated. Preferably, the concentration is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the polypeptide of the present invention. Isolated polypeptides of the present invention can be prepared, for example, by extraction from natural sources, expression of recombinant nucleic acids encoding such polypeptides, or chemical synthesis of the protein. Purity can be determined by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0358] As used herein, the term "linker" may refer to a covalent linker (e.g., a covalent bond), a nonvalent linker, a chemical group, or a molecule that connects two molecules or moieties, such as two components of a protein complex or ribonucleoprotein complex, or two domains of a fusion protein, such as, for example, a polynucleotide-programmable DNA-binding domain (e.g., dCas9) and a deaminase domain (e.g., adenosine deaminase, or adenosine deaminase and cytidine deaminase, as described, for example, in PCT / US19 / 44935). A linker can connect different components or different portions of a base editor system. For example, in some embodiments, a linker can connect the guide polynucleotide-binding domain of a polynucleotide-programmable DNA-binding domain and the catalytic domain of a deaminase. In some embodiments, a linker can connect a CRISPR polypeptide and a deaminase. In some embodiments, a linker can connect Cas9 and a deaminase. In some embodiments, a linker can connect dCas9 and a deaminase. In some embodiments, a linker can connect nCas9 and a deaminase. In some embodiments, a linker can bind the guide polynucleotide and the deaminase. In some embodiments, a linker can bind the deamination component of a base editor system and the polynucleotide-programmable nucleotide-binding component. In some embodiments, a linker can bind the RNA-binding portion of the deamination component of a base editor system and the polynucleotide-programmable nucleotide-binding component. In some embodiments, a linker can bind the RNA-binding portion of the deamination component of a base editor system and the RNA-binding portion of the polynucleotide-programmable nucleotide-binding component. A linker can be positioned between or flanking two groups, molecules, or other moieties, linking them via a covalent bond or non-covalent interaction, thereby linking the two. In some embodiments, a linker can be an organic molecule, group, polymer, or chemical moiety. In some embodiments, a linker can be a polynucleotide. In some embodiments, a linker can be a DNA linker. In some embodiments, a linker can be an RNA linker. In some embodiments, a linker can comprise an aptamer that can bind a ligand. In some embodiments, the ligand can be a carbohydrate, peptide, protein, or nucleic acid. In some embodiments, the linker can comprise an aptamer derived from a riboswitch. The riboswitch from which the aptamer is derived can be selected from the group consisting of theophylline riboswitch, thiamine pyrophosphate (TPP) riboswitch, adenosylcobalamin (AdoCbl) riboswitch, S-adenosylmethionine (SAM) riboswitch, SAH riboswitch, flavin mononucleotide (FMN) riboswitch, tetrahydrofolate riboswitch, lysine riboswitch, glycine riboswitch, purine riboswitch, GlmS riboswitch, or pre-queosine 1 (PreQ1) riboswitch. In some embodiments, the linker can comprise an aptamer that binds to a polypeptide or protein domain (e.g., a polypeptide ligand).In some embodiments, the polypeptide ligand can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile α motif, the telomerase Ku binding motif and Ku protein, the telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif. In some embodiments, the polypeptide ligand can be part of a base editor system component. For example, a nucleobase editor component can include a deaminase domain and an RNA recognition motif.

[0359] In some embodiments, the linker can be one amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker can be about 5-100 amino acids in length, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids in length. In some embodiments, the linker can be about 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 amino acids in length. Longer or shorter linkers are also contemplated.

[0360] In some embodiments, a linker joins the gRNA binding domain of an RNA-programmable nuclease (including the Cas9 nuclease domain) and the catalytic domain of a nucleic acid-editing protein (e.g., adenosine deaminase). In some embodiments, a linker joins dCas9 and a nucleic acid-editing protein. For example, a linker is positioned between or flanking two groups, molecules, or other moieties and covalently links them to each other, thereby linking the two. In some embodiments, a linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, a linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-200 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90, 95, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 175, 180, 190, or 200 amino acids in length. Longer or shorter linkers are also contemplated.

[0361] In some embodiments, the domain of the nucleobase editor is fused using a linker comprising the following amino acid sequence: SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP TSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS.

[0362] In some embodiments, the domain of the nucleobase editor is fused using a linker comprising the amino acid sequence SGSETPGTSESATPES, which can also be referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker comprises (SGGS) n 、(GGGS) n 、(GGGGS) n 、(G) n 、(EAAAK) n 、(GGS) n , SGSETPGTSESATPES, or (XP) n motif, or any combination thereof, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0363] In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence

[0364] SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS. In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0365] "Marker" means any protein or polynucleotide having a modification in expression level or activity that is associated with a disease or disorder.

[0366] As used herein, the term "mutation" refers to a residue within a sequence of, for example, a nucleic acid or amino acid sequence being replaced by another residue, or being deleted or having one or more residues inserted within the sequence. When describing a mutation herein, the original residue is generally indicated first, followed by the position of the residue within the sequence, and then the newly replaced residue is indicated. The various methods for making amino acid substitutions (mutations) provided herein are known in the art and are provided in, for example, Green and Sambrook's Molecular Cloning: A Laboratory Manual (4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). In some embodiments, the base editor of the present invention can effectively produce "intended mutations" such as point mutations in nucleic acids (e.g., nucleic acids within individual genes), without producing a significant number of unexpected mutations, such as unexpected point mutations. In some embodiments, the expected mutation is a mutation produced by a specific base editor (e.g., an adenosine base editor) bound to a guide polynucleotide (e.g., gRNA), which is explicitly designed to produce the expected mutation.

[0367] Typically, mutations created or identified in a sequence (e.g., an amino acid sequence described herein) are numbered relative to a reference (or wild-type) sequence, i.e., a sequence that does not contain the mutation. Those skilled in the art readily understand how to determine the position of a mutation in an amino acid or nucleic acid sequence relative to a reference sequence.

[0368] The term "non-conservative mutation" refers to amino acid substitutions between different groups, such as lysine for tryptophan or phenylalanine for serine, etc. In these cases, non-conservative amino acid substitutions preferably do not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, resulting in a functional variant having increased biological activity compared to the wild-type protein.

[0369] The terms "nuclear localization sequence," "nuclear localization signal," or "NLS" refer to an amino acid sequence that facilitates protein import into the cell nucleus. Nuclear localization sequences are known in the art and are described, for example, in Plank et al., International PCT Application No. PCT / EP2000 / 011690, filed November 23, 2000, published as WO / 2001 / 038547 on May 31, 2001, which is incorporated herein by reference in its entirety for its disclosure of examples of nuclear localization sequences. In other embodiments, the NLS is, for example, an optimized NLS described by Koblan et al., Nature Biotech. 2018, doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0370] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a compound comprising a nucleobase and an acidic moiety (e.g., a nucleoside, a nucleotide, or a nucleotide polymer). Typically, a polymeric nucleic acid (e.g., a nucleic acid molecule comprising three or more nucleotides) is a linear molecule in which adjacent nucleotides are linked to each other by phosphodiester linkages. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" are used interchangeably to refer to a polymer of nucleotides (e.g., a strand of at least three nucleotides). In some embodiments, "nucleic acid" includes RNA and single-stranded and / or double-stranded DNA. A nucleic acid can be naturally occurring, for example, in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, nucleic acid molecules can be non-naturally occurring molecules, such as recombinant DNA or RNA, artificial chromosomes, engineered genomes, or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids, or include non-naturally occurring nucleotides or nucleosides. In addition, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, such as analogs having non-phosphodiester backbones. Nucleic acids can be purified from natural sources, manufactured using recombinant expression systems, and optionally purified, chemically synthesized, etc. Where appropriate, for example, in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs, such as analogs having chemically modified bases or sugars, and backbone modifications. Nucleic acid sequences are in a 5' to 3' orientation unless otherwise indicated. In some embodiments, the nucleic acid is or comprises a natural nucleoside (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); a nucleoside analog (e.g., 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); -aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5′-N-phosphoimidate linkages).

[0371] The term "nucleic acid programmable DNA-binding protein" or "napDNAbp" is used interchangeably with "polynucleotide programmable nucleotide binding domain" and refers to a protein that is associated with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA) that guides the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. The Cas9 protein can be associated with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Non-limiting examples of nucleic acid-programmable DNA-binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i.Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csn2, sm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, type II Cas effector protein, type V Cas effector protein, type VI Cas effector protein, CARF, DinG, homologs thereof, or modified or engineered versions thereof; other nucleic acid programmable DNA binding proteins, although they may not be explicitly listed in the present invention, are also within the scope of the present invention. See, for example: Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct; 1: 325-336. doi: 10.1089 / crispr.2018.0033; Yan et al. “Functionally diverse type VCRISPR-Cas systems” Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, the entire contents of each of which are incorporated herein by reference.

[0372] The terms "nucleobase," "nitrogenous base," or "base," used interchangeably herein, refer to nitrogenous biological compounds that form nucleosides, which in turn become components of nucleotides. The ability of nucleobases to form base pairs and stack one on top of another directly leads to the formation of long helical structures, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The five nucleobases—adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U)—are referred to as primitive or canonical. Adenine and guanine are derived from purines, and cytosine, uracil, and thymine are derived from pyrimidines. DNA and RNA may also contain other modified (non-primitive) bases. Non-limiting examples of modified nucleobases include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydroxymethylcytosine. Hypoxanthine and xanthine can be produced by the presence of mutagens, both of which are produced by deamination (replacing an amino group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be derived from the deamination of cytosine. A "nucleoside" is composed of a nucleobase and a five-carbon sugar (ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides with modified nucleobases include inosine (I), xanthine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). A "nucleotide" is composed of a nucleobase, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group.

[0373] As used herein, the term "nucleobase editing domain" or "nucleobase editing protein" refers to a protein or enzyme that catalyzes the modification of nucleobases in RNA or DNA, such as the deamination of cytosine (or cytidine) to form uracil (or uridine) or thymine (or thymidine), and the deamination of adenine (or adenosine) to form hypoxanthine (or inosine), and the addition and insertion of non-templated nucleotides. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., an adenine deaminase or adenosine deaminase; or a cytidine deaminase or a cytosine deaminase). In some embodiments, the nucleobase editing domain is more than one deaminase domain (e.g., an adenine deaminase, or an adenosine deaminase, and a cytidine or cytosine deaminase, e.g., as described in PCT / US19 / 44935). In some embodiments, the nucleobase editing domain may be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase editing domain can be an engineered or evolved nucleobase editing domain from a naturally occurring nucleobase editing domain. The nucleobase editing domain can be from any organism, such as bacteria, humans, chimpanzees, gorillas, monkeys, cows, dogs, rats, or mice.

[0374] As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, generating, preparing, or otherwise obtaining the agent.

[0375] As used herein, "patient" or "subject" refers to a mammalian individual or individual diagnosed with, suffering from, at risk of suffering from or developing, susceptible to, or suspected of suffering from or developing a disease or disorder. In some embodiments, the term "patient" refers to a mammalian individual who has a higher than average likelihood of developing a disease or disorder. Examples of patients can be humans, non-human primates, cats, dogs, pigs, cows, cats, horses, camels, alpacas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), and other mammals that can benefit from the therapies disclosed herein. Examples of human patients can be male and / or female.

[0376] A "patient in need thereof" or "individual in need thereof" as used herein refers to a patient diagnosed with, at risk for, or suffering from, previously diagnosed as suffering from, or suspected of suffering from a disease or disorder.

[0377] The terms "pathogenic mutation," "pathogenic variant," "disease-causing mutation," "disease-causing variant," "deleterious mutation," or "predisposing mutation" refer to genetic modifications or mutations that increase an individual's susceptibility or predisposition to a disease or condition. In some embodiments, the pathogenic mutation comprises a substitution of at least one wild-type amino acid with at least one pathogenic amino acid in a protein encoded by a gene.

[0378] The terms "protein," "peptide," "polypeptide," and their grammatical equivalents are used interchangeably herein and refer to polymers of amino acid residues linked together by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide is at least three amino acids in length. A protein, peptide, or polypeptide refers to an individual protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modifications, and the like. A protein, peptide, or polypeptide may also be a single molecule or a multimolecular complex. A protein, peptide, or polypeptide may be a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. One of the proteins can be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxyl-terminal (C-terminal) protein, thereby forming an amino-terminal fusion protein or a carboxyl-terminal fusion protein, respectively. The protein can contain different domains, such as a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9, which directs the protein to bind to the target site) and a nucleic acid cleavage domain, or a catalytic domain of a nucleic acid editing protein. In some embodiments, the protein comprises a proteinaceous portion, such as an amino acid sequence that constitutes a nucleic acid binding domain, and an organic compound, such as a compound that can act as a nucleic acid cleavage agent. In some embodiments, the protein is a complex containing a nucleic acid or associated with a nucleic acid (e.g., RNA or DNA). Any protein provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins containing a peptide linker. Methods of recombinant protein expression and purification are known and include those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), the entire contents of which are incorporated herein by reference.

[0379] The polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) may comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethyl-cystine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline- 2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-diphenylmethyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, high carbon phenylalanine, and α-tert-butylglycine. Polypeptides and proteins can be associated with 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-linked and O-linked), amidation, hydroxylation, alkylation (including methylation and ethylation), ubiquitination, addition of pyrrolidone carboxylic acid, disulfide bridge formation, sulfation, myristoylation, palmitoylation, prenylation, farnesylation, geranylation, glycosylphosphatidylinositolation, lipoylation, and iodination.

