Compositions and methods for treating hemoglobinopathies

Abstract

The invention features compositions and methods for editing deleterious mutations associated with heme pathologies such as sickle cell disease (SCD). In a specific embodiment, the 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] Cross-references to related applications

[0002] This application is an International PCT application, which claims priority to and the benefits of U.S. Provisional Application Nos. 62 / 805,271 filed on February 13, 2019; 62 / 805,277 filed on February 13, 2019; 62 / 852,224 filed on May 23, 2019; 62 / 852,228 filed on May 23, 2019; 62 / 931,722 filed on November 6, 2019; 62 / 931,747 filed on November 6, 2019; 62 / 941,569 filed on November 27, 2019; and 62 / 966,526 filed on January 27, 2020, all of which are incorporated herein by reference in their entirety.

[0003] References cited

[0004] All publications, patents, and patent applications mentioned in this specification are incorporated by reference to the same extent as if each publication, patent, or patent application had been 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

[0005] The present disclosure provides compositions and methods for editing deleterious mutations associated with sickle cell disease (SCD). Specifically, the present disclosure provides a modified adenosine deaminase base editor with improved base editing to correct SCD mutations compared to a reference adenosine deaminase base editor. Background Art

[0006] Sickle cell disease (SCD) is a disorder that affects heme, the molecule in red blood cells that carries oxygen to cells throughout the body. People with this disorder have atypical heme molecules that deform red blood cells into a sickle or crescent shape. The clinical symptoms of sickle cell disease (SCD) are due to intermittent episodes of microvascular occlusion, resulting in 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. The bones / bone marrow, spleen, liver, brain, lungs, kidneys, and joints are often affected.

[0007] SCD is a genetic disorder characterized by the presence of at least one heme S allele (HbS; p.Glu6Val in HBB) and a second HBB pathogenic variant resulting in abnormal heme polymerization. HbS / S (homozygous p.Glu6Val in HBB) accounts for 60% to 70% of SCD 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. A method to edit the genetic mutations that cause SCD and other heme disorders is highly desirable. Summary of the invention

[0008] As described below, the invention features compositions and methods for editing deleterious mutations associated with sickle cell disease (SCD). In certain embodiments, the invention provides for the use of a modified adenosine deaminase base editor called "ABE8" with unprecedented potency (e.g., >60 to 70%) to correct SCD mutations.

[0009] In one embodiment, the invention features a method of 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.

[0010] 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:

[0011]

[0012] The bold sequences indicate sequences derived from Cas9, the italic sequences represent linker sequences, and the underlined sequences represent bipartite nuclear localization sequences.

[0013] The adenosine deaminase variant comprises a modification at amino acid position 82 and / or 166 in MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD.

[0014] In another embodiment, the present 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 CUUCUCCACAGGAGUCAGAU; ACUUCUCCACAGGAGUCAGAU; and GACUUCUCCACAGGAGUCAGAU. In one embodiment, the gRNA further comprises the nucleic acid sequence GUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACGAAACUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG. In another embodiment, the gRNA comprises a nucleic acid sequence selected from

[0015] CUUCUCCACAGGAGUCAGAUGUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACGAAACUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG;

[0016] ACUUCUCCACAGGAGUCAGAUGUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACGAAACUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG; and

[0017] The nucleic acid sequence of GACUUCUCCACAGGAGUCAGAUGUUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACGAAACUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG.

[0018] In another embodiment, the present invention features a cell, which 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 described in any embodiment described herein; 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. 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.

[0019] 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 of the previous embodiments or any other embodiments of the invention described in detail herein. In one embodiment, the cell is an autologous cell of the individual. In another embodiment, the cell is an allogeneic cell of the individual.

[0020] In another embodiment, the present invention provides an isolated cell or a cell population propagated or expanded from any of the foregoing embodiments or any other embodiments of the present invention described in detail herein.

[0021] In another embodiment, the present invention provides a method for manufacturing erythrocytes or progenitors thereof, which involves introducing a base editor or a polynucleotide encoding a base editor into an erythrocyte progenitor cell containing 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 cell into an erythrocyte. In one embodiment, the method involves differentiating an erythrocyte progenitor cell into one or more hematopoietic stem cells, hematopoietic progenitor cells, proerythrocytes, erythroblasts, reticulocytes, or erythrocytes. In one embodiment, the erythrocyte progenitor cell 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 cells or human cells. In another embodiment, the sickle cell disease-associated SNP is modified from A·T to G·C, causing valine in the HBB polypeptide to be changed to alanine. In another embodiment, the sickle cell disease-associated SNP causes 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, the cell is 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 a variant thereof.

[0022] In various embodiments of any or any other embodiments 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 embodiments of the invention described herein, the adenosine deaminase variant comprises a V82S modification. In various embodiments of any or any other embodiments of the invention described herein, the adenosine deaminase variant comprises a T166R modification. In various embodiments of any or any other embodiments of the invention described herein, the adenosine deaminase variant comprises V82S and T166R modifications. In various embodiments of any or any other embodiments 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 embodiments 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 3H+Y147R;V82S+Y123H+Q154R;Y147R+Q154R+Y123H;Y147R+Q154R+I76Y;Y147R+Q154R+T166R;Y123H+Y147R+Q154R+I76Y;V82S+Y123H+Y147R+Q154R;or I76Y+V82S+Y123H+Y147R+Q154R. In 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 embodiments, the adenosine deaminase variant comprises a C-terminal deletion starting from a residue selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 157.

[0023] In various embodiments of any or any other embodiments of the invention described herein, the cell is in vivo or ex vivo. In various embodiments of any or any other embodiments of the invention described herein, the A·T to G·C modification of the sickle cell disease-associated SNP changes valine to alanine in the HBB polypeptide. In various embodiments of any or any other embodiments 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 embodiments of the invention described herein, the sickle cell disease-associated SNP replaces glutamic acid with valine. In various embodiments of any or any other embodiments 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 embodiments of the invention described herein, the A·T to G·C modification of the sickle cell disease-associated SNP replaces glutamic acid with alanine.

[0024] In various embodiments of any or any other embodiment of the present 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 present 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 present 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 present 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 present 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 present invention described herein, the nickase variant comprises an amino acid substitution D10A or its corresponding amino acid substitution. In various embodiments of any or any other embodiment of the present invention described herein, the base editor further comprises a zinc finger domain. In various embodiments of any or any other embodiment of the present invention described herein, the zinc finger domain comprises the recognition helix sequence RNEHLEV, QSTTLKR, and RTEHLAR or the recognition helix sequence RGEHLRQ, QSGTLKR, and RNDKLVP. In various embodiments of any or any other embodiment of the invention described herein, the zinc finger domain is one or more zf1ra or zf1rb. In various embodiments of any or any other embodiment 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 CRISPR RNA (crRNA) and a trans-encoded small RNA (tracrRNA), wherein the crRNA comprises a nucleic acid sequence complementary to a 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 nucleic acid sequence comprised by the sgRNA is complementary to a 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 valine in the HBB polypeptide to alanine. 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, 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 replaces glutamic acid with alanine. In another embodiment, the cell is 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 a variant thereof.

[0025] In one embodiment, a method for 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 a SCD-associated single nucleotide polymorphism (SNP), thereby treating SCD in the individual.

[0026] In another embodiment, a method for 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 a SCD-associated SNP, thereby treating SCD in the individual.

[0027] 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:

[0028] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, 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.

[0029] In one embodiment of the method as 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 from 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.

[0030] In one embodiment of the method as described above, the SCD-associated SNP is located in the beta globin (HBB) gene. In one embodiment of the method as described above, the SNP causes the expression of an HBB polypeptide having a valine at amino acid position 6. In one embodiment of the method as described above, the SNP replaces glutamic acid with valine. In one embodiment of the method as 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 as described above, the A·T to G·C modification of the SNP causes the expression of an HBB polypeptide having an alanine at amino acid position 6. In one embodiment of the method as described above, the A·T to G·C modification of the SNP replaces glutamic acid with alanine.

[0031] In one embodiment of the method described above, the adenosine deaminase variant is a flexible loop, an alpha helical region, an unstructured portion, or a solvent accessible portion inserted into a Cas9 or Cas12 polypeptide. In one embodiment of the method described above, the adenosine deaminase variant is an N-terminal fragment and a C-terminal fragment flanking a Cas9 or Cas12 polypeptide. In one embodiment of the method described above, the fusion protein or ABE8 comprises a structural formula NH 2 -[N-terminal fragment of Cas9 or Cas12 polypeptide]-[adenosine deaminase variant]-[C-terminal fragment of Cas9 or Cas12 polypeptide]-COOH, wherein "]-[" in each case is an optional linker. In one embodiment, the C-terminus of the N-terminal fragment or the N-terminus of the C-terminal fragment comprises a portion of a flexible loop of the Cas9 or Cas12 polypeptide. In one embodiment, when the adenosine deaminase variant deaminates the target nucleobase, the flexible loop comprises an amino acid adjacent to the target nucleobase.

[0032] 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 SCD-associated SNP target nucleobase. In one embodiment, the deamination of the SNP target nucleobase results in replacement of the target nucleobase with a non-wild-type nucleobase, and wherein the deamination of the target nucleobase alleviates a symptom of sickle cell disease. In one embodiment, the deamination of the sickle cell disease-associated SNP results in replacement of glutamic acid with alanine.

[0033] In one embodiment of the method as 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 as described above, the N-terminal fragment or the C-terminal fragment of the Cas9 or Cas12 polypeptide is a 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 into the partial or complete deletion position 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 is a bridge between the RuvC domain and the C-terminal domain.

[0034] In one embodiment of the method as 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):

[0035]

[0036]

[0037] (Single underline: HNH domain; Double underline: RuvC domain; (Cas9 reference sequence), or its corresponding region. In certain embodiments, the Cas9 polypeptide comprises a missing amino acid 1017-1069 (which is numbered according to the Cas9 polypeptide reference sequence), or its corresponding amino acid; the Cas9 polypeptide comprises a missing amino acid 792-872 (which is numbered according to the Cas9 polypeptide reference sequence), or its corresponding amino acid; or the Cas9 polypeptide comprises a missing amino acid 792-906 (which is numbered according to the Cas9 polypeptide reference sequence) , or their corresponding amino acids. In one embodiment of the method as described above, the adenosine deaminase variant is inserted into the 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 (which are numbered according to the Cas9 reference sequence), or their corresponding amino acid positions.

[0038] In one embodiment of the method as 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 is numbered according to the Cas9 reference sequence), or the corresponding amino acid positions thereof. In one embodiment of the method as described above, the deaminase variant is inserted between the following amino acid positions: 768-769, 792-793, 1022-1023, 1026-1027, 1040-1041, 1068-1069, or 1247-1248 (which is numbered according to the Cas9 reference sequence), or its corresponding amino acid positions. In one embodiment of the method as described above, the deaminase variant is inserted between the following amino acid positions: 1016-1017, 1023-1024, 1029-1030, 1040-1041, 1069-1070, or 1247-1248 (which is numbered according to the Cas9 reference sequence), or its corresponding amino acid positions. In one embodiment of the method as described above, the adenosine deaminase variant is inserted into the locus within the 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.

[0039] 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, a 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'.

[0040] 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.

[0041] 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.

[0042] In another embodiment, a pharmaceutical composition is provided, comprising a base editing system containing a fusion protein of 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, which comprises 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 of the group consisting of the nucleic acid sequence GUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACGAAACUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG. In one embodiment, the gRNA comprises a member selected from the group consisting of CUUCUCCACAGGAGUCAGAUGUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACGAAACUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG;

[0043] ACUUCUCCACAGGAGUCAGAUGUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACGAAACUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG; and

[0044] The nucleic acid sequence of GACUUCUCCACAGGAGUCAGAUGUUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACGAAACUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG.

[0045] In one embodiment, a pharmaceutical composition is provided, which comprises 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, embodiments and implementations 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.

[0046] 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.

[0047] In another embodiment, a kit is provided, comprising a base editing system comprising a fusion protein of any of the methods, embodiments and implementation schemes described above. In one embodiment, the kit further comprises a guide RNA selected from CUUCUCCACAGGAGUCAGAU; ACUUCUCCACAGGAGUCAGAU; and

[0048] The nucleic acid sequence of the group consisting of GACUUCUCCACAGGAGUCAGAU.

[0049] In another embodiment, a kit is provided, which comprises 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, embodiments and implementation schemes 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.

[0050] In another embodiment, a kit is provided, comprising any one of the cells of the above embodiments and implementations. In one embodiment of the kit, the kit further comprises a package insert with instructions for use.

[0051] In one embodiment, a base editor system is provided herein, comprising a polynucleotide programmable DNA binding domain and at least one base editor domain comprising an adenosine deaminase variant, the variant comprising a modification at amino acid position 82 or 166 of the following sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD; and a guide RNA, wherein the guide RNA targets the base editor to perform a modification of 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 the 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.

[0052] In one embodiment, a base editor system is provided herein, comprising one or more guide RNAs and a fusion protein, the latter comprising a polynucleotide programmable DNA binding domain comprising the following sequence:

[0053]

[0054] In one embodiment, a cell is provided, comprising any one of the base editor systems described above. In some embodiments, the cell is a human cell or a mammalian cell. In some embodiments, the cell is in vitro, in vivo, or in vitro.

[0055] 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 may vary. It is generally understood by those skilled in the art that there are many variations and modifications of the present invention, all of which are encompassed within the scope thereof.

[0056] 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 related to the examples provided below. Other features and advantages of the present invention will be apparent from the detailed description and claims. Unless otherwise indicated, the operation of some embodiments disclosed herein is carried out using conventional immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA techniques, which are within the scope of the art. See, e.g., 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)).

[0057] The section headings provided herein are for organizational purposes only and are not intended to be limitations of the subject matter described.

[0058] Although various features of the present invention may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present invention is described in separate embodiments herein for purposes of illustration, the present 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.

[0059] 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 embodiments of the present invention illustrated in the following detailed description and by referring to the accompanying drawings.

[0060] definition

[0061] The following definitions are provided to supplement those in the related art and are relevant to the present application, and are not intended to bring into 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 will still be described herein. Therefore, the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the invention belongs. The following references provide general definitions of many of the terms used in connection with the present invention 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).

[0063] In this application, the singular includes the plural, unless otherwise expressly stated. It should be noted that the singular forms "a", "an", and "the" used in this specification include the plural counterparts, unless otherwise expressly stated. In this application, the use of "or" means "and / or", unless otherwise stated, and is generally understood to be inclusive. In addition, the use of the term "including" and other forms such as "include", "includes", and "included" is not limited.

[0064] 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 being implemented using any method or composition of the invention, and vice versa. In addition, the compositions of the invention can be used to achieve the methods of the invention.

[0065] The term "about" or "approximately" means that a particular value is within an acceptable error range as determined by one skilled in the art, partly depending on the manner in which the value is measured or determined, i.e., limited by the measurement system. For example, depending on the operation of the relevant art, "about" may mean within 1 or more than 1 standard deviation. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of the specified value. Alternatively, particularly for biological systems or processes, the term may mean within a certain order of magnitude, such as within 5 times or within 2 times of 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.

[0066] It is generally understood that ranges provided herein are shorthand for all values ​​within the range. For example, a range of 1 to 50 is generally understood to include any number, combination of numbers, or small range in the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0067] References throughout 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 present invention, but not necessarily in all embodiments of the present invention.

[0068] "Adenosine deaminase" means 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 deoxyinosine from deoxyadenosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine of deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) may be from any organism, such as bacteria. In some embodiments, the adenosine deaminase comprises a modification in the following sequence:

[0069] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0070] (also known as TadA*7.10).

[0071] 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 above-mentioned sequences 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.

[0072] In other embodiments, the present invention provides adenosine deaminase variants comprising deletions, such as TadA*8, comprising a C-terminal deletion starting 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 is selected from the group consisting of Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+ 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.

[0073] 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 other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each comprising a domain selected from 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.

[0074] 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 domain selected from 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.

[0075] 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.

[0076] 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:

[0077] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0078] 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.

[0079] In certain embodiments, the adenosine deaminase heterodimer comprises a TadA*8 domain and one of the following adenosine deaminase domains:

[0080] Staphylococcus aureus (S. aureus) TadA:

[0081] MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN

[0082] Bacillus subtilis (B.subtilis) TadA:

[0083] MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE

[0084] Salmonella typhimurium (S. typhimurium) TadA:

[0085] MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV

[0086] Shewanella putrefaciens (S. putrefaciens) TadA:

[0087] MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE

[0088] Haemophilus influenzae F3031 (H. influenzae) TadA:

[0089] MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTΑΗAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK

[0090] Caulobacter crescentus (C. crescentus) TadA:

[0091] MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI

[0092] Geobacter sulfurreducens (G. sulfurreducens) TadA:

[0093] MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP

[0094] TadA*7.10

[0095] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0096] 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:

[0097] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD. In some embodiments, ABE8 comprises other modifications relative to the reference sequence.

[0098] "Adenosine deaminase base editor 8 (ABE8) polynucleotide" means a polynucleotide (polynucleotide sequence) encoding an ABE8 polypeptide.

[0099] "Administering" in this context means providing one or more compositions described herein to a patient or individual. For example and without limitation, for example, administration of an injectable composition may be by intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im). One or more of these routes may be used. Parenteral administration may be, for example, by rapid bolus injection or prolonged drip infusion. Alternatively or concurrently, administration may be by oral route.

[0100] "Agent" means any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or a fragment thereof.

[0101] "Alteration" means to change (e.g., increase or decrease) the structure, expression level or activity of a gene or polypeptide, which is detected by methods known to the standard art, such as those described herein. The modification used herein includes changing the polynucleotide or polypeptide sequence or changing the expression level, such as: changing 25%, changing 40%, changing 50%, or more.

[0102] "Alteration" means to reduce, suppress, attenuate, eliminate, arrest, or stabilize the development or progression of a disease.

[0103] "Analog" means a molecule that is not identical but has similar functional or structural features. For example, a polynucleotide or polypeptide analog retains the biological activity of the corresponding natural polynucleotide or polypeptide, but has certain modifications that enhance the analog's function relative to the naturally occurring polynucleotide or polypeptide. Such 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.

[0104] "Base editor (BE)" or "nucleobase editor (NBE)" means an agent that binds to a polynucleotide and has nucleobase modification activity. In various embodiments, the base editor comprises a nucleobase modification polypeptide (e.g., a deaminase) and a nucleic acid programmable nucleotide binding domain, 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 a base (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 connected 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 connected to a guide RNA (e.g., via an RNA binding motif on a guide RNA and an RNA binding domain fused to a deaminase). In some embodiments, a domain with base editing activity can deaminize a base in a nucleic acid molecule. In some embodiments, the base editor can deaminize one or more bases in a DNA molecule. In some embodiments, the base editor can deaminize adenosine (A) in DNA. In some embodiments, the base editor is an adenosine base editor (ABE).

[0105] In some embodiments, the production of base editors (e.g., ABE8) is to select adenosine deaminase variants (e.g., TadA*8) into a backbone including a circular arrangement of Cas9 (e.g., spCAS9 or saCAS9) and a bipartite nuclear localization sequence. Circular arrangement of Cas9 is known in the art and is described in, for example, Oakes et al., Cell 176, 254–267, 2019. In the following circular arrangement examples, the bold sequence indicates a sequence derived from Cas9, the italic sequence represents a linker sequence, and the bottom line sequence represents a bipartite nuclear localization sequence.

[0106] CP5 (with MSP "NGC = Pam variant, with mutation pattern Cas9 such as: NGG" PID = protein interaction domain and "D10A" nickase):

[0107]

[0108]

[0109] In some embodiments, ABE8 is selected from: a base editor 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 monomer construct. In some embodiments, ABE8 is a heterodimer construct. In some embodiments, ABE8 comprises the sequence:

[0110] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0111] 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.

[0112] 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 higher identity to the ABE nucleic acid sequence are also included.

[0113] ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT

[0114] GACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG

[0115] TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCATTG

[0116] ACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCC

[0117] ATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGT

[0118] CAGATCCGCTAGAGATCCGCGGCCGCTAATACGACTCACTATAGGGAGAGCCGCCACCATGAAACGGACA

[0119] GCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTCTCTGAAGTCGAGTTTAGCCACGAGT

[0120] ATTGGATGAGGCACGCACTGACCCTGGCAAAGCGAGCATGGGATGAAAGAGAAGTCCCCGTGGGCGCCGT

[0121] GCTGGTGCACAACAATAGAGTGATCGGAGAGGGATGGAACAGGCCAATCGGCCGCCACGACCCTACCGCA

[0122] CACGCAGAGATCATGGCACTGAGGCAGGGAGGCCTGGTCATGCAGAATTACCGCCTGATCGATGCCACCC

[0123] TGTATGTGACACTGGAGCCATGCGTGATGTGCGCAGGAGCAATGATCCACAGCAGGATCGGAAGAGTGGT

[0124] GTTCGGAGCACGGGACGCCAAGACCGGCGCAGCAGGCTCCCTGATGGATGTGCTGCACCACCCCGGCATG

[0125] AACCACCGGGTGGAGATCACAGAGGGAATCCTGGCAGACGAGTGCGCCGCCCTGCTGAGCGATTTCTTTA

[0126] GAATGCGGAGACAGGAGATCAAGGCCCAGAAGAAGGCACAGAGCTCCACCGACTCTGGAGGATCTAGCGG

[0127] AGGATCCTCTGGAAGCGAGACACCAGGCACAAGCGAGTCCGCCACACCAGAGAGCTCCGGCGGCTCCTCC

[0128] GGAGGATCCTCTGAGGTGGAGTTTTCCCACGAGTACTGGATGAGACATGCCCTGACCCTGGCCAAGAGGG

[0129] CACGCGATGAGAGGGAGGTGCCTGTGGGAGCCGTGCTGGTGCTGAACAATAGAGTGATCGGCGAGGGCTG

[0130] GAACAGAGCCATCGGCCTGCACGACCCAACAGCCCATGCCGAAATTATGGCCCTGAGACAGGGCGGCCTG

[0131] GTCATGCAGAACTACAGACTGATTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTGATGTGCGCCG

[0132] GCGCCATGATCCACTCTAGGATCGGCCGCGTGGTGTTTGGCGTGAGGAACGCAAAAACCGGCGCCGCAGG

[0133] CTCCCTGATGGACGTGCTGCACTACCCCGGCATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCA

[0134] GATGAATGTGCCGCCCTGCTGTGCTATTTCTTTCGGATGCCTAGACAGGTGTTCAATGCTCAGAAGAAGG

[0135] CCCAGAGCTCCACCGACTCCGGAGGATCTAGCGGAGGCTCCTCTGGCTCTGAGACACCTGGCACAAGCGA

[0136] GAGCGCAACACCTGAAAGCAGCGGGGGCAGCAGCGGGGGGTCAGACAAGAAGTACAGCATCGGCCTGGCC

[0137] ATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGG

[0138] TGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGA

[0139] AACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGC

[0140] TATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGT

[0141] CCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGC

[0142] CTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGAC

[0143] CTGCGGCTGATCTATCTGGCGCTGGCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACC

[0144] TGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGA

[0145] GGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGA

[0146] CGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGGAGAAAGAATGGCCTGTTCGGAAACCTGATTGCCC

[0147] TGAGCCTGGGCCTGACCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAG

[0148] CAAGGACACCTACGACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGGCCGACCTGTTTT

[0149] CTGGCCGCCAAGAACCTGCTCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCA

[0150] AGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGC

[0151] TCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCC

[0152] GGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGG

[0153] ACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAA

[0154] CGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTAC

[0155] CCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCC

[0156] CTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAA

[0157] CTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAG

[0158] AACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGC

[0159] TGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGC

[0160] CATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAG

[0161] AAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACAT

[0162] ACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGA

[0163] AGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCC

[0164] CACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCC

[0165] GGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGG

[0166] CTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAA

[0167] GCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTA

[0168] AGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA

[0169] GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGA

[0170] ATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACA

[0171] CCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGA

[0172] ACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGAC

[0173] TCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAG

[0174] AGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTT

[0175] CGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAG

[0176] CTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACG

[0177] ACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCG

[0178] GAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAAC

[0179] GCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACA

[0180] AGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTT

[0181] CTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGG

[0182] CCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGC

[0183] GGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAA

[0184] AGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAG

[0185] TACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGT

[0186] CCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAA

[0187] TCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAG

[0188] TACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAA

[0189] ACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGG

[0190] CTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATC

[0191] GAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCT

[0192] ACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAA

[0193] TCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAA

[0194] GAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTC

[0195] AGCTGGGAGGTGACTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAG

[0196] GAAAGTCTAACCGGTCATCATCACCATCACCATTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTT

[0197] CTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC

[0198] TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGT

[0199] GGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCT

[0200] CTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGATACCGTCGACCTCTAGCTAGAGCTTGGCGTA

[0201] ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA

[0202] AGCATAAAGTGTAAAGCCTAGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGC

[0203] CCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGG

[0204] TTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGA

[0205] GCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA

[0206] TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCT

[0207] CCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA

[0208] AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGAT

[0209] ACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC

[0210] GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA

[0211] TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTA

[0212] ACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTA

[0213] CACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC

[0214] TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA

[0215] GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACACTCAGTGGAACGAAAACTC

[0216] ACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA

[0217] AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG

[0218] CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTAC

[0219] GATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA

[0220] GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCT

[0221] CCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGT

[0222] TGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCC

[0223] CAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGA

[0224] TCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTAC

[0225] TGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGT

[0226] ATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAA

[0227] AAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG

[0228] TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGA

[0229] GCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATAC

[0230] TCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATG

[0231] TATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGA

[0232] TCGGGAGATCGATCTCCCGATCCCCTAGGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAA

[0233] GCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAAC

[0234] AAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGAT

[0235] GTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCAT

[0236] TAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC

[0237] CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCAT

[0238] TGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC

[0239] "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, the base editing activity is analyzed using editing efficacy. The base editing efficacy can be measured in any suitable manner, for example: using Sanger sequencing or next-generation sequencing. In some embodiments, the base editing efficacy is to measure the percentage of total sequenced reads with nuclear base conversions performed by the base editor, 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, the base editing efficacy is to measure the percentage of total cells with nuclear base conversions performed by the base editor.

[0240] 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.

[0241] In some embodiments, the base editor system may include more than one base editing component. For example, the base editor system may include more than one deaminase. In some embodiments, the 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.

[0242] The deaminase domain and the polynucleotide programmable nucleotide binding component of the base editor system can be associated with each other using covalent or non-covalent, or associative methods and any combination of their interactions. For example: In some embodiments, the deaminase domain can target a target nucleotide sequence using a polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or connected to the deaminase domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the deaminase domain to a target nucleotide sequence by non-covalent interaction with the deaminase domain or association with the deaminase domain. For example: In some embodiments, the deaminase domain can include an additional heterologous portion or domain, which can interact, associate, or form a complex with an additional heterologous portion or domain that is part of the polynucleotide programmable nucleotide binding domain. In some embodiments, the additional heterologous portion can bind, interact, associate, or form a complex with a polypeptide. In some embodiments, the additional heterologous portion can bind, interact, associate, or form a complex with a polynucleotide. In some embodiments, the additional heterologous portion can be combined with a guide polynucleotide. In some embodiments, the additional heterologous portion can be combined with a polypeptide linker. In some embodiments, the additional heterologous portion can be combined with a polynucleotide linker. The additional heterologous portion can be a protein domain. In some embodiments, the additional heterologous portion can be a K Homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a 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.

[0243] The base editor system may further include a guide polynucleotide component. It should be understood that the components of the base editor system may be associated with each other through covalent bonds, non-covalent interactions, or any combination of their associations and interactions. In some embodiments, the deaminase domain can target the target nucleotide sequence through the guide polynucleotide. For example: In some embodiments, the deaminase domain may include an additional heterologous portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein), which can interact, associate, or form a complex with a portion or segment 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 connected to the deaminase domain. In some embodiments, the additional heterologous portion may be able to bind, interact, associate, or form a complex with a polypeptide. In some embodiments, the additional heterologous portion may be able to bind, interact, associate, or form a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be associated with a guide polynucleotide. In some embodiments, the additional heterologous portion may be associated with a polypeptide linker. In some embodiments, the additional heterologous portion may be associated with a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0244] In some embodiments, the base editor system may further include an inhibitor of a base excision repair (BER) component. It should be understood that the components of the base editor system 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 include 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 can target a target nucleotide sequence through a polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or connected to an inhibitor of BER. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or connected to a deaminase domain and an inhibitor of BER. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the inhibitor of BER to a target nucleotide sequence by non-covalent interaction or association with an inhibitor of BER. For example, in some embodiments, the BER inhibitor component may include an additional heterologous moiety or domain that can interact, 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.

[0245] In some embodiments, an inhibitor of BER can be targeted to a target nucleotide sequence via a guide polynucleotide. For example, in some embodiments, an inhibitor of BER can include 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 a guide polynucleotide (e.g., a polynucleotide motif). In some embodiments, an additional heterologous portion 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 an inhibitor of BER. In some embodiments, the additional heterologous portion may be able to bind, interact, associate, or form a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be able to bind to a guide polynucleotide. In some embodiments, the additional heterologous portion may be able to bind to a polypeptide linker. In some embodiments, the additional heterologous portion may be able to bind to a polynucleotide linker. The additional heterologous portion may 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, a 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.

[0246] "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 E7V) mutation. Examples of β-globin amino acid sequences are provided below.

[0247] 1MVHLTPEEKS AVTALWGKVN VDEVGGEALG RLLVVYPWTQ RFFESFGDLS TPDAVMGNPK

[0248] 61VKAHGKKVLG AFSDGLAHLD NLKGTFATLS ELHCDKLHVD PENFRLLGNV LVCVLAHHFG

[0249] 121KEFTPPVQAA YQKVVAGVAN ALAHKYH

[0250] "HBB polynucleotide" means a nucleic acid molecule encoding a β-globin protein or a fragment thereof. The sequence of an example of an HBB polynucleotide can be obtained from NCBI Accession No. NM_000518, provided below:

[0251] 1acatttgctt ctgacacaac tgtgttcact agcaacctca aacagacacc atggtgcatc

[0252] 61tgactcctga ggagaagtct gccgttactg ccctgtgggg caaggtgaac gtggatgaag

[0253] 121ttggtggtga ggccctgggc aggctgctgg tggtctaccc ttggacccag aggttctttg

[0254] 181agtcctttgg ggatctgtcc actcctgatg ctgttatggg caaccctaag gtgaaggctc

[0255] 241atggcaagaa agtgctcggt gcctttagtg atggcctggc tcacctggac aacctcaagg

[0256] 301gcacctttgc cacactgagt gagctgcact gtgacaagct gcacgtggat cctgagaact

[0257] 361tcaggctcct gggcaacgtg ctggtctgtg tgctggccca tcactttggc aaagaattca

[0258] 421ccccaccagt gcaggctgcc tatcagaaag tggtggctgg tgtggctaat gccctggccc

[0259] 481acaagtatca ctaagctcgc tttcttgctg tccaatttct attaaaggtt cctttgttcc

[0260] 541ctaagtccaa ctactaaact gggggatatt atgaagggcc ttgagcatct ggattctgcc

[0261] 601taataaaaaa catttatttt cattgcaa

[0262] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease that includes a Cas9 protein, or a fragment thereof (e.g., a protein that includes 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 includes a spacer, a sequence complementary to the aforementioned mobile element, 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 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 strand that is not complementary to crRNA is first cut by an endonucleolytic manner. Then 3′-5′ is trimmed in an exonucleolytic manner. DNA-binding and cleavage in nature usually require proteins and two RNAs. However, a single guide RNA ("sgRNA" or "gNRA" for short) can be engineered so that both crRNA and tracrRNA embodiments are incorporated 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 between self and non-self.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 WM, Ajdic D.J., 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 hostfactor RNase III." Deltcheva E., Chylinski K., Sharma CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are each incorporated herein by reference). Cas9 orthologous genes have been described in various species, including (but not limited to): Streptococcus pyogenes (S. pyogenes) and Streptococcus thermophilus (S. thermophilus).Other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on the present invention, and include Cas9 sequences of 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.

[0263] One example of Cas9 is Streptococcus pyogenes Cas9 (spCas9), the amino acid sequence of which is provided below:

[0264]

[0265]

[0266] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0267] 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 have been 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, that is, Cas9 is a nickase, referred to as a "nCas9" protein (for "nickase" Cas9).

[0268] 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) a gRNA binding domain of Cas9; or (2) a DNA cleavage domain of Cas9. In some embodiments, a protein comprising Cas9 or a fragment thereof is referred to as a "Cas9 variant". A Cas9 variant has 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% 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 amino acid length.

[0269] 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):

[0270]

[0271]

[0272] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0273] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence:

[0274]

[0275]

[0276]

[0277] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0278] 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):

[0279]

[0280]

[0281]

[0282] (SEQ ID NO: 1. Single underline: HNH domain; double underline: RuvC domain).

[0283] In some embodiments, Cas9 refers to Cas9 from the following: 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.

[0284] 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 NNNNGATT PAM, NNNNCCTA PAM, NNNNCCTC PAM, NNNNCCTT 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, wherein N is any of A, G, C, or T. In some embodiments, NmeCas9 is specific for NNNNCCGT PAM, NNNNCGGPAM, 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 N 4 GAT, N 4 CC, and N 4CAAA. 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 have been incorporated herein by reference in their entirety.