[0380] As used herein, the term "recombinant" in the context of a protein or nucleic acid refers to a protein or nucleic acid that does not occur in nature, but is a product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations relative to any naturally occurring sequence.

[0381] "Reduced" means a negative modification of at least 10%, 25%, 50%, 75%, or 100%.

[0382] "Reference" refers to a test or control condition. In one embodiment, the reference is a wild-type or healthy cell. In other embodiments and without limitation, the reference is an untreated cell that has not been subjected to the test condition, or a cell that has been treated with a placebo or saline, vehicle, buffer, and / or a control vector without the polynucleotide of interest.

[0383] A "reference sequence" is a designated sequence used as a basis for sequence alignment. A reference sequence can be a subset or the entirety of a particular sequence; for example, a segment of a full-length cDNA or gene sequence, or the entire cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence is typically at least about 16 amino acids, at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence is typically at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, and about 100 nucleotides, or about 300 nucleotides, or any integer therebetween. In some embodiments, the reference sequence is the wild-type sequence of a protein of interest. In other embodiments, the reference sequence is a polynucleotide sequence encoding the wild-type protein.

[0384] The terms "RNA-programmable nuclease" and "RNA-guide nuclease" are used in conjunction with (e.g., bound to or associated with) one or more RNAs that are not targets for cleavage. In some embodiments, when complexed with RNA, the RNA-programmable nuclease may be referred to as a nuclease:RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA). The gRNA may be present as a complex of two or more RNAs, or as a single RNA molecule. A gRNA present as a single RNA molecule may be referred to as a single-guide RNA (sgRNA), but "gRNA" may be used interchangeably with a gRNA present as a single molecule or as a complex of two or more molecules. Typically, a gRNA present as a single RNA species comprises two domains: (1) a domain that shares common homology with the target nucleic acid (e.g., and directs the binding of the Cas9 complex to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA provided by Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNAs (e.g., those that include domain 2) can be found in U.S. Provisional Patent Application No. 61 / 874,682, filed on September 6, 2013, entitled "Switchable Cas9 Nucleases and Uses Thereof," and U.S. Provisional Patent Application No. 61 / 874,746, filed on September 6, 2013, entitled "Delivery System For Functional Nucleases," the entire contents of which are each incorporated herein by reference. In some embodiments, a gRNA comprises two or more domains (1) and (2), and can be referred to as an "extended gRNA." For example, an extended gRNA will, for example, bind to two or more Cas9 proteins and bind to a target nucleic acid at two or more independent regions, as described herein. The gRNA comprises a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site and provides sequence specificity to the nuclease:RNA complex.

[0385] In some embodiments, the RNA-programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 (Csn1) from Streptococcus pyogenes (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes”, Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C, Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III", Deltcheva E.,Chylinski K.,SharmaCM.,Gonzales K.,Chao Y.,Pirzada ZA,Eckert MR,Vogel J.,Charpentier E.,Nature 471:602-607(2011).

[0386] Because RNA-programmable nucleases (e.g., Cas9) utilize RNA:DNA hybridization to target DNA cleavage sites, these proteins can, in principle, be targeted to any sequence specified by a guide RNA. Methods for using RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify the genome) are known in the art (see, e.g., Cong, L. et al., Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, WY et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference).

[0387] The term "single nucleotide polymorphism (SNP)" refers to a single nucleotide change occurring at a specific position in a gene, where each change occurs to a significant degree (e.g., >1%) within a population. For example, at a specific base position in a human genome, a C nucleotide may occur in most individuals, but in a minority of individuals, an A nucleotide may occupy that position. This indicates a SNP at that specific position, and the two possible nucleotide changes, C or A, are called alleles at that position. SNPs represent fundamental differences in disease susceptibility. Disease severity and our body's response to treatment are also indicative of genetic variation. SNPs can occur within the coding region of a gene, within non-coding regions, or within intergenic regions (the region between genes). In some embodiments, due to the degeneracy of the genetic code, SNPs within coding sequences do not necessarily alter the amino acid sequence of the resulting protein. There are two types of SNPs within coding regions: synonymous and nonsynonymous. Synonymous SNPs do not affect the protein sequence, while nonsynonymous SNPs do alter the protein's amino acid sequence. There are two types of nonsynonymous SNPs: missense and nonsense. SNPs that are not in protein coding regions can still affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNA. Gene expression affected by such SNPs is called eSNP (expression SNP) and can be upstream or downstream of the gene. Single nucleotide variants (SNVs) are changes in a single nucleotide, with no frequency restrictions and can come from somatic cells. Somatic single nucleotide changes can also be called single nucleotide modifications.

[0388] "Specific binding" refers to a nucleic acid molecule, polypeptide, or complex thereof (e.g., a nucleic acid programmable DNA binding domain and a guide nucleic acid), compound, or molecule that recognizes and binds to a polypeptide and / or nucleic acid molecule of the present invention but is substantially unable to recognize and bind to other molecules in a sample (e.g., a biological sample).

[0389] Nucleic acid molecules suitable for use in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but generally exhibit substantial identity. Polynucleotides that are "substantially identical" to an endogenous sequence can generally hybridize to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules suitable for use in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but generally exhibit substantial identity. Polynucleotides that are "substantially identical" to an endogenous sequence can generally hybridize to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" refers to the pairing of complementary polynucleotide sequences (e.g., the genes described herein), or portions thereof, under conditions of varying stringency to form a double-stranded molecule (see, for example, Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0390] 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, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of an organic solvent (e.g., formamide), while high stringency hybridization can be achieved in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions typically include temperatures of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Other parameters that can be varied, such as hybridization time, detergent (e.g., sodium dodecyl sulfate (SDS)) concentration, and the inclusion or exclusion of vector DNA, are well within the skill of those skilled in the art. Various degrees of stringency can be achieved by combining these various conditions as desired. In one embodiment, hybridization is performed at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization is performed 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 is performed at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Variations on these conditions are readily apparent to those skilled in the art.

[0391] For most applications, the wash step following the hybridization method also varies in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As described above, wash stringency can be increased by lowering the salt concentration or increasing the temperature. For example, the stringent salt concentration for the wash step is preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash step generally include at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In one embodiment, the wash step is performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash step is performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash step is performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Other variations on these conditions will be readily apparent to those skilled in the art. Hybridization temperatures are known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0392] "Split" means to divide into two or more segments.

[0393] "Split Cas9 protein" or "split Cas9" means that the Cas9 protein is provided as an N-terminal fragment and a C-terminal fragment encoded by two separate nucleotide sequences. 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 split into two fragments within the disordered region of the protein, as described, for example, in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014; or in Jiang et al. (2016) Science 351: 867-871. PDB file: 5F9R, each of which is incorporated herein by reference. In some embodiments, the protein is split into two fragments at any C, T, A, or S within the region between approximately amino acids A292-G364, F445-K483, or E565-T637 of SpCas9, or at corresponding positions in any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other napDNAbp. In some embodiments, the protein is split into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of splitting a protein into two fragments is referred to as "splitting" the protein.

[0394] In other embodiments, the N-terminal portion of the Cas9 protein comprises amino acids 1-573 or 1-637 of Streptococcus pyogenes (S. pyogenes) Cas9 wild-type (SpCas9) (NCBI reference sequence: NC_002737.2, Uniprot reference sequence: Q99ZW2), or corresponding positions / mutations thereof, and the C-terminal portion of the Cas9 protein comprises a portion of amino acids 574-1368 or 638-1368 of the SpCas9 wild-type.

[0395] The C-terminal portion of the split-Cas9 protein is joined to the N-terminal portion of the split-Cas9 protein to form the complete Cas9 protein. In some embodiments, the C-terminal portion of the Cas9 protein begins where the N-terminal portion of the Cas9 protein ends. Thus, in some embodiments, the C-terminal portion of the split-Cas9 protein comprises amino acids (551-651)-1368 of spCas9. "(551-651)-1368" means starting with amino acids 551-651 (inclusive) and ending at amino acid 1368.For example, the C-terminal portion of the split Cas9 can include any portion of amino acids 551-1368, 552-1368, 553-1368, 554-1368, 555-1368, 556-1368, 557-1368, 558-1368, 559-1368, 560-1368, 561-1368, 562-1368, 563-1368, 564-1368, 565-1368, 566-1368, 567-1368, 568-1368, 569-1368, 570-1368, 571-1368, 572-1368, 573- 1368, 574-1368, 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 68, 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, 634-1368, 635-1368, 636-1368, 637-1368, 638-1368, 639-1368, 640-1368, 641-1368, 642-1368, 643-1368, 644-1368, 645-1368, 646-1368, 647-1368, 648-1368, 649-1368, 650-1368, or 651-1368. In some embodiments, the C-terminal portion of the split-Cas9 protein comprises a portion of amino acids 574-1368 or 638-1368 of SpCas9.

[0396] "Subject" means a mammal, including but not limited to a human or non-human mammal, such as a cow, horse, dog, sheep, or cat. Subjects include livestock, domestic animals raised to provide labor and produce consumer goods, such as food, including but not limited to cattle, goats, chickens, horses, pigs, rabbits, and sheep.

[0397] "Substantially identical" means that the polypeptide or nucleic acid molecule has at least 50% identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). In one embodiment, such sequences are at least 60%, 80%, 85%, 90%, 95%, or even 99% identical to the sequence used for comparison at the amino acid level or nucleic acid level.

[0398] Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). These programs match identical or similar sequences by specifying the degree of homology for various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions of the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. An example of a method for determining identity can be using the BLAST program, which is used by e -3 to e -100 For example, when using COBALT, the following parameters are used:

[0399] a) Parallelism parameters: gap penalty -11, -1, and end gap penalty -5, -1,

[0400] b) CDD parameters: RPS BLAST was used; Blast E-value was 0.003; conserved amino acid positions were searched and duplicate counting was enabled; and

[0401] c) Query clustering parameters: Enable query clustering; character size 4; maximum cluster distance 0.8; general letters.

[0402] Use EMBOSS needles, for example, according to the following parameters:

[0403] a) Matrix: BLOSUM62;

[0404] b) GAP OPEN: 10;

[0405] c) GAP EXTEND: 0.5;

[0406] d) Output format: one pair;

[0407] e) End gap penalty: None;

[0408] f) END GAP OPEN: 10; and

[0409] g) END GAP EXTEND: 0.5.

[0410] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a base editor. In one embodiment, the target site is deaminated by a deaminase or a fusion protein comprising a deaminase (e.g., adenine deaminase).

[0411] As used herein, the terms "treat," "treating," "treatment," and similar terms refer to the reduction or alleviation of a disease, disorder, and / or symptom associated therewith or the achievement of a desired pharmacological and / or physiological effect. It is generally understood that, although not excluded, the treatment of a disease or condition does not require the complete elimination of the disease, disorder, and / or symptom associated therewith. In some embodiments, the effect is therapeutic, that is, without limitation, the effect is the partial or complete reduction, elimination, abrogation, alleviation, alleviation, reduction in severity of, or cure of the disease and / or symptoms attributable to the disease. In some embodiments, the effect is prophylactic, that is, the effect protects or prevents the occurrence or recurrence of a disease or condition. Thus, the methods of the present invention comprise administering a therapeutically effective amount of a composition described herein. In some embodiments, the disease or condition is sickle cell disease (SCD) or β-thalassemia.

[0412] "Uracilase inhibitor" or "UGI" refers to 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 glycosidase and prevents the removal of uracil residues from DNA. In one embodiment, the UGI is a protein, fragment, or domain thereof that inhibits the uracil-DNA glycosidase base-excision repair enzyme. In some embodiments, the UGI domain comprises wild-type UGI or a modified form thereof. In some embodiments, the UGI domain comprises a fragment of the amino acid sequence exemplified below. In some embodiments, the UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the UGI sequence exemplified below. In some embodiments, the UGI comprises an amino acid sequence that is homologous to the UGI amino acid sequence exemplified below or a fragment thereof. In some embodiments, the UGI, or a portion thereof, is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a wild-type UGI or UGI sequence, or a portion thereof, as shown below. An example of a UGI comprises the following amino acid sequence:

[0413] >splP14739IUNGI_BPPB2 uracil-DNA glycosidase inhibitor

[0414] MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAY DESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML.

[0415] The term "vector" refers to a means for introducing a nucleic acid sequence into a cell, which results in a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, and episomes. An "expression vector" is a nucleic acid sequence comprising a nucleotide sequence intended for expression in a recipient cell. An expression vector may include additional nucleic acid sequences that promote and / or facilitate expression of the introduced sequence, such as initiation, termination, enhancer, promoter, and secretion sequences.

[0416] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0417] DNA editing has emerged as a viable approach for modifying disease at the genetic level by correcting pathogenic mutations. Until recently, all DNA editing platforms were designed to induce DNA double-strand breaks (DSBs) at defined genomic sites, with the resulting products determined semi-stochastically by endogenous DNA repair pathways, resulting in a complex array of genetic products. While precise, user-defined repair outcomes can be achieved through the homology-directed repair (HDR) pathway, numerous challenges hinder efficient repair using HDR in therapeutically relevant cell types. In practice, this pathway is less efficient than the competing and error-prone non-homologous end joining pathway. Furthermore, HDR is strictly restricted to the G1 and S phases of the cell cycle, preventing precise repair of DSBs in post-mitotic cells. Consequently, efficiently modifying genomic sequences in these populations in a user-defined, programmable manner has proven difficult or impossible. BRIEF DESCRIPTION OF THE DRAWINGS

[0418] Figure 1A-1C Depict the plasmid. Figure 1A An expression vector encoding the TadA7.10-dCas9 base editor. Figure 1B The present invention is a plasmid comprising a nucleic acid molecule encoding proteins that confer resistance to chloramphenicol (CamR) and spectinomycin (SpectR). The plasmid also comprises a kanamycin resistance gene that is abolished by two point mutations. Figure 1C The plasmid comprises a nucleic acid molecule encoding proteins that confer chloramphenicol resistance (CamR) and spectinomycin resistance (SpectR). The plasmid also comprises a kanamycin resistance gene that has been abolished by three point mutations.

[0419] Figure 2 Images of bacterial colonies transduced with the expression vector depicted in Figure 1, which includes a defective kanamycin resistance gene, are shown. Vectors containing ABE7.10 variants were generated using error-prone PCR. Bacterial cells expressing these "evolved" ABE7.10 variants were selected for kanamycin resistance using increasing concentrations of kanamycin. Bacteria expressing ABE7.10 variants and possessing adenosine deaminase activity can correct the mutation introduced into the kanamycin resistance gene, thereby restoring kanamycin resistance. Kanamycin-resistant cells were selected for further analysis.

[0420] Figure 3A and Figure 3B We demonstrate editing of the regulatory region of the heme subunit gamma (HGB1) locus, a medically relevant site for upregulation of fetal hemoglobin. Figure 3A FIG2 is a diagram showing a portion of the regulatory region of the HGB1 gene. Figure 3BThe potency and specificity of the adenosine deaminase variants listed in Table 15 were quantified. The editing method was analyzed in HEK293T cells at the heme subunit gamma 1 (HGB1) locus, a therapeutically relevant site for upregulating fetal hemoglobin. The figure above shows the nucleotide residues in the targeted region of the HGB1 gene regulatory sequence. A5, A8, A9, and A11 represent edited adenosine residues in HGB1.

[0421] Figure 4 This figure illustrates the relative effectiveness of adenosine base editors containing dCas9 that recognize atypical PAM sequences. The top panel shows the coding sequence for the heme subunit. The bottom panel demonstrates the performance of adenosine deaminase variant base editors using guide RNAs of varying lengths.

[0422] Figure 5 Figure 1 illustrates the potency and specificity of ABE8. The percentage of editing at the intended target nucleotide and unintended target nucleotides (bystanders) was quantified.

[0423] Figure 6 Figure 1 illustrates the potency and specificity of ABE8. The percentage of editing at the intended target nucleotide and unintended target nucleotides (bystanders) was quantified.

[0424] Figures 7A-7C Graph and bar graph depicting neutralization of A·T to G·C conversion and phenotypic consequences in primary cells. Figure 7A A diagram of the embryonic, fetal, and adult globin genes located on chromosome 11 is shown, indicating the HBG1 / 2HPFH site where a single base editor introduces a double helix edit. Figure 7B This graphic illustrates DNA editing efficiency in CD34+ cells. It shows A·T to G·C conversion at the -198HBG1 / 2 promoter site in ABE-treated CD34+ cells from two separate donors. NGS analysis was performed 48 and 144 hours after treatment. The -198HBG1 / 2 target sequence is as follows: A7 is bold and double-underlined. Plot the percentages from A·T to G·C for A7. Figure 7C Figure 1 is a graph reflecting the expression percentage of γ-globin / α-globin in erythrocytes derived from ABE-edited cells. Figure 7C The percentage of γ-globin formed is shown as a ratio relative to α-globin. Figure 7B and Figure 7C The values ​​are from two different donors after ABE treatment and red blood cell differentiation. Figure 7B As observed, the editing efficiency of ABE8 at the -198HBG1 / 2 promoter target site was 2-3 times higher than that at earlier time points (48 hr). Figure 7CAs observed, ABE8 editing in CD34+ cells increased γ-globin production in differentiated erythrocytes by approximately 1.4-fold. For example, the ABE8.13-d base editor resulted in 55% γ-globin / α-globin expression.

[0425] Figure 8A and Figure 8B Depicts A·T to G·C conversion in CD34+ cells at the -198 promoter site upstream of HBG1 / 2 after ABE8 treatment. Figure 8A Figure 2. Heat map depicting the frequency of A to G editing of ABE8 in CD34+ cells from two donors, 48 ​​and 144 hours after editor treatment; donor 2 is heterozygous for sickle cell disease. Figure 8B Graphs represent the distribution of total sequenced reads containing only the A7 edit or the combined (A7+A8) edits.

[0426] Figure 9 A heat map depicting the frequency of indels (INDELs) at the -198 site of the γ-globin promoter in CD34+ cells following ABE8 treatment. Frequencies from two donors are shown at the 48-hour and 144-hour time points. At the HBG1 / 2 -198 promoter target site described herein, complete A·T to G·C conversion occurs, creating a 10-nt poly-G stripe. Because such homopolymer manipulation often increases the rate of PCR- and sequencing-induced errors, an increased frequency of indels (INDELs) is observed at this site.

[0427] Figure 10 Ultra-high performance liquid chromatography (UHPLC) UV-Vis trace (220 nm) of untreated differentiated CD34+ cells (Donor 1) and integration of the globin chain phase are shown.

[0428] Figure 11 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE7.10-m and the integration of the globin chain phase.

[0429] Figure 12 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE7.10-d and the integration of the globin chain phase.

[0430] Figure 13 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.8-m and the integration of the globin chain phase.

[0431] Figure 14Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.8-d and the integration of the globin chain phase.

[0432] Figure 15 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.13-m and the integration of the globin chain phase.

[0433] Figure 16 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.13-d and the integration of the globin chain phase.

[0434] Figure 17 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.17-m and the integration of the globin chain phase.

[0435] Figure 18 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.17-d and the integration of the globin chain phase.

[0436] Figure 19 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.20-m and the integration of the globin chain phase.

[0437] Figure 20 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.20-d and the integration of the globin chain phase.

[0438] Figure 21 Depicted is the UHPLC UV-Vis trace (220 nm) of untreated differentiated CD34+ cells (Donor 2) and the integration of the globin chain phase. Note: Donor 2 is heterozygous for sickle cell disease.

[0439] Figure 22 Depicted is the UHPLC UV-Vis trace (220 nm) and integration of the globin chain phase of differentiated CD34+ cells (Donor 2) treated with ABE7.10-m. Note: Donor 2 is heterozygous for sickle cell disease.

[0440] Figure 23Depicted is the UHPLC UV-Vis trace (220 nm) and integration of the globin chain phase of differentiated CD34+ cells (Donor 2) treated with ABE7.10-d. Note: Donor 2 is heterozygous for sickle cell disease.

[0441] Figure 24 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE8.8-m and the integration of the globin chain phase. Note: Donor 2 is heterozygous for sickle cell disease.

[0442] Figure 25 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE8.8-d and the integration of the globin chain phase. Note: Donor 2 is heterozygous for sickle cell disease.

[0443] Figure 26 Depicted is the UHPLC UV-Vis trace (220 nm) and integration of the globin chain phase of differentiated CD34+ cells (Donor 2) treated with ABE8.13-m. Note: Donor 2 is heterozygous for sickle cell disease.

[0444] Figure 27 Depicted is the UHPLC UV-Vis trace (220 nm) and integration of the globin chain phase of differentiated CD34+ cells (Donor 2) treated with ABE8.13-d. Note: Donor 2 is heterozygous for sickle cell disease.

[0445] Figure 28 Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.17-m and the integration of the globin chain phase.

[0446] Figure 29 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE8.17-d and the integration of the globin chain phase. Note: Donor 2 is heterozygous for sickle cell disease.

[0447] Figure 30A and Figure 30B Depicted are UHPLC UV-Vis traces (220 nm) of differentiated CD34+ cells treated with ABE8 and the integration of the globin chain phase. Figure 30A Depicts the UHPLC UV-Vis trace (220 nm) and integration of the globin chain phase of differentiated CD34+ cells (Donor 2) treated with ABE8.20-m. Note: Donor 2 is heterozygous for sickle cell disease. Figure 30BDepicted is the UHPLC UV-Vis trace (220 nm) and integration of the globin chain phase of differentiated CD34+ cells (Donor 2) treated with ABE8.20-d. Note: Donor 2 is heterozygous for sickle cell disease.

[0448] Figures 31A-31E Depicts editing at two independent sites using ABE8.8, achieving 90% editing before enucleation on day 11 after erythroid differentiation, and approximately 60% editing of gamma globin on day 18 after erythroid differentiation, exceeding either alpha globin or total beta family globin. Figure 31A Graph depicting the average ABE8.8 editing in two healthy donors from two independent experiments. Editing efficiency was measured using primers that discriminate between HBG1 and HBG2. Figure 31B Graph depicting the mean of two independent experiments from one healthy donor. Editing efficiency was measured using primers that recognize both HBG1 and HBG2. Figure 31C Schematic diagram depicting editing of ABE8.8 in a donor with a heterozygous E6V mutation. Figure 31D and Figure 31E Graph depicting the increase in γ-globin in ABE8.8-edited cells.

[0449] Figure 32A and Figure 32B Depicted are the percentage of editing that corrects the sickle cell mutation using the ABE variant. Figure 32A Figure 2: Schematic diagram of screening different editor variants with approximately 70% editing in SCD patient fibroblasts. Figure 32B Figure 3. Schematic diagram of CD34 cells from a healthy donor being edited with a lead ABE variant targeting a synonymous mutation A13 in an adjacent proline within the editing window and acting as a surrogate for editing SCD mutations. The ABE8 variant showed an average editing frequency of approximately 40% at surrogate A13.

[0450] Figure 33A and Figure 33B Figure 2. RNA amplicon sequencing assay for the editing of cellular A-formation-I in RNA associated with ABE treatment. Individual data points are shown, and error bars represent the standard deviation (sd) of n = 3 biological replicates performed independently on different days. Figure 33A Graph depicting the frequency of A-to-I editing in target RNA amplicons for the core ABE 8 construct compared to ABE7 and Cas9(D10A) nickase controls. Figure 33B Graph depicting the frequency of A-to-I editing in target RNA amplicons for ABE8 harboring mutations reported to ameliorate off-target RNA editing.

[0451] Figure 34A and Figure 34BThe following diagrams and UPHLC chromatography tracks are presented for editing of SCD CD34+ cells. CD34+ cells from SCD patients were transfected with ABE8.8 mRNA and sgRNA (HBG1 / 2, 50nM) using electroporation. The edited cells differentiated into erythrocytes in vitro. Next-generation genome sequencing (NGS) was used to measure the editing rate at the HBG1 / 2 promoter. Figure 34A As shown, 16.5% editing by the ABE8.8 base editor was observed 48 hours after differentiation, and 89.2% editing was observed on day 14 after differentiation. Figure 34B Shown are bystander edit disintegration at 48 hours and 14 days after differentiation.

[0452] Figures 35A-35D Show the UPHLC chromatographic trace of the globin stage and Figure 34A and Figure 34B A diagram illustrating the functional upregulation of HbF and downregulation of HbS in edited SCD CD34+ cells is shown. Edited SCD CD34+ cells were differentiated into erythroid cells, and the globin stage was analyzed on day 18 of differentiation. Figure 35A The presented track shows the globin stage of erythroid cells differentiated from unedited SCD CD34+ cells. Figure 35B The presented trajectory shows the globin stage of erythroid cells differentiated from edited SCDCD34+ cells. Figure 35C It was shown that 63.2% of the gamma globin stage was detected in erythroid cells differentiated from edited SCD CD34+ cells compared to unedited cells. Figure 35D The results showed that compared with unedited cells, the S-globin expression of SCD CD34+ cells differentiated from edited cells decreased from 86% to 32.9%. Upregulating fetal hemoglobin is a promising approach for the treatment of SCD and beta-thalassemia.

[0453] Figures 36A-36C The ribbon structure, target sequence, and diagrams related to the generation of variants of ABE editors for editing atypical Cas9 NGG PAM sequences are shown. An ABE base editor comprising a modified SpCas9 including an MQKFRAER amino acid substitution and specificity for the altered PAM 5'-NGC-3' described herein was designed. Figure 36A ), the sickle allele within the editing window of ABE can be targeted ("target A"), e.g. Figure 36B As shown, this provides the ability to directly edit this position in the target site, which is normally inaccessible using traditional spCas9. Figure 36CA graphical illustration of the base editing activity of a variant editor containing the MQKFRAER amino acid substitution is shown. It can recognize the target site and convert the nucleobase A to the nucleobase T (A·T), achieving the desired correction Val->Ala. Each variant is plotted on the x-axis, with "Pro->Pro" representing the leftmost bar; "Val->Ala" representing the middle bar; and "Ser->Pro" representing the rightmost bar.