[0285] One 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:

[0286] 1maafkpnpin yilgldigia svgwamveid edenpiclid lgvrvferae vpktgdslam

[0287] 61arrrlarsvrr ltrrrahrll rarrllkreg vlqaadfden glikslpntp wqlraaaldr

[0288] 121kltplewsav llhlikhrgy lsqrkneget adkelgallk gvadnahalq tgdfrtpael

[0289] 181alnkfekesg hirnqrgdys htfsrkdlqa elillfekqk efgnphvsgg lkegietllm

[0290] 241tqrpalsgda vqkmlghctf epaepkaakn tytaerfiwl tklnnlrile qgserpltdt

[0291] 301eratlmdepy rkskltyaqa rkllgledta ffkglrygkd naeastlmem kayhaisral

[0292] 361ekeglkdkks plnlspelqd eigtafslfk tdeditgrlk driqpeilea llkhisfdkf

[0293] 421vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee kiylppipad eirnpvvlra

[0294] 481lsqarkving vvrrygspar ihietarevg ksfkdrkeie krqeenrkdr ekaaakfrey

[0295] 541fpnfvgepks kdilklrlye qqhgkclysg keinlgrlne kgyveidhal pfsrtwddsf

[0296] 601nnkvlvlgse nqnkgnqtpy eyfngkdnsr ewqefkarve tsrfprskkq rillqkfded

[0297] 661gfkernlndt ryvnrflcqf vadrmrltgk gkkrvfasng qitnllrgfw glrkvraend

[0298] 721rhhaldavvv acstvamqqk itrfvrykem nafdgktidk etgevlhqkt hfpqpweffa

[0299] 781qevmirvfgk pdgkpefeea dtpeklrtll aeklssrpea vheyvtplfv srapnrkmsg

[0300] 841qghmetvksa krldegvsvl rvpltqlklk dlekmvnrer epklyealka rleahkddpa

[0301] 901kafaepfyky dkagnrtqqv kavrveqvqk tgvwvrnhng iadnatmvrv dvfekgdkyy

[0302] 961lvpiyswqva kgilpdravv qgkdeedwql iddsfnfkfs lhpndlvevi tkkarmfgyf

[0303] 1021aschrgtgni nirihdldhk igkngilegi gvktalsfqk yqidelgkei rpcrlkkrpp

[0304] 1081vr

[0305] 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:

[0306] 1maafkpnpin yilgldigia svgwamveid eeenpirlid lgvrvferae vpktgdslam

[0307] 61arrrlarsvrr ltrrrahrll rarrllkreg vlqaadfden glikslpntp wqlraaaldr

[0308] 121kltplewsav llhlikhrgy lsqrkneget adkelgallk gvannahalq tgdfrtpael

[0309] 181alnkfekesg hirnqrgdys htfsrkdlqa elillfekqk efgnphvsgg lkegietllm

[0310] 241tqrpalsgda vqkmlghctf epaepkaakn tytaerfiwl tklnnlrile qgserpltdt

[0311] 301eratlmdepy rkskltyaqa rkllgledta ffkglrygkd naeastlmem kayhaisral

[0312] 361ekeglkdkks plnlsselqd eigtafslfk tdeditgrlk drvqpeilea llkhisfdkf

[0313] 421vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee kiylppipad eirnpvvlra <h2 style=";text-align:left;direction:ltr">

[0314] <h2 style=";text-align:left;direction:ltr"> 481lsqarkving vvrrygspar ihietarevg ksfkdrkeie krqeenrkdr ekaaakfrey<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0315] <h2 style=";text-align:left;direction:ltr"> 541fpnfvgepks kdilklrlye qqhgkclysg keinlvrlne kgyveidhal pfsrtwddsf<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0316] <h2 style=";text-align:left;direction:ltr"> 601nnkvlvlgse nqnkgnqtpy ​​eyfngkdnsr ewqefkarve tsrfprskkq rillqkfded<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0317] <h2 style=";text-align:left;direction:ltr"> 661gfkecnlndt ryvnrflcqf vadhilltgk gkrrvfasng qitnllrgfw glrkvraend<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0318] <h2 style=";text-align:left;direction:ltr"> 721rhhaldavvv acstvamqqk itrfvrykem nafdgktidk etgkvlhqkt hfpqpweffa<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0319] <h2 style=";text-align:left;direction:ltr"> 781qevmirvfgk pdgkpefeea dtpeklrtll aeklssrpea vheyvtplfv srapnrkmsg<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0320] <h2 style=";text-align:left;direction:ltr"> 841ahkdtlrsak rfvkhnekis vkrvwlteik ladlenmvny kngreielye alkarleayg<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0321] <h2 style=";text-align:left;direction:ltr"> 901gnakqafdpk dnpfykkggq lvkavrvekt qesgvllnkk naytiadngd mvrvdvfckv<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0322] <h2 style=";text-align:left;direction:ltr"> 961dkkgknqyfi vpiyawqvae nilpdidckg yriddsytfc fslhkydlia fqkdekskve<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0323] <h2 style=";text-align:left;direction:ltr"> 1021fayyincdss ngrfylawhd kgskeqqfri stqnlvliqk yqvnelgkei rpcrlkkrpp<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0324] <h2 style=";text-align:left;direction:ltr"> 1081vr<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0325] 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 the 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):

[0326]

[0327]

[0328] (Single bottom line: HNH domain; double bottom line: RuvC domain).

[0329] 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.

[0330] In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which, for example, result in nuclease-inactive Cas9 (dCas9). Such mutations, for example, include other amino acid substitutions in D10 and H840, or include 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, which 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, by about 100 amino acids or more.

[0331] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of a Cas9 protein, for example, one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but only comprise one or more fragments thereof. Examples of amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and those skilled in the art will appreciate additional suitable sequences of Cas9 domains and fragments.

[0332] 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.

[0333] Catalytically inactive Cas9 (dCas9) example:

[0334]

[0335] Catalytic Cas9 nickase (nCas9) example:

[0336]

[0337] Examples of catalytically active Cas9:

[0338]

[0339] In some embodiments, Cas9 refers to Cas9 from an archaea (e.g., nanoarchaea), which belongs to the unicellular prokaryotic microbial domain and the unicellular prokaryotic microbial kingdom. In some embodiments, Cas9 refers to CasX or CasY, which has been 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 have been incorporated herein by reference. Using genome-resolved metagenomics, many CRISPR-Cas systems have been identified, including the first reported Cas9 in the archaeal domain of life. This diverse Cas9 protein has appeared in the little-studied nanoarchaea, which is part of an active CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, were found, 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-guided DNA binding proteins can be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present invention.

[0340] 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, such as SpCas9 variants, are specific for NRNH PAMs, wherein R is A or G and H is A, C, or T. In some embodiments, SpCas9 variants are specific for 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.

[0341]

[0342]

[0343] (Single bottom line: HNH domain; double bottom line: RuvC domain).

[0344] 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 Fig.49 .

[0345] Table A

[0346]

[0347] Table B

[0348]

[0349]

[0350] Table C

[0351]

[0352] Table D

[0353]

[0354]

[0355] In certain embodiments, napDNAbp suitable for the methods of the present invention include circular arrangements known in the art and described in, for example, Oakes et al., Cell 176, 254–267, 2019. An example of a circular arrangement is as follows, wherein the bold sequence indicates a sequence derived from Cas9, the italic sequence represents a linker sequence, and the bottom line sequence represents a bipartite nuclear localization sequence,

[0356] CP5 (with MSP "NGC = Pam variant, with mutation pattern Cas9 such as: NGG" PID = protein interaction domain and "D10A" nickase):

[0357]

[0358]

[0359] 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).

[0360] 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 natural CasX or CasY protein. In some embodiments, the napDNAbp is a natural CasX or CasY protein. In some embodiments, the amino acid sequence of 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.

[0361] Cas12b / C2c1(uniprot.org / uniprot / T0D7A2#2)

[0362] 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

[0363] MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECDKTAEECKAELLERLRARQVENGHRGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKEKAETRKSADRTADVLRALADFGLKPLMRVYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQEYAKLVEQKNRFEQKNFVGQEHLVHLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSDKVFEKWGKLAPDAPFDLYDAEIKNVQRRNTRRFGSHDLFAKLAEPEYQALWREDASFLTRYAVYNSILRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGERRHAIRFHKLLKVENGVAREVDDVTVPISMSEQLDNLLPRDPNEPIALYFRDYGAEQHFTGEFGGAKIQCRRDQLAHMHRRRGARDVYLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPFFFPIKGNDNLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPVDAANHMTPDWREAFENELQKLKSLHGICSDKEWMDAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYAKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELINQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCTQEHNPEPFPWWLNKFVVEHTLDACPLRADDLIPTGEGEIFVSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRLRCDWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYERERGKKRRKVFAQEKLSEEEAELLVEADEAREKSVVLMRDPSGIINRGNWTRQKEFWSMV NQRIEGYLVKQIRSRVPLQDSACENTGDI

[0364] CasX (uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53)

[0365] >tr|F0NN87|F0NN87_SULIH CRISPR - associated Casx protein OS = Sulfolobus islandicus (strain HVE10 / 4) GN = SiH_0402 PE = 4 SV = 1

[0366] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0367] >tr|F0NH53|F0NH53_SULIR CRISPR - associated protein, Casx OS = Sulfolobus islandicus (strain REY15A) GN = SiRe_0771 PE = 4 SV = 1

[0368] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTSNIILPLSGNDKNPWTETLKCYNFPTTTVALSEVFKNFSQVKECEEVSAPSFVKPFEYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0369] δ- proteobacteria (Deltaproteobacteria) CasX

[0370] MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQK WYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0371] CasY(ncbi.nlm.nih.gov / protein / APG80656.1)

[0372] >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria bacteria]

[0373]

[0374] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid by another amino acid with common properties. A functional way to define common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Based on such analyses, groups of amino acids can be defined, within which the amino acids can be preferentially exchanged with each other, and thus their effects on the overall protein structure are most similar to each other (Schulz, GE and Schirmer, RH, i.e., supra). Non-limiting examples of conservative mutations include amino acid substitutions of amino acids, such as: substitution of arginine with lysine and vice versa, thereby maintaining a positive charge; substitution of aspartic acid with glutamic acid and vice versa, thereby maintaining a negative charge; substitution of threonine with serine, thereby maintaining a free -OH; and substitution of aspartic acid with glutamic acid, thereby maintaining a free -NH 2 .

[0375] The terms "coding sequence" or "protein coding sequence" used interchangeably herein refer to a segment of a polynucleotide that encodes a protein. The region or sequence is bounded by a start codon near the 5' end and a stop codon at the receiving 3' end. A coding sequence may also be referred to as an open reading frame.

[0376] 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, adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) can be from any organism, such as bacteria. In some embodiments, the adenosine deaminase is from a bacterium, such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Caulobacter crescentus.

[0377] 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 native deaminase from an organism such as a 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, the deaminase domain is 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 have been incorporated herein by reference.

[0378] "Detection" refers to determining the presence, absence or amount of an analyte to be detected. In one embodiment, sequence modifications in polynucleotides or polypeptides are detected. In another embodiment, the presence of insertions / deletions (indels) are detected.

[0379] "Detectable label" means that the composition, when linked to a molecule of interest, can be detected by microscopy, photochemistry, biochemistry, immunochemistry, or chemical means. For example, suitable labels include radioactive isotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, high electron density reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin, or haptens.

[0380] "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.

[0381] The term "effective amount" as used herein refers to the amount of a bioactive agent sufficient to induce a desired biological response. The effective amount of the active compound (group) used to treat a disease in the operation of the present invention will vary with the method of administration, the age, weight, and general health of the individual. The appropriate dosage and dosage course will ultimately be determined by the participating physician or veterinarian. Such amounts are referred to as "effective" amounts. In a specific embodiment, the effective amount is the amount of the base editor system of the present invention (e.g., a fusion protein comprising a programmable DNA binding protein, a nuclear base editor, and a gRNA) sufficient to modify the SCD mutation in the cell to achieve a medical effect (e.g., reducing or controlling the individual's SCD or its symptoms or symptoms). Such medical effects do not necessarily require full modification of the SCD of all cells of a tissue or organ, but only about 1%, 5%, 10%, 25%, 50%, 75% or more cells in the tissue or organ cells. In one embodiment, the effective amount is sufficient to alleviate one or more SCD symptoms, including anemia and ischemia.

[0382] By "fragment" is meant 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 may 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.