[0454] Figure 37 Figures, target site sequences, and tables are provided for the generation of additional adenosine deaminase variants in which the linker to TadA has been removed and placed closer to the Cas9 complex. These variants have improved efficacy in editing a model cell line (HEK293T) expressing the sickle allele target site. The term "ISLAY" or "IBE" refers to base editors in which the TadA adenosine deaminase has been inserted into the Cas9 sequence, for example: ISLAY1 V1015, ISLAY2 I1022, ISLAY3 I1029, ISLAY4 E1040, ISLAY5 E1058, ISLAY6 G1347, ISLAY7 E1054, ISLAY8 E1026, and ISLAY9 Q768, as shown in Table 14A below. The target site, PAM site, and corresponding amino acid sequence in the nucleic acid sequence are shown on the right side of the figure. In the table, "Cp5" (MSP552) refers to ABE8 in the backbone, which includes a circular arrangement of Cas9, having the following amino acid sequence and described below.

[0455]

[0456] The 20nt guide sgRNA (1000ng) used in the experiment, spCas9-MQKFRAER, which is specific for NGC PAM, was used in triplicate to transform HEK293T cells (2x10 5 cells / well).

[0457] Figure 38 and Figure 39 A schematic diagram of different adenosine deaminase ISLAY variants demonstrating enhanced editing of target sites (e.g. Figure 37 The diagram shown in the middle figure is a comparison with another ABE editor (ABE7.10) having a linker with a TadA domain.

[0458] Figure 40 The percentage of base editing achieved in CD34+ cells expressing the SCD target site is shown, along with a table showing the edited nucleic acid and amino acid changes. CD34+ cells from a heterozygous sickle trait patient were treated with an ABE editor, and editing at the target site (9G) was measured, converting the nucleobase A to the nucleobase T to achieve the desired correction Val>Ala. Ninety-six hours after electroporation of CD34+ cells, the variant ABE editor achieved over 50% editing of the sickle cell allele. This was maintained after in vitro differentiation of the cells into erythroid cells (IVD), as over 60% editing was observed in differentiated erythroid cells (heterozygous for sickle trait) by 12 days post-erythroid differentiation. The diagram for the Editor_nM mRNA_[sgRNA]:[mRNA]_Timepoint evaluation is based on the use of a 21nt gRNA.

[0459] Figure 41A and Figure 41B High performance liquid chromatography (UHPLC) chromatographic traces and LC-MS results associated with the detection of unique beta-globin species in edited heterozygous HbS (beta-globin in sickle cells) differentiated erythrocytes are presented. Prior to these experiments and analyses, practitioners in the art were generally unable to successfully distinguish and separate the HbG Makassar variant globin from the HbS sickle globin variant using traditional methods. Here, a UHPLC method was developed and used to distinguish these two different globin variants in cells (e.g., CD34+ cells) from SCD patients that had been edited using the ABE8 editor described herein. After editing, CD34+ cells from heterozygous HbSS samples can be detected using UHPLC based on molecular weight corresponding to the different beta globin (Hb) variants ( Figure 41A). The editing peaks analyzed by liquid chromatography-mass spectrometry (LC-MS) showed a charge envelope indicative of an independent new β-globin variant (Makassar variant) ( Figure 41B ).

[0460] Figure 42 A table of base editor and sgRNA sequences for base editing SCD samples with HbS globin variants is presented to correct the HbG Makassar variant globin. The ABE8 mutation was introduced into the lead editor candidate, and sgRNAs of different lengths (21nt, 20nt, 19nt protospacers) were analyzed to detect whether they improve on-target editing while reducing potentially harmful 1G editing (conversion of Ser10Pro). "A" nucleotides in bold / italic / underlined indicate sickle substitutions. Lowercase letters in the sgRNA / protospacer sequence indicate 2′-O-methylated nucleobases. Lowercase "s" in the sgRNA / protospacer sequence indicates phosphorothioate.

[0461] Figure 43A and Figure 43B The results show that the 9G target site (or 9G and other sites) in CD34+ cells (heterozygous sickle cell trait samples) was transformed with different ABE editors 48h after electroporation ( Figure 43A ) or differentiated erythrocytes in vitro (heterozygous sickle trait samples) after 7 days of differentiation ( Figure 43B ). While additional mutations did not significantly improve on-target editing, four editors demonstrated comparable on-target editing efficacy. A 20nt sgRNA length resulted in lower rates of undesired 1G bystander edits. In these plots, either Editor_sgRNA nt (Editor_sgRNA nt) or Editor_100nM mRNA_μM sgRNA (Editor_100nM mRNA_μM sgRNA) (20nt) was evaluated. Nearly 80% editing was maintained throughout in vitro erythroid differentiation.

[0462] Figure 44A and Figure 44B A bar graph and table showing the edited nucleic acid sequence and corresponding amino acid sequence transitions associated with total base editing at position 9G of HbS in a homozygous SCD (HbSS) sample are presented. The cells were obtained from whole blood (non-mobilized) samples of SCD (HbSS) patients and base edited using ABE variant base editors. Figure 44ACD34+ cells (~200,000 cells, homozygous SCD samples) were electroporated with 50 nM of the ABE variant editor (MSP619 (ISLAY5)) at a 100:1 ratio (2 μg mRNA, 4.1 μg sgRNA (21 nt)). The ABE variant base editor achieved approximately 65% ​​editing at position 9G on day 7 after electroporation and approximately 60% editing at position 9G on day 14 after electroporation. Figure 44B CD34+ cells (~200,000 cells, homozygous SCD samples) were electroporated with 30 nM of the ABE variant editor (MSP616(ISLAY2)) at a 200:1 ratio (1.3 μg mRNA, 4.95 μg sgRNA (21 nt)). The ABE variant base editor achieved at least approximately 50% editing at position 9G in erythrocytes on days 7 and 14 after electroporation. Figure 45 Shown is the UHPLC chromatographic trace after UHPLC analysis, which shows clear separation and differentiation of HbS type and HbG Makassar variant type after base editing using ABE variant base editor in homozygous HbSS cells obtained from an SCD patient sample. Figure 46A and Figure 46B UHPLC chromatographic traces and LC-MS results are shown for the detection of separate β-globin species in edited heterozygous HbS (β-globin in sickle cells) differentiated erythrocytes. Figure 41A and Figure 41B In the edited heterozygous HbSS samples, the different β-globin (Hb) variants corresponding to those with Val→Ala substitutions can be detected based on their molecular weight ( Figure 46A ). Edited peaks of the LC-MS trace show a charged mantle indicating a novel β-globin variant ( Figure 46B ). Figure 47 The UHPLC chromatograms and LC-MS results of HbSS(SCD) samples that have been base edited ("HbSS-Edited") or not ("HbSS-Unedited") are shown. As shown in the UHPLC chromatograms in the upper and middle figures, the HbG Makassar globin variant is separated from the HbS(SCD) globin type (10.03min) at 9.81 minutes based on the difference in UHPLC elution time. The other globin types are easily distinguished. In the LC-MS in the lower figure, the Makassar HbG variant and the HbS type of globin have different and distinguishable characteristics. Similar Figure 41A 、 Figure 41B 、 Figure 45 、 Figure 46A and Figure 46BThe results presented show that UHPLC and LC-MS analysis of cells from SCD (HbSS) red blood cell samples edited with the ABE variant base editors described herein clearly identified and separated the HbG Makassar variant and the HbS (SCD) globin variant in the samples, thereby providing a useful tool for identifying true SCD (HbS) patients and reducing or preventing misdiagnosis of patients who instead present with the HbG Makassar globin variant SCD (HbSS). Figures 48A-48C A bar graph representing the relative area under the peaks of UHPLC chromatographic data is presented. The area under the peaks was used to quantify the total change in the levels of different β-globin variants in homozygous SCD samples that had been base-edited using the ABE variants of the present invention (base editor MSP619, 50 nM mRNA, 5000 nM sgRNA (21 nt)). The results presented indicate that the degree of conversion to HbS variant globin is positively correlated with asymptomatic HbG-Makassar globin. Figure 49 This table depicts all possible PAM-accessible Cas9 variants within the NRNN PAM interval. Only Cas9 variants that are required to recognize three or fewer of the specified nucleotides in their PAMs are listed. Non-G PAM variants include SpCas9-NRRH, SpCas9-NRTH, and SpCas9-NRCH. (Miller, SM et al., Continuous evolution of SpCas9 variants compatible with non-G PAMs, Nat. Biotechnol. (2020), ( / / doi.org / 10.1038 / s41587-020-0412-8), the contents of which are incorporated herein by reference in their entirety. DETAILED DESCRIPTION

[0463] As described below, the present invention features compositions and methods for modifying mutations associated with sickle cell disease (SCD). In some embodiments, the editing corrects a deleterious mutation, such that the edited polynucleotide is distinguishable from a wild-type reference polynucleotide sequence. In another embodiment, the editing corrects a deleterious mutation, such that the edited polynucleotide comprises a benign mutation.

[0464] HBB gene editing

[0465] As described herein, the compositions and methods of the present invention are suitable and advantageous for use in treating sickle cell disease (SCD), which is caused by a Glu→Val mutation at the sixth amino acid position in the β-globin protein encoded by the HBB gene. Despite significant advances in gene editing, precise correction of the diseased HBB gene to restore the Val→Glu position remains unavailable, and this is currently unattainable using CRISPR / Cas nucleases or CRISPR / Cas base editing methods.

[0466] Genome editing using CRISPR / Cas nucleases to replace the affected nucleotide in the HBB gene requires cleavage of the genome DNA. However, cleavage of the genome DNA increases the risk of indels, potentially leading to unintended and undesirable consequences, including premature stop codons and altered codon reading frames. Furthermore, the creation of double-strand breaks within the β-globin locus has the potential to fundamentally modify the locus through recombination events. The β-globin locus contains a cluster of globin genes that share sequence identity with another 5'-ε-; Gγ-; Aγ-; δ-; and β-globin-3'. Due to the structure of the β-globin locus, recombination repair of double-strand breaks within the locus could potentially result in gene loss of sequences intercalated between globin genes, such as between the δ- and β-globin genes.

[0467] Unintended modification of gene loci also carries the risk of causing thalassemia. CRISPR / Cas base editing also guarantees its ability to produce precise modifications at the nucleobase stage. However, precise modification of Val→Glu(G T G→G A G) A T·A to A·T inversion editor is required, which is currently known not to exist. In addition, the specificity of CRISPR / Cas base editing is due in part to the limited window of editable nucleotides created by the R-loop formed when CRISPR / Cas binds to DNA. Therefore, CRISPR / Cas targeting must occur at or near the sickle cell locus in order for base editing to be possible, and optimal editing within the window may have additional sequence requirements. One requirement for CRISPR / Cas targeting is the presence of a protospacer adjacent motif (PAM) flanking the targeted site. For example, many base editors are based on SpCas9, which requires an NGG PAM. Even though it is hypothesized that T·A can be inverted to A·T, these SpCas9 base editors do not have an NGG PAM that would place the target "A" at the desired position. Although many new CRISPR / Cas proteins have been discovered or generated to expand the collection of available PAMs, the requirement for a PAM remains a limiting factor in the ability to direct CRISPR / Cas base editors to specific nucleotides anywhere in the genome.

[0468] The present invention is based, at least in part, on several discoveries described herein that address the aforementioned challenges and provide genome editing methods for treating sickle cell anemia. In one embodiment, the present invention is based, in part, on the ability to replace the valine at amino acid position 6, which causes sickle cell disease, with alanine, thereby generating an Hb variant (HbMakassar) that does not produce a sickle cell phenotype. Although precise correction (GTG→GAG) is not possible without a T·A to A·T inverted base editor, experiments conducted herein have found that using an A·T to G·C base editor (ABE) can generate a Val→Ala (GTG→GCG) substitution (i.e., the HbMakassar variant). This was achieved, in part, by developing novel base editors and novel base editing strategies provided herein. For example, novel ABE base editors (i.e., having an adenosine deaminase domain) utilize flanking sequences (e.g., PAM sequences; zinc finger binding sequences) for optimal base editing at sickle cell target sites.

[0469] Thus, the present invention includes compositions and methods for base editing thymidine (T) to cytidine (C) in the codon for the 6th amino acid of the sickle cell variant of the β-globin protein (sickle HbS; E6V), thereby replacing valine at this amino acid position with alanine (V6A). Substituting valine at position 6 of HbS with alanine produces a β-globin protein variant that lacks a sickle cell phenotype (e.g., lacks the potential for polymerization as in pathogenic variant HbS). Thus, the compositions and methods of the present invention are useful for treating sickle cell disease (SCD).

[0470] Nucleobase editors

[0471] Disclosed herein is a base editor or a nuclear base editor for editing, modifying or modifying a target nucleotide sequence of a polynucleotide (e.g., an HBB polynucleotide). This invention describes a nuclear base editor or a base editor comprising a polynucleotide programmable nucleotide binding domain and a nuclear base editing domain (e.g., an adenosine deaminase). The polynucleotide programmable nucleotide binding domain, when coupled to a bound guide polynucleotide (e.g., a gRNA), can specifically bind to a target polynucleotide sequence (i.e., through complementary base pairing between the bases of the bound guide nucleic acid and the bases of the target polynucleotide sequence), thereby allowing the base editor to lock onto the target nucleic acid sequence to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid.