[0383] "Guide RNA" or "gRNA" means a polynucleotide that can be specific to 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 in a single RNA molecule can be referred to as a single guide RNA (sgRNA), but "gRNA" can be used interchangeably with a guide RNA in a single molecule or in a complex of two or more molecules. Typically, a gRNA in a single RNA species comprises two domains: (1) a domain that has common homology with the target nucleic acid (e.g., and dominates 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 has been incorporated herein by reference. Other gRNA examples (e.g., those including domain 2) can be found in U.S. Provisional Patent Application USSN 61 / 874,682, filed on September 6, 2013, entitled "Switchable Cas9 Nucleases and Uses Thereof"; and U.S. Provisional Patent Application USSN 61 / 874,746, filed on September 6, 2013, entitled "Delivery System For Functional Nucleases", the entire disclosures of which have been incorporated herein by reference. In some embodiments, the 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, which mediates the binding of the nuclease / RNA complex to the target site, providing sequence specificity of the nuclease: RNA complex. Those skilled in the art generally understand that RNA polynucleotide sequences, such as gRNA sequences, include the nucleobase uracil (U), which is a pyrimidine derivative, rather than the nucleobase thymine (T) included in the DNA polynucleotide sequence. In RNA, uracil is base-paired with adenine and replaces thymine during DNA transcription.

[0384] "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 residues are usually replaced by alanyl residues. 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 processes such as isoelectric focusing, heme electrophoresis separation 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).

[0385] "Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0386] 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 inhibitors of base repair 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 a catalytically inactive EndoV or a catalytically inactive hAAG. In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, the base repair inhibitor is a catalytically inactive EndoV or a catalytically inactive hAAG.

[0387] 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 a wild-type UGI or a fragment of wild-type UGI. In some embodiments, the UGI protein provided herein includes a fragment of UGI and a protein homologous to UGI or a fragment of UGI. 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 inactivated inosine-specific nuclease" or an "inactivated inosine-specific nuclease". Without being limited by any particular theory, a catalytically inactivated inosine glycosidase (e.g., alkyl adenine glycosidase (AAG)) can bind to inosine, but will not produce abasic sites or exclude inosine, thereby sterically blocking the newly formed inosine moiety from DNA damage / repair mechanisms. In some embodiments, a catalytically inactivated inosine-specific nuclease can bind to inosine in nucleic acids, but will not cleave nucleic acids. Non-limiting examples of catalytically inactive inosine-specific nucleases include catalytically inactive alkyl adenosyl glycosidases (AAG nucleases), e.g., from humans, and catalytically inactive endonucleases V (EndoV nucleases), e.g., from Escherichia coli (E. coli). In some embodiments, the catalytically inactive AAG nuclease comprises an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0388] By "increase" is meant a positive change of at least 10%, 25%, 50%, 75%, or 100%.

[0389] "Inteins (inteins)" are fragments of proteins that can self-excise and join other fragments (exteins) using peptide bonds in a process called protein splicing. Inteins are also called "protein introns". The process of inteins self-excising and joining other protein parts is referred to herein as "protein splicing" or "intein-mediated protein splicing". In some embodiments, the precursor protein intein (the protein containing the intein before intein-mediated protein splicing) comes 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 (which is 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".

[0390] Other intein systems may also be employed. For example, synthetic inteins based on the dnaE intein: pairs of inteins 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 pairs of inteins 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. Pat. No. 8,394,604, incorporated herein by reference).

[0391] Examples of nucleotide and amino acid sequences of inteins are provided.

[0392] DnaE Intein-N DNA:

[0393] TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTTGCCAATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCGATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGGGGA GAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAGGGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTTCCTAAT

[0394] DnaE Intein-N Protein:

[0395] CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN

[0396] DnaE intein-C DNA:

[0397] ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGATATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAGCTTCTAAT

[0398] Intein-C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN

[0399] Cfa-N DNA:

[0400] TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCCTATTGGAAAGATTGTCGAAGAGAGAATTGAATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGGCACAATCGCGGCGAACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACGAGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAATAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTGCCA

[0401] Cfa-N protein:

[0402] CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP

[0403] Cfa-C DNA:

[0404] ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAGGAAAGTAAAGATAATATCTCGAAAAAGTCTTGGTACCCAAAATGTCTATGATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTAGCCAGCAAC

[0405] Cfa-C protein:

[0406] MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN

[0407] Intein-N and intein-C may be fused to the N-terminal portion of the split Cas9 and the C-terminal portion of the split Cas9, respectively, for joining the N-terminal portion of the split Cas9 and the C-terminal portion of the split Cas9. For example, in some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of the split Cas9, i.e., forming the structural formula N--[N-terminal portion of the split Cas9]-[intein-N]--C. In some embodiments, intein-C is fused to the N-terminus of the C-terminal portion of the split Cas9, i.e., forming the structural formula N-[intein-C]--[C-terminal portion of the 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.

[0408] The terms "isolated", "purified", or "biologically pure" refer to the exclusion of various materials that normally occur naturally with a component to varying degrees. "Isolated" represents a degree of separation from the original source or environment. "Purified" represents a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials so that any impurities cannot substantially affect the biological properties of the protein or cause other adverse consequences. That is, the nucleic acid or peptide of the present invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or if it is free of chemical precursors or other chemicals when chemically synthesized. Purity and homology are generally determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can represent that a nucleic acid or protein produces essentially a single band on an electrophoretic gel. Proteins that can be modified, for example, by phosphorylation or glycosylation, different modifications can produce different isolated proteins that can be separated and purified.

[0409] "Isolated polynucleotide" means that the nucleic acid (e.g., DNA) flanking a gene has been excluded from the natural genome of the organism from which the nucleic acid molecule of the present 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 the like and other sequences that are separate molecules that are independent of each other (e.g., cDNA or genome or cDNA fragments generated by PCR or restriction endonuclease digestion). In addition, the term includes RNA molecules transcribed from DNA molecules, and recombinant DNA that becomes part of a hybrid gene encoding an additional polypeptide sequence.

[0410] "Isolated polypeptide" means a polypeptide of the invention that has been separated from naturally associated components. Generally, a polypeptide is isolated when at least 60% by weight of the proteins and natural organic molecules with which it is naturally associated have been excluded. 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 invention. The isolated polypeptide of the invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide, or by chemically synthesizing the protein. Purity can be determined by any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0411] The term "linker" as used herein 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 a 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, e.g., described in PCT / US19 / 44935). The linker may join different components or different parts of a base editor system. For example, in some embodiments, the linker may join a guide polynucleotide binding domain of a polynucleotide programmable nucleotide binding domain and a catalytic domain of a deaminase. In some embodiments, the linker may join a CRISPR polypeptide and a deaminase. In some embodiments, the linker may join Cas9 and a deaminase. In some embodiments, the linker may join dCas9 and a deaminase. In some embodiments, the linker may join nCas9 and a deaminase. In some embodiments, the linker can be joined to the guide polynucleotide and the deaminase. In some embodiments, the linker can be joined to the deamination component of the base editor system and the polynucleotide programmable nucleotide binding component. In some embodiments, the linker can be joined to the RNA-binding portion of the deamination component of the base editor system and the polynucleotide programmable nucleotide binding component. In some embodiments, the linker can be joined to the RNA-binding portion of the deamination component of the base editor system and the RNA-binding portion of the polynucleotide programmable nucleotide binding component. The linker can be located between two groups, molecules, or other moieties or flanked by them, linked to each other via covalent bonds or non-covalent interactions, thereby linking the two. In some embodiments, the linker can be an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, the linker can include an aptamer that can bind to a ligand. In some embodiments, the ligand can be a carbohydrate, a peptide, a protein, or a nucleic acid. In some embodiments, the linker may comprise an aptamer derived from a riboswitch. The riboswitch from which the aptamer is derived may be selected from a theophylline riboswitch, a 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 Q nucleoside precursor (pre-queosine 1) (PreQ1) riboswitch. In some embodiments, the linker may comprise an aptamer that binds to a polypeptide or protein domain (e.g., a polypeptide ligand).In some embodiments, the polypeptide ligand may be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and a Ku protein, a telomerase Sm7 binding motif and a Sm7 protein, or an RNA recognition motif. In some embodiments, the polypeptide ligand may be part of a base editor system component. For example, a nucleobase editing component may include a deaminase domain and an RNA recognition motif.

[0412] 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.

[0413] In some embodiments, the 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, the linker joins dCas9 and a nucleic acid editing protein. For example, a linker is located between or flanked by two groups, molecules, or other moieties and is covalently linked to each other, thereby linking the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the 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.

[0414] In some embodiments, the domain of the nucleobase editor is fused using a linker comprising the following amino acid sequence: SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS.

[0415] 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 A 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.

[0416] 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

[0417] SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS. In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0418] "Marker" means any protein or polynucleotide having a modification in expression level or activity that is associated with a disease or disorder.

[0419] The term "mutation" as used herein refers to a residue in a sequence of, for example, a nucleic acid or amino acid sequence being replaced by another residue, or being deleted or inserting one or more residues in the sequence. When describing a mutation herein, the original residue is usually indicated first, followed by the position of the residue in the sequence, and then the newly replaced residue is indicated. The various different methods for making amino acid substitutions (mutations) provided herein are known in the art and are provided in, for example, Molecular Cloning: A Laboratory Manual (4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012) by Green and Sambrook. In some embodiments, the base editor of the present invention can effectively produce "intended mutations (intended mutations)" in nucleic acids (e.g., nucleic acids in individual genes), such as point mutations, 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) combined with a guide polynucleotide (e.g., gRNA), which is explicitly designed to produce the expected mutation.

[0420] 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.

[0421] The term "non-conservative mutation" refers to amino acid substitutions between different groups, such as: substitution of tryptophan by lysine, or substitution of serine by phenylalanine, etc. In this case, 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, so that the biological activity of the functional variant is improved compared to the wild-type protein.

[0422] The term "nuclear localization sequence", "nuclear localization signal", or "NLS" refers to an amino acid sequence that facilitates protein import into the nucleus. Nuclear localization sequences are known in the art and are described, for example, in Plank et al., International PCT Application PCT / EP2000 / 011690, filed on November 23, 2000, published as WO / 2001 / 038547 on May 31, 2001, which disclosures of examples of nuclear localization sequences are incorporated herein by reference in their entirety. In other embodiments, the NLS is, for example, an optimized NLS described by Koblan et al. in Nature Biotech. 2018, doi: 10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0423] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to compounds 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 3 or more nucleotides) is a linear molecule in which adjacent nucleotides are linked to each other using 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. Nucleic acids may be naturally occurring in the context of, for example, a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecules. On the other hand, nucleic acid molecules can be non-naturally occurring molecules, such as recombinant DNA or RNA, artificial chromosomes, engineered genomes, or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids, or include non-naturally occurring nucleotides or nucleosides. In addition, the terms "nucleic acid", "DNA", "RNA", and / or similar terms include nucleic acid analogs, such as analogs with non-phosphodiester backbones. Nucleic acids can be purified from natural sources, manufactured using recombinant expression systems, and can be purified, chemically synthesized, etc. as needed. If appropriate, for example, taking chemically synthesized molecules as an example, nucleic acids can include nucleoside analogs, such as analogs with chemically modified bases or sugars, and backbone modifications. Nucleic acid sequences are in the 5' to 3' direction unless otherwise specified. 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, 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-imidophosphoryl linkages).

[0424] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" is used interchangeably with "polynucleotide programmable nucleotide binding domain" and refers to a protein associated with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA) that guides 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, 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 may not be explicitly listed in the present invention, but 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 which have been incorporated herein by reference.

[0425] The terms "nucleobase", "nitrogenous base" or "base" used interchangeably herein refer to nitrogen-containing 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 forms long helical structures, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Five nucleobases - adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) - are called primitive or classical. 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 the amino group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can come from the deamination of cytosine. "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). "Nucleotide" is composed of a nucleobase, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group.