[0472] Polynucleotide programmable nucleotide binding domain

[0473] It should be understood that a polynucleotide-programmable nucleotide-binding domain may also include a nucleic acid-programmable protein that binds to RNA. For example, a polynucleotide-programmable nucleotide-binding domain may be associated with a nucleic acid that directs the polynucleotide-programmable nucleotide-binding domain to RNA. Other nucleic acid-programmable DNA-binding proteins are also within the scope of the present invention but are not explicitly listed herein.

[0474] The polynucleotide programmable nucleotide binding domain of the base editor itself may comprise one or more domains. For example: the polynucleotide programmable nucleotide binding domain may comprise one or more nuclease domains. In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain may comprise an endonuclease or an exonuclease. The term "exonuclease" herein refers to a protein or polypeptide that can digest a nucleic acid (e.g., RNA or DNA) from a free terminal, and the term "endonuclease" refers to a protein or polypeptide that can catalyze (e.g., cleave) an internal region in a nucleic acid (e.g., DNA or RNA). In some embodiments, an endonuclease can cleave a single strand of a double-stranded nucleic acid. In some embodiments, an endonuclease can cleave both strands of a double-stranded nucleic acid molecule. In some embodiments, the polynucleotide programmable nucleotide binding domain may be a deoxyribonuclease. In some embodiments, the polynucleotide programmable nucleotide binding domain may be a ribonuclease.

[0475] In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain can cut the zero (zero) strand, one strand, or both strands of the target polynucleotide. In some embodiments, the polynucleotide programmable nucleotide binding domain may include a nickase domain. The term "nickase" herein refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain that can only cleave one of the two strands of a double-stranded nucleic acid molecule (e.g., DNA). In some embodiments, a nickase can be introduced into an active polynucleotide programmable nucleotide binding domain by one or more mutations, while being derived from a complete catalytically active (e.g., natural) type of a polynucleotide programmable nucleotide binding domain. For example, if the polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain may include a D10A mutation and a histidine at position 840. In these examples, residue H840 retains catalytic activity and thereby cleaves a single strand of the nucleic acid double helix. In another example, the Cas9-derived nickase domain may include an H840A mutation, while the amino acid residue at position 10 remains D. In some embodiments, the nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide-programmable nucleotide-binding domain by removing all or part of the nuclease domain that is not required for nickase activity. For example, if the polynucleotide-programmable nucleotide-binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can comprise a deletion of all or part of the RuvC domain or the HNH domain.

[0476] The amino acid sequence of an example of a catalytically active Cas9 is as follows:

[0477]

[0478] A base editor comprising a polynucleotide programmable nucleotide binding domain containing a nickase domain can therefore produce single-stranded DNA breaks (nicks) on a specific polynucleotide target sequence (e.g., determined by the complementary sequence of the bound guide nucleic acid). In some embodiments, one strand of the nucleic acid double-helix target polynucleotide sequence cleaved by a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain) is a strand that will not be edited by the base editor (i.e., the strand cleaved by the base editor is opposite to the strand containing the base to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain) can cleave a strand of the DNA molecule that is targeted for editing. In this case, the strand that is not targeted will not be cleaved.

[0479] Also provided herein is a base editor comprising a catalytically inactivated (i.e., incapable of cleaving a target polynucleotide sequence) polynucleotide programmable nucleotide binding domain. The terms "catalytically inactivated" and "nuclease inactivated" are used interchangeably herein and refer to a polynucleotide programmable nucleotide binding domain having one or more mutations and / or deletions, resulting in its inability to cleave a strand of nucleic acid. In some embodiments, the catalytically inactivated polynucleotide programmable nucleotide binding domain base editor lacks nuclease activity due to specific point mutations in one or more nuclease domains. For example, taking a base editor comprising a Cas9 domain as an example, Cas9 may comprise both a D10A mutation and an H840A mutation. These mutations inactivate both nuclease domains, thereby resulting in a loss of nuclease activity. In other embodiments, the catalytically inactivated polynucleotide programmable nucleotide binding domain may comprise one or more deletions of all or a portion of the catalytic domain (e.g., RuvC1 and / or HNH domain). In other embodiments, the catalytically inactivated polynucleotide programmable nucleotide binding domain comprises a point mutation (e.g., D10A or H840A) and a deletion of all or a portion of the nuclease domain.

[0480] It is also contemplated herein that mutations of catalytically inactivated polynucleotide programmable nucleotide binding domains can be generated from previously functional polynucleotide programmable nucleotide binding domains. For example, using catalytically inactivated Cas9 ("dCas9") as an example, variants are provided having mutations other than D10A and H840A, which result in nuclease-inactivated Cas9. Such mutations, for example, include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). Other suitable nuclease-inactivating dCas9 domains are known to those skilled in the art based on the present invention and are within the scope of the present invention. Examples of such other suitable nuclease-inactivating Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9):833-838, the entire contents of which are incorporated herein by reference).

[0481] Non-limiting examples of polynucleotide programmable nucleotide-binding domains that can be incorporated into base editors include CRISPR protein-derived domains, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some embodiments, a base editor comprises a polynucleotide-programmable nucleotide-binding domain comprising a natural or modified protein or portion thereof, which, via a bound guide nucleic acid, can bind to a nucleic acid sequence during CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-mediated modification of the nucleic acid. These proteins are referred to herein as "CRISPR proteins." Thus, disclosed herein is a base editor comprising a polynucleotide-programmable nucleotide-binding domain comprising all or a portion of a CRISPR protein (i.e., a base editor comprising all or a portion of a CRISPR protein as a domain, also referred to as a "CRISPR protein-derived domain" of the base editor). The CRISPR protein-derived domain incorporated into the base editor can be modified relative to a wild-type or natural CRISPR protein. For example, a CRISPR protein-derived domain, as described below, can comprise one or more mutations, insertions, deletions, rearrangements, and / or recombination relative to a wild-type or natural CRISPR protein.

[0482] CRISPR is an acquired immune system that provides protection against mobile genetic factors (viruses, transposable elements, and conjugative plasmids). The CRISPR cluster contains a spacer, a sequence complementary to the aforementioned mobile factor, and a target invasive nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In the type II CRISPR system, the correction processing of pre-crRNA requires trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. TracrRNA acts as a guide for ribonuclease 3 to assist pre-crRNA processing. Subsequently, Cas9 / crRNA / tracrRNA cleaves linear or circular dsDN targets complementary to the spacer in an endonucleolytic manner. The target chain that is not complementary to the crRNA is first cut in an endonucleolytic manner, and then trimmed 3′-5′ in an exonucleolytic manner. In nature, DNA-binding and cleavage usually require proteins and two types of RNA. However, a single guide RNA ("sgRNA" or simply "gRNA") can be engineered to incorporate both crRNA and tracrRNA into a single RNA species. See, for example, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif (PAM or protospacer adjacent motif) within the CRISPR repeat sequence to help distinguish between self and non-self repeats.

[0483] In some embodiments, the methods described herein can utilize engineered Cas proteins. Guide RNAs (gRNAs) are short synthetic RNAs composed of the backbone sequence necessary for Cas binding and a user-defined spacer of ~20 nucleotides that specifies the genomic target to be modified. Thus, one skilled in the art can alter the genomic target specificity of a Cas protein, which is determined in part by the degree of specificity of the gRNA targeting sequence for that genomic target relative to the rest of the genome.

[0484] In some embodiments, the gRNA backbone sequence is as follows: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAUAAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU.

[0485] In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is an endonuclease (e.g., a deoxyribonuclease or a ribonuclease) that can bind to a target polynucleotide when bound to a bound guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is a nickase that can bind to a target polynucleotide when bound to a bound guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is a catalytic inactivation domain that can bind to a target polynucleotide when bound to a bound guide nucleic acid. In some embodiments, the target polynucleotide bound by the CRISPR protein-derived domain of the base editor is DNA. In some embodiments, the target polynucleotide bound by the CRISPR protein-derived domain of the base editor is RNA.

[0486] Cas proteins that can be used herein include class 1 and class 2, and non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4 , Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i, CARF, DinG, homologs thereof, or modified forms thereof. Unmodified CRISPR enzymes can have DNA cleavage activity, such as Cas9, which has two functional endonuclease domains: RuvC and HNH. The CRISPR enzyme can predominantly cleave a target sequence, such as one or both strands within the target sequence and / or within the complement of the target sequence. For example, the CRISPR enzyme can predominantly cleave one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence.

[0487] Vectors encoding CRISPR enzymes that have been mutated relative to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide comprising the target sequence, can be used. Cas9 can refer to a polypeptide having at least or about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to an example wild-type Cas9 polypeptide (e.g., Cas9 from Streptococcus pyogenes). Cas9 can refer to a polypeptide having at most or at most about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to a wild-type Cas9 polypeptide example (e.g., from Streptococcus pyogenes). Cas9 can refer to a wild-type or modified form of a Cas9 protein, which can include amino acid changes, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0488] In some embodiments, the CRISPR protein-derived domain of the base editor may include all or part of Cas9 from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense, China (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021847.1); 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 Staphylococcus aureus.

[0489] Cas9 domains of nucleobase editors

[0490] The Cas9 nuclease sequence and structure are known to those skilled in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C, Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski J., et al., “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” (See, for example, "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," by Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E., Science 337:816-821 (2012), each of which is hereby incorporated by reference in its entirety.) Cas9 orthologs have been described in various species, including, but not limited to, Streptococcus pyogenes and Streptococcus thermophilus.Other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art in light of the present disclosure, and include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0491] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting examples of Cas9 domains are provided herein. The Cas9 domain may be a nuclease active Cas9 domain, a nuclease inactivation Cas9 domain (dCas9), or a Cas9 nickase (nCas9). In some embodiments, the Cas9 domain is a nuclease active domain. For example: the Cas9 domain may be a Cas9 domain that can cut two strands of a double-helical nucleic acid (e.g., two strands of a double-helical DNA molecule). In some embodiments, the Cas9 domain comprises any one of the amino acid sequences shown herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences shown herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of the amino acid sequences disclosed herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any one of the amino acid sequences disclosed herein.

[0492] In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, the protein comprises one of the two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or a fragment thereof are referred to as "Cas9 variants." Cas9 variants and Cas9 or a fragment thereof share common homology. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 variant can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the corresponding wild-type Cas9. In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0493] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but rather comprise only one or more fragments thereof. Examples of amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and those skilled in the art generally understand the sequences of other suitable Cas9 domains and fragments.

[0494] The Cas9 protein can be associated with a guide RNA that directs the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Examples of nucleic acid-programmable DNA-binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b / C2C1, and Cas12c / C2C3.

[0495] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI reference sequence: NC_017053.1, nucleotide and amino acid sequences are as follows):

[0496] ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGAT GATTATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAAT CTTATAGGGGGCTCTTTTATTTGGCAGTGGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGT AGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAA GTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGT CATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTG CGAAAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATG ATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTA TTTATCCAGTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTAGAGTAGAT GCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCC GGTGAGAAGAGAAATGGCTTGTTTGGGAATCTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAA TCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGAT AATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCT ATTTTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATT AAGCGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAA AAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGC CAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTG AAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATT CACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGT GAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGT CGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGAT AAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAA GTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATAT GTTACTGAGGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTC TTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTT GATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGCGCCTACCATGATTTGCTA AAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTA ACATTGACCTTATTTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGAT GATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATT AATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAAT CGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGATATTCAAAGGCACAGGTG TCTGGACAAGGCCATAGTTTACATGAACAGATTGCTAACTTAGCTGGCAGTCCTGCTATTAAAAAAGGT ATTTTACAGACTGTAAAAATTGTTGATGAACTGGTCAAAGTAATGGGGCATAAGCCAGAAAATATCGTT ATTGAAATGGCACGTGAAAATCAGACAACTCAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGA ATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTG CAAAATGAAAAGCTCTATCTCTATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGAT ATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCAATA GACAATAAGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTA GTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGAT AATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTG GTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGAT GAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGA AAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAAT GCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTAT AAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATAT TTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAA CGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACA GTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTC TCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCA AAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAA GGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTT GAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAA CTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTA CAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAA AAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTA GATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAA GTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTA TTTACGTTGACGAATCTTGGAGCTCCCGCTTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGA TATACGTCTACAAAAGAAGTTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACA CGCATTGATTTGAGTCAGCTAGGAGGTGACTGA

[0497]

[0498] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0499] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence:

[0500] ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGAT GAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAAT CTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGG AGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAA GTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGG CACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTC AGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATG ATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTG TTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGAT GCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCC GGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAG TCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGAC AATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCA ATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATC AAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAG AAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGGAGT CAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTA AAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGACTTTCGACAACGGTAGCATTCCACATCAAATC CACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGT GAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTACTATGTGGGACCCCTGGCCCGAGGGAACTCT CGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGAT AAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCACTTTGACAAGAATTTACCGAACGAAAAA GTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTAT GTCACTGAGGGCATGCGTAAACCCGCCTTTTCTAAGCGGAGAACAGAAAGCAATAGTAGATCTGTTA TTCAAGACCAACCGCAAAGTGACAGTTAAGCATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTC GATTCTGTCGAGATCTCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTA AAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTG ACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGAC GATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATC AACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAAT AGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTT TCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGC ATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATT GTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAG AGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAA TTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTG GACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCA ATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAA GTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTC GATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAG CTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAGATTCCCGAATGAATACGAAATAC GACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTC AGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTT AATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGAT TACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAA TACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGC AAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCG ACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGG TTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGAC CCGAAAGGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAG AAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCT TTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATT AAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAG CTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTAC GAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTAT CTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGAC AAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCAT TTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAA CGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAA ACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGAC TACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCA GGA