[0426] 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: deamination of cytosine (or cytidine) to form uracil (or uridine) or thymine (or thymidine), and deamination of adenine (or adenosine) to form hypoxanthine (or inosine), and addition and insertion of non-templated nucleotides. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., adenine deaminase or adenosine deaminase; or cytidine deaminase or cytosine deaminase). In some embodiments, the nucleobase editing domain is more than one deaminase domain (e.g., adenine deaminase, or adenosine deaminase, and cytidine or cytosine deaminase, for example, 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 is from any organism, such as bacteria, humans, chimpanzees, gorillas, monkeys, cows, dogs, rats, or mice.

[0427] As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, generating, preparing, or otherwise obtaining the agent.

[0428] 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 with a higher than average probability of developing a disease or disorder. Patient examples may 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 may benefit from the therapies disclosed herein. Human patient examples may be male and / or female.

[0429] A "patient in need thereof" or "individual in need thereof" refers herein to a patient diagnosed with, at risk for or suffering from, previously diagnosed with, or suspected of suffering from a disease or disorder.

[0430] The term "pathogenic mutation", "pathogenic variant", "disease-causing mutation", "disease-causing variant", "deleterious mutation", or "predisposing mutation" refers to a genetic modification or mutation that increases 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.

[0431] The terms "protein", "peptide", "polypeptide" and their grammatical equivalents are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. Such 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, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multimolecular complex. A protein, peptide, or polypeptide may be a fragment of a naturally occurring protein or peptide only. 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 manufactured by any method known in the art. For example, the protein provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising a peptide linker. Methods of recombinant protein expression and purification are well 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.

[0432] The polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) may be modified to include synthetic amino acids replacing 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 pyrrolidonecarboxylic acid, disulfide bridge formation, sulfation, myristoylation, palmitoylation, prenylation, farnesylation, geranylation, glycosylphosphatidylinositolization, lipoylation, and iodination.

[0433] The term "recombinant" as used herein in the context of proteins or nucleic acids refers to proteins or nucleic acids that do not occur naturally, but are the product of human engineering. For example: In some embodiments, the amino acid or nucleotide sequence comprised by the recombinant protein or nucleic acid molecule 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.

[0434] By "reduced" is meant a negative modification of at least 10%, 25%, 50%, 75%, or 100%.

[0435] "Reference" means a reference 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, a vehicle, a buffer, and / or a control vector without the polynucleotide of interest.

[0436] A "reference sequence" is a designated sequence used as a basis for sequence alignment. A reference sequence may be a subset or all of a particular sequence; for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of a 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 a 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 near or 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 a wild-type protein.

[0437] The terms "RNA-programmable nuclease" and "RNA-guide nuclease" are used together 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 can be referred to as a nuclease: RNA complex. Typically, the bound RNA is referred to as 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 has 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 USSN 61 / 874,682, filed on September 6, 2013, entitled "Switchable Cas9 Nucleases and Uses Thereof", and U.S. Provisional Patent Application USSN 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, the 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 target nucleic acids 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, providing sequence specificity of the nuclease:RNA complex.

[0438] In some embodiments, the RNA-programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, for example: Cas9 (Csn1) from Streptococcus pyogenes (Streptococcus pyogenes) (see, e.g., "Complete genome sequence of an Ml 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).

[0439] Since RNA-programmable nucleases such as Cas9 utilize RNA:DNA hybridization to target DNA cleavage sites, these proteins can in principle be targeted to any sequence specified by the guide RNA. Methods for using RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify 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 which have been incorporated herein by reference respectively).

[0440] The term "single nucleotide polymorphism (SNP)" is a change in a single nucleotide that occurs at a specific position in a genome, where each change occurs to some significant degree in a population (e.g., >1%). For example, at a specific base position in a human genome, a C nucleotide may appear in most individuals, but the position in a few individuals is occupied by an A. This indicates that there is a SNP at this specific position, and the two possible nucleotide changes, C or A, are called alleles at this position. SNPs are the basic differences in susceptibility to disease. The severity of disease and our body's response to treatment are also manifestations of genetic changes. SNPs can fall within the coding region of a gene, in a non-coding region of a gene, or in an intergenic region (the region between genes). In some embodiments, because the genetic code is degenerate, SNPs within the coding sequence do not necessarily change the amino acid sequence of the protein produced. There are two types of SNPs within the coding region: synonymous and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, while non-synonymous SNPs change the amino acid sequence of the protein. There are two types of non-synonymous SNPs: missense and nonsense. SNPs that are not 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, without any frequency restrictions and can come from body cells. Body single nucleotide changes can also be called single nucleotide modifications.

[0441] "Specific binding" means 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).

[0442] Nucleic acid molecules suitable for the methods of the present invention include any nucleic acid molecules encoding polypeptides of the present invention or fragments thereof. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but generally have substantial identity. Polynucleotides having "substantial identity" to the endogenous sequence can generally hybridize with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules suitable for the methods of the present invention include any nucleic acid molecules encoding polypeptides of the present invention or fragments thereof. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but generally have substantial identity. Polynucleotides having "substantial identity" to the endogenous sequence can generally hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridization" means pairing to form a double-stranded molecule between complementary polynucleotide sequences (e.g., the genes described herein), or portions thereof, under various stringent conditions (see, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152: 399; Kimmel, AR (1987) Methods Enzymol. 152: 507).

[0443] 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 a temperature of at least about 30° C., more preferably at least about 37° C., and most preferably at least about 42° C. Other parameters that vary, such as hybridization time, detergent (e.g., sodium dodecyl sulfate (SDS)) concentration, and inclusion or exclusion of vector DNA are well known to those skilled in the art. Various degrees of stringency can be achieved by combining these various conditions as needed. In one embodiment, the hybridization is performed at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, the 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, the 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 generally understood by those skilled in the art.

[0444] For most purposes, the washing step after the hybridization method also varies in stringency. Washing stringency conditions can be defined by salt concentration and temperature. As described above, washing stringency can be increased by reducing the salt concentration or increasing the temperature. For example: the stringent salt concentration of the washing step is preferably less than about 30mM NaCl and 3mM trisodium citrate, and most preferably less than about 15mM NaCl and 1.5mM trisodium citrate. The stringent temperature conditions of the washing 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 washing step will be carried out at 25°C in 30mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step will be carried out at 42°C in 15mM NaCl, 1.5mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash step will be performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Other variations on these conditions are 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.

[0445] "Split" means to divide into two or more segments.

[0446] "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. The polypeptides corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein can be spliced ​​into a "reconstituted" Cas9 protein. In a specific embodiment, the Cas9 protein is divided into two fragments within the disordered region of the protein, for example: described in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014; or described in Jiang et al. (2016) Science 351: 867-871. PDB file: 5F9R, the contents of which have been incorporated herein by reference. In some embodiments, the protein is divided into two fragments at any C, T, A, or S in the region between about amino acids A292-G364, F445-K483, or E565-T637 of SpCas9, or in the corresponding positions of any other Cas9, Cas9 variants (e.g., nCas9, dCas9), or other napDNAbp. In some embodiments, the protein is divided into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of dividing a protein into two fragments is referred to as "splitting" the protein.

[0447] In other embodiments, the N-terminal portion of the Cas9 protein comprises amino acids 1-573 or 1-637 of the 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.

[0448] The C-terminal portion of the split Cas9 and the N-terminal portion of the split Cas9 are joined to form a 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 comprises amino acids (551-651)-1368 of spCas9. "(551-651)-1368" means starting with amino acids between amino acids 551-651 (inclusive) and ending at amino acid 1368.For example, the C-terminal portion of the split Cas9 may include any portion of amino acids 551-1368, 552-1368, 553-1368, 554-1368, 555-1368, 556-1368, 557-1368, 558-1368, 559-1368, 560-1368, 561-1368, 562-1368, 563-1368, 564-1368, 565-1368, 566-1368, 567-1368, 568-1368, 569-1368, 570-1368, 571-1368, 572-1368, 573-1368 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.

[0449] "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 consumer products, such as food, including but not limited to cattle, goats, chickens, horses, pigs, rabbits, and sheep.

[0450] "Substantially identical" means that the polypeptide or nucleic acid molecule has at least 50% identity to a reference amino acid sequence (e.g., any amino acid sequence described herein) or nucleic acid sequence (e.g., any nucleic acid sequence described herein). In one embodiment, such sequences and sequences used for comparison are at least 60%, 80% or 85%, 90%, 95% or even 99% identical at the amino acid level or nucleic acid.

[0451] Sequence identity is often 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). Such software matches identical or similar sequences by specifying degrees of homology with various substitutions, deletions, and / or other modifications. Conservative substitutions often 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 represented by the sequence identity. -3 To e -100 The probability score between them indicates highly correlated sequences. For example, when using COBALT, the following parameters are used:

[0452] a) Parallelism parameters: gap penalty -11, -1, and end gap penalty -5, -1,

[0453] b) CDD parameters: RPS BLAST was turned on; Blast E-value was 0.003; conserved amino acid positions were searched and duplicate counting was turned on, and

[0454] c) Query clustering parameters: use query clustering on; character size 4; maximum cluster distance 0.8; general letters.

[0455] Use EMBOSS needles, for example, according to the following parameters:

[0456] a) Matrix: BLOSUM62;

[0457] b) GAP OPEN: 10;

[0458] c) GAP EXTEND: 0.5;

[0459] d) Output format: a pair;

[0460] e) End gap penalty: None;

[0461] f) END GAP OPEN: 10; and

[0462] g) END GAP EXTEND: 0.5.

[0463] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a nucleobase editor. In one embodiment, the target site is deaminated by a deaminase or a fusion protein comprising a deaminase (eg, adenine deaminase).

[0464] 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 obtaining of a desired pharmacological and / or physiological effect. It is generally understood that, although not excluded, the treatment of a disease or disorder 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 a partial or complete reduction, elimination, abrogation, alleviation, alleviation, reduction in severity or cure of the disease and / or symptoms attributed to the disease. In some embodiments, the effect is preventive, that is, the effect protects or prevents the occurrence or recurrence of a disease or disorder. Therefore, the methods of the present invention include administering a medically effective amount of a composition described herein. In some embodiments, the disease or disorder is sickle cell disease (SCD) or β-thalassemia.

[0465] "Uracil glycosidase inhibitor" or "UGI" means an agent that inhibits the uracil-excision repair system. In one embodiment, the agent is a protein or fragment thereof that binds to the host uracil-DNA glycosidase and prevents the uracil residue from being separated from the DNA. In one embodiment, UGI is a protein, fragment, or domain that can inhibit the uracil-DNA glycosidase base-excision repair enzyme. In some embodiments, the UGI domain comprises a 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 amino acid sequence contained in the UGI fragment 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, UGI comprises an amino acid sequence that is homologous to the UGI amino acid sequence or a fragment thereof exemplified below. In some embodiments, 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:

[0466] >splP14739IUNGI_BPPB2 Uracil-DNA glycosidase inhibitor

[0467] MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML.

[0468] The term "vector" refers to a means for introducing a nucleic acid sequence into a cell, which produces 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 to be expressed 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.

[0469] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0470] DNA editing has emerged as a viable approach to modify disease at the genetic level by correcting pathogenic mutations. Until recently, all DNA editing platforms have the ability to induce DNA double-strand breaks (DSBs) at defined genomic sites, with the product outcomes determined in a semi-stochastic manner by endogenous DNA repair pathways, resulting in a complex population of genetic products. While precise user-defined repair outcomes can be achieved via the homology-directed repair (HDR) pathway, a number of challenges prevent efficient repair using HDR in medically relevant cell types. In fact, this pathway is less efficient than the competing and error-prone non-homologous end joining pathway. Furthermore, HDR is extremely restricted to the G1 and S phases of the cell cycle, preventing accurate repair of DSBs in post-mitotic cells. As a result, it has proven difficult or impossible to efficiently modify genomic sequences in such populations in a user-defined, programmable manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0471] Figures 1A-1C Depict the plasmid. Figure 1A An expression vector encoding the TadA7.10-dCas9 base editor. Figure 1B The plasmid comprises a nucleic acid molecule encoding a protein conferring chloramphenicol resistance (CamR) and spectinomycin resistance (SpectR). The plasmid also comprises a kanamycin resistance gene abolished by two point mutations. Figure 1C The plasmid comprises a nucleic acid molecule encoding a protein conferring chloramphenicol resistance (CamR) and spectinomycin resistance (SpectR). The plasmid also comprises a kanamycin resistance gene abolished by three point mutations.