[0501]

[0502]

[0503] (Single underline: HNH domain; double underline: RuvC domain)

[0504] In some embodiments, the 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):

[0505] ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGAT GAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAAT CTTATAGGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGT AGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAA GTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGT CATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTG CGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATG ATTAAGTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTA TTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGAT GCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCC GGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAA TCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAGATACTTACGATGATGATTTAGAT AATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCT ATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTTATCAGCTTCAATGATT AAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAA AAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGC CAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTG AAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATT CACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGT GAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGT CGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGAT AAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAA GTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATAT GTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTC TTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTT GATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTA AAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTA ACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGAT GATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATT AATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAAT CGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTG TCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGT ATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATC GTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAA CGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAA TTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTA GATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCA ATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAA GTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTT GATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAA TTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATAC GATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTC CGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTA AATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGAT TATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAA TATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGC AAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCC ACATGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGA TTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGAT CCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAA AAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCC TTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAGACTTAATCATT AAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAA TTACAAAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTAT GAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAACAACAATTGTTTGTGGAGCAGCATAAGCATTAT TTGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGAT AAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCAT TTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAA CGATATACGTCTACAAAAGAAGTTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAA ACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGA

[0506]

[0507]

[0508] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0509] In some embodiments, Cas9 is Cas9 from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense, China (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021847.1); Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis I (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1), or Neisseria meningitidis (NCBI Ref: YP_002342100.1), or Cas9 from any other organism.

[0510] It should be understood that additional Cas9 proteins (e.g., nuclease-inactivated Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including variants and homologs thereof, are within the scope of the present invention. Examples of Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is a nuclease-inactivated Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease-active Cas9.

[0511] In some embodiments, the Cas9 domain is a nuclease-inactivating Cas9 domain (dCas9). For example, the dCas9 domain may bind to a double-helical nucleic acid molecule (e.g., via a gRNA molecule) and will not cleave either chain of the double-helical nucleic acid molecule. In some embodiments, the nuclease-inactivating dCas9 domain comprises a D10X mutation and an H840X mutation of the amino acid sequence shown herein, or a corresponding mutation of any amino acid sequence provided herein, wherein X is any amino acid change. In some embodiments, the nuclease-inactivating dCas9 domain comprises a D10A mutation and an H840A mutation of the amino acid sequence shown herein, or a corresponding mutation of any amino acid sequence provided herein. In one example, the nuclease-inactivating Cas9 domain comprises the amino acid sequence shown in the cloning vector pPlatTET-gRNA2 (accession number BAV54124).

[0512] The amino acid sequence of a catalytically inactive Cas9 (dCas9) example is as follows: MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0513] (See, for example, 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 content of which is incorporated herein by reference).

[0514] Other suitable nuclease-inactivating dCas9 domains are known to those skilled in the art based on the present invention and are within the scope of the present invention. Examples of such other suitable nuclease-inactivating Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9):833-838, the entire contents of which are incorporated herein by reference).

[0515] In some embodiments, the Cas9 nuclease has an inactivated (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase, referred to as an "nCas9" protein (for "nickase" Cas9). Nuclease-inactivated Cas9 proteins can be used interchangeably with "dCas9" proteins (for nuclease-"inactivated" Cas9) or catalytically inactivated Cas9. Methods for generating Cas9 proteins (or fragments thereof) with inactivated DNA cleavage domains are known (see, e.g., Jinek et al., Science. 337:816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152(5):1173-83, each of which is incorporated herein by reference in its entirety). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the complementary strand of the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337:816-821 (2012); Qi et al., Cell. 28;152(5):1173-83 (2013)).

[0516] In some embodiments, the dCas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the dCas9 domains provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of the amino acid sequences disclosed herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any one of the amino acid sequences disclosed herein.

[0517] In some embodiments, the dCas9 corresponds to or comprises a portion or all of a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises the D10A and H840A mutations or corresponding mutations in another Cas9.

[0518] In some embodiments, the dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A):

[0519]

[0520]

[0521] (Single underline: HNH domain; double underline: RuvC domain).

[0522] In some embodiments, the Cas9 domain comprises a D10A mutation, and the residue at position 840 retains a histidine in the amino acid sequences provided above, or at the corresponding position in any of the amino acid sequences provided herein.

[0523] In other embodiments, dCas9 variants are provided that have mutations other than D10A and H840A, which, for example, result in nuclease-inactive Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, the provided dCas9 variants or homologs are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, dCas9 variants are provided having shorter or longer amino acid sequences that differ by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, or by about 100 or more amino acids.

[0524] In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase may be a Cas9 protein that can cleave only one strand of a double-helical nucleic acid molecule (e.g., a double-helical DNA molecule). In some embodiments, the Cas9 nickase cleaves the target strand of the double-helical nucleic acid molecule, meaning that the strand cleaved by the Cas9 nickase is base-paired (complementary) with the gRNA (e.g., sgRNA) that has been bound to Cas9. In some embodiments, the Cas9 nickase comprises a D10A mutation and has a histidine at position 840. In some embodiments, the Cas9 nickase cleaves the non-target, non-base-edited strand of the double-helical nucleic acid molecule, meaning that the strand cleaved by the Cas9 nickase does not base-pair with the gRNA (e.g., sgRNA) that has been bound to Cas9. In some embodiments, the Cas9 nickase comprises an H840A mutation and has an aspartic acid residue at position 10, or a corresponding mutation. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the Cas9 nickases provided herein. Other suitable Cas9 nickases are known to those skilled in the art based on the present invention and are within the scope of the present invention.

[0525] The amino acid sequence of an example catalytic Cas9 nickase (nCas9) is as follows:

[0526] In some embodiments, Cas9 refers to Cas9 from an archaeon (e.g., nanoarchaea), which belongs to the domain and kingdom Unicellular Prokaryotes. In some embodiments, the programmable nucleotide-binding protein may be CasX or CasY, as 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, the entire contents of which are incorporated herein by reference. Using genome-resolved metagenomics, numerous CRISPR-Cas systems have been identified, including Cas9, which was first reported in the Archaeal domain of life. These diverse Cas9 proteins have emerged in the little-studied Nanoarchaea as part of an active CRISPR-Cas system. Two previously unknown systems, CRISPR-CasX and CRISPR-CasY, have been discovered in bacteria and are the most robust systems discovered to date. In some embodiments, in the base editor system described herein, Cas9 is replaced by CasX or a variant of CasX. In some embodiments, in the base editor system described herein, Cas9 is replaced by CasY or a variant of CasY. It should be understood that other RNA-guided DNA-binding proteins can be used as nucleic acid programmable DNA-binding proteins (napDNAbp) and are within the scope of the present invention. In some embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) of any fusion protein provided herein may be CasX or CasY protein. In some embodiments, napDNAbp is CasX protein. In some embodiments, napDNAbp is CasY protein. In some embodiments, napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a native CasX or CasY protein. In some embodiments, the programmable nucleotide-binding protein is a native CasX or CasY protein. In some embodiments, the programmable nucleotide-binding protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any CasX or CasY protein described herein. It will be appreciated that CasX and CasY from other bacterial species may also be employed according to the present invention.An exemplary CasX ((uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53)tr|F0NN87|F0NN87_SULIHCRISPR-associatedCasx protein OS=Sulfolobus islandicus (strain HVE10 / 4) GN=SiH_0402PE=4SV=1) amino acid sequence is as follows: MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGE. An exemplary CasX (>tr|F0NH53|F0NH53_SULIR CRISPR-associated protein, Casx OS=Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4SV=1) amino acid sequence is as follows: MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTSNIILPLSGNDKNPWTETLKCYNFPTTTVALSEVFKNFSQVKECEEVSAPSFVKPFEYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGE. δ- proteobacteria (Deltaproteobacteria) CasX MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQ KWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA An exemplary CasY ((ncbi.nlm.nih.gov / protein / APG80656.1)>APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria bacteria]) amino acid sequence is as follows:

[0527] The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Upon target binding, Cas9 undergoes a conformational change that positions the nuclease domains to cleave the opposite strand of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (~3-4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired by one of two general repair pathways: (1) the efficient but error-prone nonhomologous end joining (NHEJ) pathway; or (2) the less efficient but higher-fidelity homology-directed repair (HDR) pathway.

[0528] The "efficacy" of non-homologous end joining (NHEJ) and / or homology-guided repair (HDR) can be calculated using any convenient method. For example, in some embodiments, the efficacy is expressed as a percentage of successful HDR. For example, a surveyor nuclease assay can be used to generate cleavage products, and the percentage is calculated using the ratio of products to substrate. For example, a surveyor nuclease assay can be used to directly cleave DNA containing newly integrated restriction enzyme sequences generated as a result of successful HDR. The more substrate that is cleaved, the higher the HDR percentage (the higher the efficacy of HDR). In an exemplary embodiment, the following formula can be used to calculate the ratio (percentage) of HDR [(cleavage product) / (substrate plus cleavage product)] (for example, (b+c) / (a+b+c), where "a" is the band density of the DNA substrate, and "b" and "c" are cleavage products).

[0529] In some embodiments, efficacy can be expressed as a percentage of successful NHEJ. For example, a T7 endonuclease I assay can be used to generate cleavage products, and the ratio of product to substrate can be used to calculate the percentage of NHEJ. T7 endonuclease I cleaves mismatched duplex DNA resulting from hybridization of wild-type and mutant DNA strands (NHEJ generates small random insertions or deletions (indels) at the original break site). More cleavages indicate a higher percentage of NHEJ (higher NHEJ efficacy). In an exemplary embodiment, the ratio (percentage) of NHEJ can be calculated using the following formula: (1-(1-(b+c) / (a+b+c)) 1 / 2 )×100, where “a” is the band density of the DNA substrate, and “b” and “c” are the cleavage products (Ran et al., Cell. 2013 Sep. 12; 154(6): 1380-9; and Ran et al., Nat Protoc. 2013 Nov.; 8(11): 2281–2308).

[0530] The NHEJ repair pathway is the most active repair mechanism, frequently causing small nucleotide insertions or deletions at DSB sites. The stochastic nature of NHEJ-mediated DSB repair has important practical implications, as cell populations expressing Cas9 and gRNA or guide polynucleotides can generate divergent mutations. In most cases, NHEJ generates small insertions or deletions in the target DNA, resulting in amino acid deletions, insertions, or frameshift mutations, leading to the premature formation of stop codons within the open reading frame (ORF) of the target gene. The ideal end result is a loss-of-function mutation within the target gene.

[0531] Although NHEJ-mediated DSB repair often disrupts the open reading frame of a gene, homology-guided repair (HDR) can be used to generate specific nucleotide changes, ranging from single nucleotide changes to large blocks, such as the addition of a fluorophore or tag. To utilize HDR for gene editing, a DNA repair template containing the desired sequence can be delivered to a cell type equipped with a gRNA (group) and Cas9 or a Cas9 nickase. The repair template can contain the desired edit and additional homology sequences immediately upstream and downstream of the target (referred to as the left and right homology arms). The length of each homology arm can be determined by the size of the change to be introduced, with larger blocks requiring longer homology arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. Even in cells expressing Cas9, gRNA, and an exogenous repair template, the efficacy of HDR is generally low (<10% of modified alleles). The efficacy of HDR can be enhanced by cell synchronization, as HDR occurs during the S and G2 phases of the cell cycle. Chemical or genetic suppressors of genes involved in NHEJ can also increase the frequency of HDR.

[0532] In some embodiments, Cas9 is a modified Cas9. A given gRNA targeting sequence may have additional sites throughout the genome with partial homology. These sites are called off-target sites and must be considered when designing the gRNA. In addition to optimizing gRNA design, CRISPR specificity can also be improved by modifying Cas9. Cas9 generates double-strand breaks (DSBs) by combining the activities of two nuclease domains, RuvC and HNH. Cas9 nickase (which is the D10A mutant of SpCas9) retains one nuclease domain and generates DNA nicks instead of DSBs. The nickase system can also be combined with HDR-mediated gene editing for specific gene editing.

[0533] In some embodiments, Cas9 is a variant Cas9 protein. The amino acid sequence of the variant Cas9 polypeptide differs from the amino acid sequence of the wild-type Cas9 protein by one amino acid (e.g., deletion, insertion, substitution, fusion). In some examples, the amino acid changes (e.g., deletion, insertion, or substitution) of the variant Cas9 polypeptide reduce the nuclease activity of the Cas9 polypeptide. For example, in some examples, the variant Cas9 polypeptide has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 protein. In some embodiments, the variant Cas9 protein has no substantial nuclease activity. When the individual Cas9 protein is a variant Cas9 protein without substantial nuclease activity, it can be referred to as "dCas9".

[0534] In some embodiments, the variant Cas9 protein has reduced nuclease activity. For example, the variant Cas9 protein has less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the endonuclease activity of a wild-type Cas9 protein (e.g., a wild-type Cas9 protein).

[0535] In some embodiments, the variant Cas9 protein can cleave the complementary strand of the guide target sequence, but the ability to cleave the non-complementary strand of the double-stranded guide target sequence is reduced. For example, the variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, the variant Cas9 protein has D10A (aspartic acid at position 10 becomes an alanine amino acid), so that the complementary strand of the double-stranded guide target sequence can be cleaved, but the ability to cleave the non-complementary strand of the double-stranded guide target sequence is reduced (so when the variant Cas9 protein cleaves the double-stranded target nucleic acid, it will cause a single-strand break (SSB) instead of a double-strand break (DSB)) (see, for example: Jinek et al., Science. 2012 Aug. 17; 337(6096): 816-21).

[0536] In some embodiments, the variant Cas9 protein can cleave the non-complementary strand of a double-stranded guide target sequence, but has a reduced ability to cleave the complementary strand of the guide target sequence. For example, the variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif). As a non-limiting example, in some embodiments, the variant Cas9 protein has an H840A (histidine at position 840 becomes alanine amino acid) mutation, thereby cleaving the non-complementary strand of the guide target sequence, but has a reduced ability to cleave the complementary strand of the guide target sequence (thus, when the variant Cas9 protein cleaves a double-stranded guide target sequence, it generates an SSB instead of a DSB). Such Cas9 proteins have a reduced ability to cleave a guide target sequence (e.g., a single-stranded guide target sequence), but retain the ability to bind to the guide target sequence (e.g., a single-stranded guide target sequence).

[0537] In some embodiments, the variant Cas9 protein has reduced ability to cleave both the complementary and non-complementary strands of double-stranded target DNA. As a non-limiting example, in some embodiments, the variant Cas9 protein carries both the D10A and H840A mutations, thereby reducing the ability of the polypeptide to cleave both the complementary and non-complementary strands of double-stranded target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0538] As another non-limiting example, in some embodiments, a variant Cas9 protein has W476A and W1126A mutations, thereby reducing the ability of the polypeptide to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0539] As another non-limiting example, in some embodiments, the variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, thereby reducing the ability of the polypeptide to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0540] As another non-limiting example, in some embodiments, the variant Cas9 protein has H840A, W476A, and W1126A mutations, thereby reducing the ability of the polypeptide to cleave target DNA. These Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, the variant Cas9 protein has H840A, D10A, W476A, and W1126A mutations, thereby reducing the ability of the polypeptide to cleave target DNA. These Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). In some embodiments, the variant Cas9 has restored the catalytic His residue (A840H) at position 840 of the Cas9 HNH domain.

[0541] As another non-limiting example, in some embodiments, the variant Cas9 protein has H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, thereby reducing the ability of the polypeptide to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, the variant Cas9 protein has D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, thereby reducing the ability of the polypeptide to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA). In some embodiments, when a variant Cas9 protein has W476A and W1126A mutations, or when a variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, the variant Cas9 protein does not effectively bind to a PAM sequence. Thus, in some instances, when such variant Cas9 proteins are used in a binding method, the method does not require a PAM sequence. In other words, in some embodiments, when such variant Cas9 proteins are used in a binding method, the method may include a guide RNA, but the method can be performed without a PAM sequence (so that binding specificity is provided by the targeting segment of the guide RNA). Other residues can be mutated to achieve the above-described effects (i.e., to inactivate one or the other nuclease portion). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 may be altered (i.e., substituted). In addition, mutations other than alanine substitutions are also suitable.

[0542] In some embodiments, the variant Cas9 protein has reduced catalytic activity (for example, when the Cas9 protein has D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 mutations, such as D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A), the variant Cas9 protein still binds to the target DNA in a site-specific manner (because it is still guided to the target DNA sequence by the guide RNA), as long as it still retains the ability to interact with the guide RNA.

[0543] 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.

[0544] In some embodiments, a modified SpCas9 comprising amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and having specificity for the altered PAM 5′-NGC-3′ is employed.

[0545] Alternatives to the Streptococcus pyogenes (S. pyogenes) Cas9 include RNA-guided endonucleases from the Cpf1 family, which exhibit cleavage activity in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA-editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of the class II CRISPR / Cas system. This postnatal immune mechanism is present in Prevotella and Francisella bacteria. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses the guide RNA to locate and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the limitations of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, Cpf1-mediated DNA cleavage results in a double-strand break with a short 3′ overhang. Cpf1's staggered cleavage pattern opens the possibility of directional gene delivery, similar to traditional restriction enzyme selection methods, which can improve gene editing efficiency. Like the aforementioned Cas9 variants and orthologs, Cpf1 can also expand the number of sites targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM site preferred by SpCas9. The Cpf1 locus comprises a mixed α / β domain, RuvC-I, followed by a helical region, RuvC-II, and zinc finger-like domains. The Cpf1 protein possesses a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, Cpf1 lacks the HNH endonuclease domain, and the N-terminus of Cpf1 lacks the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain architecture suggests that Cpf1 possesses unique functionality, placing it within a Class 2, Type V CRISPR system. The Cas1, Cas2, and Cas4 proteins encoded by the Cpf1 locus are more similar to Type I and Type III systems than to Type II systems. Functional Cpf1 does not require transactivating CRISPR RNA (tracrRNA), so only CRISPR (crRNA) is required. This is advantageous for genome editing because Cpf1 is not only smaller than Cas9, but also has a smaller sgRNA molecule (approximately half the number of nucleotides of Cas9). In contrast to the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex cleaves the target DNA or RNA by recognizing the protospacer-adjacent motif 5'-YTN-3'. After recognizing the PAM, Cpf1 introduces a sticky-end-like DNA double-strand break containing an overhang of 4 or 5 nucleotides.

[0546] Cas12 domain of nucleobase editors

[0547] Microbial CRISPR-Cas systems are generally divided into Type 1 and Type 2 systems. Type 1 systems have multi-subunit effector complexes, while Type 2 systems have single protein effectors. For example, Cas9 and Cpf1 are Type 2 effectors, although they have different types (Type II and Type V, respectively). In addition to Cpf1, Type 2, Type V CRISPR-Cas systems also include Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. See, for example, Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems” Mol. Cell, 2015 Nov. 5; 60(3): 385-397; Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR Journal, 2018, 1(5): 325-336; and Yan et al., “Functionally Diverse Type VCRISPR-Cas Systems” Science, 2019 Jan. 4; 363: 88-91; the entire contents of each of which are incorporated herein by reference. Type V Cas proteins contain a RuvC (or RuvC-like) endonuclease domain. Although the production of mature CRISPR RNA (crRNA) is generally independent of tracrRNA, for example, Cas12b / C2c1 requires tracrRNA to produce crRNA. Cas12b / C2c1 relies on both crRNA and tracrRNA to cleave DNA.

[0548] The nucleic acid programmable DNA binding proteins contemplated by the present invention include Cas proteins belonging to Class 2 V-type (Cas12 proteins). 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, homologs thereof, or modified forms thereof. The Cas12 protein employed herein may also be referred to as a Cas12 nuclease, a Cas12 domain, or a Cas12 protein domain. In some embodiments, the Cas12 protein of the present invention comprises an amino acid sequence interspersed with an internal fusion protein domain (e.g., a deaminase domain).

[0549] In some embodiments, the Cas12 domain is a nuclease inactivation Cas12 domain or a Cas12 nickase. In some embodiments, the Cas12 domain is a nuclease active domain. For example: the Cas12 domain may be a Cas12 domain that cuts one chain in a double-helical nucleic acid (e.g., a double-helical DNA molecule). In some embodiments, the Cas12 domain comprises any one of the amino acid sequences shown herein. In some embodiments, the Cas12 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences shown herein. In some embodiments, the Cas12 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of the amino acid sequences shown herein. In some embodiments, the Cas12 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical consecutive amino acid residues compared to any one of the amino acid sequences shown herein.

[0550] In some embodiments, proteins comprising fragments of Cas12 are provided. For example, in some embodiments, the protein comprises one or two Cas12 domains: (1) the gRNA binding domain of Cas12; or (2) the DNA cleavage domain of Cas12. In some embodiments, proteins comprising Cas12 or a fragment thereof are therefore referred to as "Cas12 variants." Cas12 variants and Cas12 or a fragment thereof share common homology. For example, a Cas12 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas12. In some embodiments, the Cas12 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas12. In some embodiments, the Cas12 variant comprises a fragment of Cas12 (e.g., a gRNA binding domain or a DNA cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas12. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the corresponding wild-type Cas12 amino acid length. 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.

[0551] In some embodiments, Cas12 corresponds to or comprises a portion or all of a Cas12 amino acid sequence having one or more mutations that modify the nuclease activity of Cas12. Such mutations include, for example, amino acid substitutions within the RuvC nuclease domain of Cas12. In some embodiments, the variants or homologs of Cas12 provided are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas12. In some embodiments, the Cas12 variants provided have shorter or longer amino acid sequences that differ by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, or by about 100 or more amino acids.

[0552] In some embodiments, the Cas12 fusion proteins provided herein comprise the full-length amino acid sequence of a Cas12 protein, for example, one of the Cas12 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas12 sequence, but only comprise one or more fragments thereof. Examples of amino acid sequences of suitable Cas12 domains provided herein and other suitable Cas12 domain sequences and fragments are generally understood by those skilled in the art.

[0553] Typically, the second type V Cas protein has a single functional RuvC endonuclease domain (see, for example, Chen et al., "CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity." Science 360:436-439 (2018)). In some cases, the Cas12 protein is a variant of the Cas12b protein. (See Strecker et al., Nature Communications, 2019, 10 (1): Art. No.: 212). In one embodiment, the amino acid sequence of the variant Cas12 polypeptide has 1, 2, 3, 4, 5 or more amino acid differences (e.g., deletions, insertions, substitutions, fusions) when compared to the amino acid sequence of the wild-type Cas12 protein. In some cases, the amino acid changes (e.g., deletions, insertions, or substitutions) of the variant Cas12 polypeptide reduce the activity of the Cas12 polypeptide. For example, in some cases, the variant Cas12 is a Cas12b polypeptide having less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nickase activity of the corresponding wild-type Cas12b protein. In some cases, the variant Cas12b protein has no substantial nickase activity.

[0554] In some cases, the variant Cas12b protein has reduced nickase activity. For example, the variant Cas12b protein has less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the nickase activity of the wild-type Cas12b protein.

[0555] In some embodiments, the Cas12 protein comprises an RNA-guided endonuclease from the Cas12a / Cpf1 family that exhibits activity in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of the class II CRISPR / Cas system. This latter immune mechanism is present in Prevotella and Francisella bacteria. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, which overcomes some limitations of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, Cpf1-mediated DNA cleavage results in a double-strand break with a short 3′ overhang. Cpf1's staggered cleavage pattern opens the possibility of directional gene delivery, similar to traditional restriction enzyme selection methods, which can improve gene editing efficiency. Like the aforementioned Cas9 variants and orthologs, Cpf1 also expands the number of sites available for CRISPR targeting in AT-rich regions or AT-rich genomes that lack the NGG PAM site preferred by SpCas9. The Cpf1 locus comprises a mixed α / β domain, followed by RuvC-I and RuvC-II helical regions, and zinc finger-like domains. The Cpf1 protein possesses a RuvC-like endonuclease domain, similar to the RuvC domain of Cas9. Furthermore, unlike Cas9, Cpf1 lacks the HNH endonuclease domain, and the Cpf1 N-terminus lacks the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain architecture suggests that Cpf1 possesses unique functionality, placing it within a Class II, Type V CRISPR system. The Cas1, Cas2, and Cas4 proteins encoded by the Cpf1 locus are more similar to type I and type III systems than to type II systems. Functional Cpf1 does not require transactivating CRISPR RNA (tracrRNA), so only CRISPR (crRNA) is required. This is beneficial for genome editing because Cpf1 is not only smaller than Cas9, but also has a smaller sgRNA molecule (approximately half the number of nucleotides of Cas9). In contrast to Cas9's targeting of G-rich PAMs, the Cpf1-crRNA complex cleaves target DNA or RNA by discriminating between the protospacer-adjacent motifs 5'-YTN-3' or 5'-TTTN-3'. After discriminating the PAM, Cpf1 introduces a sticky-end-like DNA double-strand break containing a 4- or 5-nucleotide overhang.