[0472] Figure 2 An image of a bacterial colony transduced with the expression vector depicted in Figure 1, which includes a defective kanamycin resistance gene, is shown. The vector containing the ABE7.10 variant was 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 the ABE7.10 variants and having 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.

[0473] Figure 3A and 3B Editing of the regulatory region of the heme subunit gamma (HGB1) locus, a medically relevant site for upregulation of fetal hemoglobin, is demonstrated. Figure 3A 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 on the heme subunit gamma 1 (HGB1) locus of HEK293T cells, which is a medically relevant site for upregulating fetal hemoglobin. The figure above shows the nucleotide residues in the target region of the HGB1 gene regulatory sequence. A5, A8, A9, and A11 represent edited adenosine residues in HGB1.

[0474] Figure 4 Illustrating 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 efficacy of adenosine deaminase variant base editors using guide RNAs of varying lengths.

[0475] Figure 5 Figure 1 is a diagram illustrating the potency and specificity of ABE8. The percentage of editing at the intended target nucleotide and the unintended target nucleotide (bystander) was quantified.

[0476] Figure 6 Figure 1 is a diagram illustrating the potency and specificity of ABE8. The percentage of editing at the intended target nucleotide and the unintended target nucleotide (bystander) was quantified.

[0477] Figures 7A-7C Schematic and bar graphs depicting neutralization of A·T to G·C conversion and phenotypic consequences in primary cells. Fig. 7A Diagram showing the embryonic, fetal, and adult globin genes located on chromosome 11, indicating the HBG1 / 2HPFH site where a single base editor introduces a double helix edit. Figure 7B To show the efficacy of DNA editing in CD34+ cells. It shows the 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 hours and 144 hours after treatment. The -198HBG1 / 2 target sequence is as follows: A7 is bold and has a double underline. 7 Plot the percentage of A·T to G·C. Figure 7C Figure 1 is a graph reflecting the percentage of γ-globin / α-globin expression 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 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 the formation of γ-globin in differentiated erythrocytes by about 1.4-fold. For example, the ABE8.13-d base editor resulted in 55% γ-globin / α-globin expression.

[0478] Fig. 8A and 8B Depicts the A·T to G·C conversion of CD34+ cells treated with ABE8 at the -198 promoter site upstream of HBG1 / 2. Fig. 8A Figure 2. Heat map depicting the frequency of A to G editing of ABE8 in CD34+ cells from two donors, 48 ​​and 144 h after editor treatment, where donor 2 was heterozygous for sickle cell disease. Figure 8B Graphs representing the distribution of total sequenced reads containing only the A7 edit or the combined (A7+A8) edits.

[0479] Fig. 9 Heatmap depicting the frequency of insertion / deletions (INDELs) in CD34+ cells at the -198 site of the γ-globin promoter after ABE8 treatment. Frequencies from two donors are shown at the 48h and 144h time points. Complete A·T to G·C conversion creates a 10nt poly-G stripe at the HBG1 / 2-198 promoter target site described herein. Since the manipulation of such homopolymers often increases the proportion of PCR- and sequencing-induced errors, an increased insertion / deletion (INDEL) frequency is observed at this site.

[0480] Fig.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.

[0481] Fig.11 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE7.10-m and the integration of the globin chain phase.

[0482] Fig.12 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE7.10-d and the integration of the globin chain phase.

[0483] Fig.13 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.8-m and the integration of the globin chain phase.

[0484] Fig.14Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.8-d and the integration of the globin chain phase.

[0485] Fig.15 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.13-m and the integration of the globin chain phase.

[0486] Fig.16 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.13-d and the integration of the globin chain phase.

[0487] Fig.17 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.17-m and the integration of the globin chain phase.

[0488] Fig.18 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.17-d and the integration of the globin chain phase.

[0489] Fig.19 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.20-m and the integration of the globin chain phase.

[0490] Fig. 20 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.20-d and the integration of the globin chain phase.

[0491] Fig.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.

[0492] Fig. 22 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE7.10-m and the integration of the globin chain phase. Note: Donor 2 is heterozygous for sickle cell disease.

[0493] Fig.23Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE7.10-d and the integration of the globin chain phase. Note: Donor 2 is heterozygous for sickle cell disease.

[0494] Fig.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.

[0495] Fig.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.

[0496] Fig.26 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE8.13-m and the integration of the globin chain phase. Note: Donor 2 is heterozygous for sickle cell disease.

[0497] Fig. 27 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE8.13-d and the integration of the globin chain phase. Note: Donor 2 is heterozygous for sickle cell disease.

[0498] Fig.28 Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 1) treated with ABE8.17-m and the integration of the globin chain phase.

[0499] Fig.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.

[0500] Fig. 30A and 30B Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells treated with ABE8 and the integration of the globin chain phase. Fig. 30A Depicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE8.20-m and the integration of the globin chain phase. Note: Donor 2 is heterozygous for sickle cell disease. Fig. 30BDepicted is the UHPLC UV-Vis trace (220 nm) of differentiated CD34+ cells (Donor 2) treated with ABE8.20-d and the integration of the globin chain phase. Note: Donor 2 is heterozygous for sickle cell disease.

[0501] Figures 31A-31E Depicts editing at two independent sites using ABE8.8, achieving 90% editing before enucleation at day 11 after erythroid differentiation, and approximately 60% gamma globin, exceeding alpha globin or total beta family globin, at day 18 after erythroid differentiation. Fig.31A Graph depicting average ABE8.8 editing in 2 healthy donors in 2 independent experiments. Editing efficiency was measured using primers that discriminate between HBG1 and HBG2. Fig.31B Graph depicting the mean of 2 independent experiments from 1 healthy donor. Editing efficiency was measured using primers that recognize both HBG1 and HBG2. Fig.31C Schematic diagram depicting editing of ABE8.8 in a donor with a heterozygous E6V mutation. Fig.31D and 31E Graph depicting increase in γ-globin in ABE8.8-edited cells.

[0502] Fig.32A and 32B Depicted are the percentage of editing using ABE variants to correct sickle cell mutations. Fig.32A To illustrate the schematic of screening different editor variants with approximately 70% editing in SCD patient fibroblasts. Fig.32B Figure 3. Schematic representation of CD34 cells from a healthy donor edited by a lead ABE variant targeting a synonymous mutation A13 in an adjacent proline that remains within the editing window and serves as a surrogate for editing SCD mutations. The ABE8 variant showed an average editing frequency of about 40% at surrogate A13.

[0503] Fig.33A and 33B Figure 2. RNA amplicon sequencing to detect cellular A-formation-I editing in RNA associated with ABE treatment. Individual data points are shown, and error bars represent standard deviation sd of n = 3 biological replicates performed independently on different days. Fig.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. Fig.33B Graph depicting the frequency of A-to-I editing in target RNA amplicons for ABE8 with mutations reported to improve RNA off-target editing.

[0504] Fig.34A and 34BThe diagram and UPHLC chromatographic traces related to editing of SCD CD34+ cells are shown. CD34+ cells from SCD patients were transfected with ABE8.8 mRNA and sgRNA (HBG1 / 2, 50nM) using electroporation. The edited cells differentiated into red blood cells in vitro. Next-Genome Sequencing (NGS) was used to measure the editing rate at the HBG1 / 2 promoter. Fig.34A As shown, 16.5% editing by the ABE8.8 base editor was observed at 48 hours after differentiation, and 89.2% editing was observed at 14 days after differentiation. Fig.34B Shown are bystander editing disintegration at 48 hours and 14 days after differentiation.

[0505] Figures 35A-35D The UPHLC chromatographic trace of the globin phase and the Fig.34A and 34B A diagram showing the functions of 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 at day 18 after differentiation. Fig.35A The presented track shows the globin stage of erythroid cells differentiated from unedited SCDCD34+ cells. Fig.35B The presented track shows the globin stage of erythroid cells differentiated from edited SCD CD34+ cells. Fig.35C It was shown that 63.2% of the γ-globin stage was detected in erythroid cells differentiated from edited SCDCD34+ cells compared to unedited cells. Fig.35D The results showed that S-globin levels in edited SCD CD34+ cells decreased from 86% to 32.9% compared to unedited cells. Upregulation of fetal hemoglobin is a promising approach for the treatment of SCD and beta-thalassemia.

[0506] Figures 36A-36C Ribbon structures, target sequences, and diagrams are shown and generated for variants of ABE editors for editing atypical Cas9 NGG PAM sequences. Design of 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 Fig.36A ), the sickle allele within the editing window of ABE can be targeted ("target A"), e.g. Fig.36B As shown, this provides the ability to directly edit this position in the target site, which is not normally accessible using traditional spCas9. Fig.36CA graphical representation of the base editing activity of a variant editor comprising the MQKFRAER amino acid substitution is shown, which can recognize the target site and convert nucleobase A to 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.

[0507] Fig.37 Diagrams, target site sequences and tables related to the generation of additional adenosine deaminase variants are shown, in which the linker to TadA has been excluded 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 ISLAY9Q768, as shown in Table 14A below. On the right side of the figure, the target site, PAM site and corresponding amino acid sequence in the nucleic acid sequence are shown. In the table, "Cp5" (MSP552) refers to ABE8 in the backbone, which includes a circular arrangement of Cas9, has the following amino acid sequence and is described below.

[0508]

[0509] 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).

[0510] Fig.38 and 39 A schematic diagram of different adenosine deaminase ISLAY variants demonstrating enhanced editing of target sites (e.g. Fig.37 The diagram shown in the middle figure is a comparison with another ABE editor (ABE7.10) having a linker with a TadA domain.

[0511] Fig.40 The percentage of base editing achieved in CD34+ cells expressing the SCD target site is shown, and the table shows the edited nucleic acid and amino acid changes. CD34+ cells from a heterozygous sickle trait patient were treated with the ABE editor and editing of the target site (9G) was measured, i.e., converting nucleobase A to nucleobase T to achieve the desired correction Val>Ala. At 96 hours post 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 cells into erythroid cells (IVD), as over 60% editing was shown in differentiated erythroid cells (heterozygous for sickle trait) at 12 days post erythroid differentiation. The diagram of Editor_nM mRNA_[sgRNA]:[mRNA]_Timepoint was evaluated using a 21nt gRNA.

[0512] Fig.41A and 41B High performance liquid chromatography (UHPLC) chromatographic traces and LC-MS results associated with the detection of unique β-globin species in edited heterozygous HbS (β-globin in sickle cells) differentiated erythrocytes are presented. Prior to such experiments and analyses, practitioners in the art were generally unable to successfully distinguish and separate HbG Makassar variant globin from the HbS sickle globin variant using traditional methods. A UHPLC method was developed and used to distinguish these two different globin variants in cells (e.g., CD34+ cells) from SCD patients that have 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 β-globin (Hb) variants ( Fig.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) ( Fig.41B ).