[0556] In some embodiments of the present invention, vectors encoding CRISPR enzymes that have been mutated in relation to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide comprising a target sequence. Cas12 can refer to a peptide having at least or about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to an exemplary wild-type Cas12 polypeptide (e.g., Cas12 from Bacillus hisashii). Cas12 can refer to a polypeptide having at most or about at most 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to an exemplary wild-type Cas12 polypeptide (e.g., polypeptides from Bacillus hisashii (BhCas12b), Bacillus sp. V3-13 (BvCas12b), and Alicyclobacillus acidiphilus (AaCas12b). Cas12 can refer to a wild-type or modified form of a Cas12 protein, which can comprise amino acid changes, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0557] Nucleic acid programmable DNA binding protein

[0558] Some embodiments of the present invention provide fusion proteins comprising a domain that can serve as a nucleic acid-programmable DNA-binding protein, which can be used to guide proteins such as base editors to specific nucleic acid (e.g., DNA or RNA) sequences. In certain embodiments, the fusion protein comprises a nucleic acid-programmable DNA-binding protein domain and a deaminase domain. Non-limiting examples of nucleic acid-programmable DNA-binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a type II Cas effector protein, a type V Cas effector protein, a type VI Cas effector protein, CARF, DinG, a homolog thereof, or a modified or engineered form thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of the invention, but may not be explicitly listed in this disclosure.See, for example: Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct; 1: 325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, the contents of which are each incorporated herein by reference in their entirety.

[0559] An example of a nucleic acid-programmable DNA-binding protein with a different PAM specificity than Cas9 is the clustered regularly interspaced short palindromic repeats from Prevotella and Francisella 1 (Cpf1). Similar to Cas9, Cpf1 is also a class II CRISPR effector. Cpf1 has been shown to mediate robust DNA interference, a feature distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking a tracrRNA and utilizes T-rich protospacer-adjacent motifs (TTN, TTTN, or YTN). Furthermore, Cpf1 cleaves DNA via staggered DNA double-strand breaks. Of the 16 Cpf1-f family proteins, two from the genera Acidaminococcus and Lachnospiraceae have been shown to possess potent genome-editing activity in human cells. The Cpf1 protein is known in the art and has been described previously, for example, in Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; the entire contents of which are incorporated herein by reference.

[0560] Suitable for use in the present compositions and methods are nuclease-inactive Cpf1 (dCpf1) variants that utilize a programmable DNA-binding protein domain as a guide nucleotide sequence. The Cpf1 protein possesses a RuvC-like endonuclease domain similar to the RuvC domain of Cas9, but lacks the HNH endonuclease domain, and the N-terminus of Cpf1 lacks the α-helical recognition lobe of Cas9. As shown in Zetsche et al., Cell, 163, 759-771, 2015 (incorporated herein by reference), the RuvC-like domain of Cpf1 is responsible for cleaving both DNA strands, and inactivation of the RuvC-like domain inactivates Cpf1 nuclease activity. For example, mutations corresponding to D917A, E1006A, or D1255A in Francisella novicida Cpf1 inactivate Cpf1 nuclease activity. In some embodiments, dCpf1 of the present invention comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It is understood that any mutation that inactivates the RuvC domain of Cpf1, such as a substitution mutation, deletion, or insertion, can be used according to the present invention.

[0561] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In some embodiments, the Cpf1 protein is a nuclease-inactivated Cpf1 (dCpf1). In some embodiments, Cpf1, nCpf1, or dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the Cpf1 sequence disclosed herein. In some embodiments, dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the Cpf1 sequence disclosed herein and comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It will be appreciated that Cpf1 from other bacterial species may also be used according to the present invention.

[0562] Wild-type Francisella novicida Cpf1 (D917, E1006, and D1255 are bold and underlined):

[0563]

[0564] Francisella novicida Cpf1 D917A (A917, E1006, and D1255 are bold and underlined):

[0565]

[0566] Francisella novicida Cpf1 E1006A (D917, A1006, and D1255 are bold and underlined):

[0567]

[0568] Francisella novicida Cpf1 D1255A (D917, E1006, and A1255 are bold and underlined)

[0569]

[0570] Francisella novicida Cpf1 D917A / E1006A (A917, A1006, and D1255 are bold and underlined):

[0571]

[0572] Francisella novicida Cpf1 D917A / D1255A (A917, E1006, and A1255 are bold and underlined):

[0573]

[0574] Francisella novicida Cpf1 E1006A / D1255A (D917, A1006, and A1255 are bold and underlined):

[0575]

[0576] Francisella novicida Cpf1D917A / E1006A / D1255A (A917, A1006, and A1255 are bold and underlined):

[0577]

[0578] In some embodiments, one of the Cas9 domains present in the fusion protein can be replaced with a programmable DNA-binding protein domain of a guide nucleotide sequence that does not require a PAM sequence.

[0579] In some embodiments, the Cas9 domain is a Cas9 domain from Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domain is a nuclease-active SaCas9, a nuclease-inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, SaCas9 comprises an N579A mutation, or a corresponding mutation in any of the amino acid sequences provided herein.

[0580] In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having an atypical PAM. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a NNGRRT or NNGRRT PAM sequence. In some embodiments, the SaCas9 domain comprises one or more E781X, N967X, and R1014X mutations, or corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more E781K, N967K, and R1014H mutations, or one or more corresponding mutations in any amino acid sequence provided herein. In some embodiments, the SaCas9 domain comprises E781K, N967K, or R1014H mutations, or corresponding mutations in any amino acid sequence provided herein.

[0581] SaCas9 sequence example:

[0582]

[0583] Residue N579, which is underlined and in bold, can be mutated (e.g., to form A579) to produce the SaCas9 nickase.

[0584] SaCas9n sequence example:

[0585]

[0586]

[0587] The above residue A579, which can generate the SaCas9 nickase from N579 mutation, is underlined and in bold.

[0588] SaKKH Cas9 Example:

[0589]

[0590] The above residue A579, which can generate the SaCas9 nickase from N579 mutation, is underlined and in bold. The residues K781, K967, and H1014, which can generate the SaKKH Cas9 from E781, N967, and R1014 mutations, are underlined and in italics.

[0591] In some embodiments, the napDNAbp is arranged in a circular pattern. In the following sequences, plain text represents adenosine deaminase sequences, bold sequences indicate sequences derived from Cas9, italic sequences represent linker sequences, and underlined sequences represent bipartite nuclear localization sequences.

[0592] CP5 (with MSP "NGC" PID and "D10A" nickase):

[0593]

[0594]

[0595] In some embodiments, the nucleic acid-programmable DNA-binding protein (napDNAbp) is the single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, but are not limited to, Cas9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Generally, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while Class 2 systems have single protein effectors. For example, Cas9 and Cpf1 are Class 2 effectors. In addition to Cas9 and Cpf1, Shmakov et al. have described three different Class 2 CRISPR-Cas systems (Cas12b / C2c1 and Cas12c / C2c3) in "Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems", Mol. Cell, 2015 Nov. 5; 60(3): 385-397, the entire contents of which have been incorporated herein by reference. The effectors of t...

Claims

1. A method for editing the promoter of hemoglobin subunit γ1 and / or 2 in a cell in vitro or in vitro, the method comprising contacting the cell with a guide RNA and a fusion protein, the fusion protein consisting of a Cas9 domain, one or two adenosine deaminase domains, one or more linkers joining different components of the fusion protein, and a nuclear localization sequence, wherein: One of the one or two adenosine deaminase domains is an adenosine deaminase variant domain consisting of the amino acid sequence of SEQ ID NO:2 or a fragment thereof lacking only the N-terminal methionine, having modifications relative to SEQ ID NO:2, wherein the modifications consist of changes or combinations of changes selected from the following relative to SEQ ID NO:2: Y147T; Y147R; Y147T and Q154R; Y147T and Q154S; Y147R, Q154R, and Y123H; Y147R, Q154R, and I76Y; Y147R, Q154R and T166R; or Y123H, Y147R, Q154R, and I76Y; Wherein, the guide RNA targets the fusion protein to achieve deamination of the nucleobases of the promoter of hemoglobin subunit γ1 and / or 2 in the cell.

2. The method according to claim 1, wherein: The modification to SEQ ID NO: 2 as a reference consists of the Y147R change to SEQ ID NO: 2 as a reference.

3. The method according to claim 1, wherein: The modification to SEQ ID NO: 2 is made by one or more of the following combinations of changes to SEQ ID NO: 2: composition: Y147T and Q154R; Y147T and Q154S; Y147R, Q154R, and Y123H; Y147R, Q154R, and I76Y; Y147R, Q154R and T166R; or Y123H, Y147R, Q154R and I76Y.

4. The method according to claim 1, wherein: The modifications based on SEQ ID NO: 2 consist of the following changes based on SEQ ID NO: 2: Y147R, Q154R and Y123H.

5. The method according to claim 1, wherein: One of the one or two adenosine deaminase domains is a wild-type adenosine deaminase domain or a fragment thereof lacking only the N-terminal methionine.

6. The method according to claim 1, wherein: Deamination of the nucleobase generates a 10-nt poly-G sequence in the hemoglobin subunit gamma 1 and / or 2 promoter.

7. The method according to claim 1, wherein: Deamination of the nucleobase disrupts the binding of the repressor to the promoter of hemoglobin subunit gamma 1 and / or 2.

8. The method according to claim 1, wherein: Deamination of the nucleobase affects an increase in gamma globin expression.

9. The method according to claim 1, wherein: The Cas9 domain is a death Cas9 or a nickase Cas9.

10. The method according to claim 9, wherein: The Cas9 domain is capable of programmable DNA binding and is selected from the group consisting of Streptococcus pyogenes Cas9, Staphylococcus aureus Cas9 and Streptococcus thermophilus Cas9.

11. The method according to claim 1, wherein: The Cas9 domain consists of the amino acid sequence of SEQ ID NO:

3.

12. An in vitro or in vitro method for producing red blood cells or progenitor cells thereof, comprising: (a) Introduction of erythroid progenitor cells: (i) a fusion protein or a polynucleotide encoding the fusion protein, wherein the fusion protein consists of the fusion protein according to claim 1, and (ii) one or more guide polynucleotides, wherein the one or more guide polynucleotides target the fusion protein to achieve a change in the nucleobases of the hemoglobin subunit γ1 and / or 2 promoter region from A*T to G*C; and (b) differentiating the erythroid progenitor cells into erythroid cells.

13. An in vitro or in vitro method for editing the promoter of hemoglobin subunit γ1 and / or 2 in a cell, the method comprising contacting the cell with a guide RNA and a fusion protein consisting of a Cas9 domain, one or two adenosine deaminase domains, one or more linkers joining different components of the fusion protein, and a nuclear localization sequence, wherein: One of the one or two adenosine deaminase domains is an adenosine deaminase variant domain consisting of the amino acid sequence of SEQ ID NO:2 or a fragment thereof lacking only the N-terminal methionine, having modifications relative to SEQ ID NO:2, wherein the modifications consist of changes or combinations of changes selected from the following relative to SEQ ID NO:2: Y147T; Y147R; Y147T and Q154R; Y147T and Q154S; Y147R, Q154R, and Y123H; Y147R, Q154R, and I76Y; Y147R, Q154R and T166R; or Y123H, Y147R, Q154R, and I76Y; wherein the guide RNA targets the fusion protein to achieve deamination of the nucleobases of the hemoglobin subunit γ1 and / or 2 promoter in the cell, and wherein the method comprises introducing a change from A*T to G*C at position -198 of the hemoglobin subunit γ1 and / or 2 promoter.

14. The method according to claim 13, wherein: The modification to SEQ ID NO: 2 as a reference consists of the Y147R change to SEQ ID NO: 2 as a reference.

15. The method according to claim 13, wherein: The modification to SEQ ID NO: 2 is made by one or more of the following combinations of changes to SEQ ID NO: 2: composition: Y147T and Q154R; Y147T and Q154S; Y147R, Q154R, and Y123H; Y147R, Q154R, and I76Y; Y147R, Q154R and T166R; or Y123H, Y147R, Q154R and I76Y.

16. The method according to claim 13, wherein: The modifications based on SEQ ID NO: 2 consist of the following changes based on SEQ ID NO: 2: Y147R, Q154R and Y123H.

17. The method according to claim 13, wherein: One of the one or two adenosine deaminase domains is a wild-type adenosine deaminase domain or a fragment thereof lacking only the N-terminal methionine.

18. The method according to claim 13, wherein: Deamination of the nucleobase generates a 10-nt poly-G sequence in the hemoglobin subunit gamma 1 and / or 2 promoter.

19. The method according to claim 13, wherein: Deamination of the nucleobase disrupts the binding of the repressor to the promoter of hemoglobin subunit gamma 1 and / or 2.

20. The method according to claim 13, wherein: Deamination of the nucleobase results in increased expression of gamma globin.

21. The method according to claim 13, wherein: The Cas9 domain is a death Cas9 or a nickase Cas9.

22. The method according to claim 21, wherein: The Cas9 domain is capable of programmable DNA binding and is selected from the group consisting of Streptococcus pyogenes Cas9, Staphylococcus aureus Cas9 and Streptococcus thermophilus Cas9.

23. The method according to claim 13, wherein: The Cas9 domain consists of the amino acid sequence of SEQ ID NO:

3.

24. An in vitro or in vitro method for producing red blood cells or progenitor cells thereof, comprising: (a) Introduction of erythroid progenitor cells: (i) a fusion protein or a polynucleotide encoding the fusion protein, wherein the fusion protein consists of the fusion protein according to claim 13, and (ii) one or more guide polynucleotides, wherein the one or more guide polynucleotides target the fusion protein to achieve a change in the nucleobases of the promoter region of hemoglobin subunit gamma 1 and / or 2 from A*T to G*C; and (b) differentiating the erythroid progenitor cells into erythroid cells.

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