[0513] Fig.42 Table of base editor and sgRNA sequences for base editing SCD samples with HbS globin variants is shown to correct the HbG Makassar variant globin. ABE8 mutations were introduced into lead editor candidates and sgRNAs of varying lengths (21nt, 20nt, 19nt protospacers) were analyzed to detect whether on-target editing was improved while reducing potentially deleterious 1G edits (conversion of Ser10Pro). "A" nucleotides in bold / italic / underlined indicate sickle substitutions. Lowercase letters in sgRNA / protospacer sequences indicate nucleobases that were 2′-O-methylated. Lowercase "s" in sgRNA / protospacer sequences indicate phosphorothioates.

[0514] Fig.43A and 43B The 9G target site (or 9G and other sites) in CD34+ cells (heterozygous sickle cell trait samples) was shown after electroporation with different ABE editors 48h ( Fig.43A ) or differentiated erythrocytes in vitro (heterozygous sickle trait samples) after 7 days of differentiation ( Fig.43B ). Although additional mutations did not significantly improve on-target editing, 4 editors demonstrated comparable on-target editing efficacy. 20nt sgRNA length can achieve lower undesired 1G bystander edits. In these plots, Editor_sgRNA nt (Editor_sgRNA nt) or Editor_100nM mRNA_μM sgRNA (Editor_100nM mRNA_μM sgRNA) (20nt) were evaluated. Nearly 80% editing was maintained throughout in vitro erythroid differentiation.

[0515] Fig.44A and 44B A bar graph and table showing edited nucleic acid sequences and corresponding amino acid sequence transitions associated with total base editing of position 9G of HbS in a homozygous SCD (HbSS) sample are shown. The cells were whole blood (non-mobilized) samples obtained from SCD (HbSS) patients and were base edited using ABE variant base editors. Fig.44A: CD34+ cells (~200,000 cells, homozygous SCD samples) were electroporated with 50 nM ABE variant editor (MSP619 (ISLAY5)) at a ratio of 100:1 (2 μg mRNA, 4.1 μg sgRNA (21 nt)). The ABE variant base editor achieved about 65% editing at position 9G of the cells on day 7 after electroporation, and about 60% editing at position 9G on day 14 after electroporation. Fig.44B : CD34+ cells (~200,000 cells, homozygous SCD samples) were electroporated with 30nM ABE variant editor (MSP616 (ISLAY2)) at a ratio of 200:1 (1.3μg mRNA, 4.95μg sgRNA (21nt)). The ABE variant base editor achieved at least about 50% editing at position 9G of red blood cells on days 7 and 14 after electroporation.

[0516] Fig.45 Shown is a UHPLC chromatographic trace after UHPLC analysis, which shows clear separation and differentiation of HbS type and HbGMakassar variant type after base editing using ABE variant base editor in homozygous HbSS cells obtained from SCD patient samples.

[0517] Fig.46A and 46B UHPLC chromatographic traces and LC-MS results related to the detection of separate β-globin species in edited heterozygous HbS (β-globin in sickle cells) differentiated erythrocytes are shown. Fig.41A and 41B In the edited heterozygous HbSS samples, the different β-globin (Hb) variants corresponding to them with Val→Ala substitutions can be detected based on molecular weight ( Fig.46A ). Edited peaks of the LC-MS trace show a charged mantle indicating a novel β-globin variant ( Fig.46B ).

[0518] Fig.47UHPLC chromatographic traces 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 upper and middle UHPLC chromatograms, based on the difference in UHPLC elution time, the HbG Makassar globin variant was separated from the HbS(SCD) globin type (10.03min) at 9.81 minutes. Other globin types are easily distinguished. In the lower LC-MS, the Makassar HbG variant and HbS type of globin have different and distinguishable characteristics. Similar Fig.41A , 41B , 45, 46A and 46B show that UHPLC and LC-MS analysis of cells from SCD (HbSS) red blood cell samples edited by the ABE variant base editors described herein clearly identify and separate 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 SCD (HbSS) patients who instead present with the HbG Makassar globin variant.

[0519] Figures 48A-48C A bar graph representing the relative area under the peak of the UHPLC chromatographic data is presented. The area under the peak is used to quantify the total change in the content of different β-globin variants in homozygous SCD samples that have been base edited using the ABE variants of the present invention (base editor MSP619, 50nM mRNA, 5000nM sgRNA (21nt)). The results presented suggest that the degree of conversion of HbS variant globin is positively correlated with asymptomatic HbG-Makassar globin.

[0520] Fig.49 Table depicting Cas9 variants that can access all possible PAMs within the NRNN PAM interval. Only Cas9 variants that require recognition of three or fewer 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 have been incorporated herein by reference in their entirety. DETAILED DESCRIPTION

[0521] As described below, the 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.

[0522] HBB gene editing

[0523] 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 in the 6th amino acid of the β-globin protein encoded by the HBB gene. Despite the current advances in the field of gene editing, it is still not possible to precisely correct the diseased HBB gene back to Val→Glu, and this is not currently possible using CRISPR / Cas nucleases or CRISPR / Cas base editing methods.

[0524] Genome editing using CRISPR / Cas nuclease approaches 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 generating base insertions / deletions, which may lead to unexpected and undesirable consequences, including the generation of premature stop codons, changes in the codon reading frame, etc. In addition, the generation of double-strand breaks in 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 with sequence identity to another-5'-ε-; Gγ-; Aγ-; δ-; and β-globin-3'. Due to the structure of the β-globin locus, recombinational repair of double-strand breaks within the locus has the potential to cause gene loss of sequences interspersed between globin genes, such as between the δ- and β-globin genes.

[0525] 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 correction of Val→Glu (G T G→G AG) 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 site in order for base editing to be possible, and optimal editing within the window may have additional sequence requirements. One of the requirements 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 NGGPAM. Even though it is hypothesized that T·A may be inverted to A·T, this SpCas9 base editor does not have the presence of 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 accessible PAMs, the need for PAMs remains a limiting factor in the ability to direct CRISPR / Cas base editors to specific nucleotides at any position in the genome.

[0526] 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 that causes sickle cell disease with alanine, thereby generating an Hb variant that does not produce a sickle cell phenotype (HbMakassar). Although precise correction is not possible without a T·A to A·T inverted base editor (G T G→G A G). However, the experiments conducted in this paper have found that the base editor (ABE) using A·T to form G·C can produce Val→Ala (G T G→G C G) replacement (i.e., Hb Makassar variant). This is achieved in part by developing novel base editors and novel base editing strategies provided herein. For example, novel ABE base editors (i.e., with adenosine deaminase domains) utilize flanking sequences (e.g., PAM sequences; zinc finger binding sequences) for optimal base editing at sickle cell target sites.

[0527] 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). Substitution of 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 to polymerize as in pathogenic variant HbS). Thus, the compositions and methods of the present invention are useful for treating sickle cell disease (SCD).

[0528] Nucleobase editors

[0529] 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 article 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 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.

[0530] Polynucleotide programmable nucleotide binding domain

[0531] 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 in the present invention.

[0532] The polynucleotide programmable nucleotide binding domain of the base editor itself may include one or more domains. For example: the polynucleotide programmable nucleotide binding domain may include one or more nuclease domains. In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain may include 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.

[0533] In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain can cut the zero (zero) strand, one strand, or two 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, which 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 through one or more mutations, and is derived from a complete catalytic activity (e.g., natural) type of a polynucleotide programmable nucleotide binding domain. For example: if a 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 a nucleic acid double helix. In another example, a 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., natural) 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 may comprise a deletion of all or part of the RuvC domain or the HNH domain.

[0534] The amino acid sequence of an example of a catalytically active Cas9 is as follows:

[0535]

[0536] A base editor comprising a polynucleotide programmable nucleotide binding domain containing a nickase domain can therefore produce a single-stranded DNA break (nick) 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 a DNA molecule that is targeted for editing. In this case, the strand that is not targeted will not be cleaved.

[0537] 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, referring 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, a 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 causing a loss of nuclease activity. In other embodiments, a catalytically inactivated polynucleotide programmable nucleotide binding domain may comprise one or more deletions of all or a portion of a catalytic domain (e.g., RuvC1 and / or HNH domain). In other embodiments, a 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 a nuclease domain.

[0538] It is also contemplated herein that mutations of a catalytically inactivated polynucleotide programmable nucleotide binding domain can be generated from a previously functional polynucleotide programmable nucleotide binding domain. For example, taking a catalytically inactivated Cas9 ("dCas9") as an example, variants having mutations other than D10A and H840A are provided, which result in a 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 at the HNH nuclease subdomain and / or the RuvC1 subdomain). Other suitable nuclease-inactivating dCas9 domains are 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).

[0539] 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, the base editor comprises a polynucleotide programmable nucleotide binding domain comprising a natural or modified protein or a portion thereof, which can bind to a nucleic acid sequence during CRISPR (i.e., Clustered Regularly Interspaced Short Palindromic Repeats)-mediated modification of the nucleic acid through a bound guide nucleic acid. These proteins are referred to herein as "CRISPR proteins." Therefore, disclosed herein is a base editor comprising a polynucleotide programmable nucleotide binding domain, wherein the domain comprises all or a portion of a CRISPR protein (i.e., the base editor comprises 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 compared to a wild-type or natural CRISPR protein. For example, the CRISPR protein-derived domain described below can comprise one or more mutations, insertions, deletions, rearrangements and / or recombination relative to a wild-type or natural CRISPR protein.

[0540] 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 serves 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 crRNA is first cut in an endonucleolytic manner, and then 3′-5′ is trimmed in an exonucleolytic manner. In nature, DNA-binding and cleavage generally require proteins and two types of RNA. However, a single guide RNA ("sgRNA" or "gRNA" for short) can be engineered to incorporate both crRNA and tracrRNA embodiments into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif (PAM or protospacer adjacent motif) in the CRISPR repeat sequence to help distinguish between self and non-self.

[0541] In some embodiments, the methods described herein can utilize engineered Cas proteins. Guide RNAs (gRNAs) are short synthetic RNAs that consist of a backbone sequence necessary for Cas-binding and a user-defined ~20 nucleotide spacer that specifies the genome target to be modified. Thus, one skilled in the art can alter the genome target specific for a Cas protein, which is determined in part by the degree of specificity of the gRNA targeting sequence for the genome target versus the rest of the genome.

[0542] In some embodiments, the gRNA backbone sequence is as follows: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAUAAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU.

[0543] 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.

[0544] 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. CRISPR enzymes can predominantly cleave a target sequence, such as one or both strands within a target sequence and / or within the complement of a target sequence. For example, a 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 in a target sequence.

[0545] Vectors encoding CRISPR enzymes that have been mutated relative to the corresponding wild-type enzymes can be used, so that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide comprising the target sequence. 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 a wild-type Cas9 polypeptide example (e.g., Cas9 from S. 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 with a wild-type Cas9 polypeptide example (e.g., from S. 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.

[0546] In some embodiments, the CRISPR protein-derived domain of the base editor may include all or part of Cas9 from the following: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium 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.

[0547] Cas9 domains of nucleobase editors

[0548] 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 et al., Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821 (2012), the entire contents of which are each incorporated herein by reference). Cas9 orthologous genes have been described in various species, including (but not limited to): Streptococcus pyogenes (S. pyogenes) and Streptococcus thermophilus (S. thermophilus).Other suitable Cas9 nucleases and sequences are known to those skilled in the art based on the present invention, and include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun and Charpentier in "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737; the entire contents of which have been incorporated herein by reference.

[0549] 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% consistent with any 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 consecutive amino acid residues compared to any of the amino acid sequences disclosed herein.

[0550] In some embodiments, a protein comprising a fragment of Cas9 is 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, a protein comprising Cas9 or a fragment thereof is referred to as a "Cas9 variant". A Cas9 variant and Cas9 or a fragment thereof have common homology. For example, a Cas9 variant and a wild-type Cas9 are 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. 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), so that the fragment and the corresponding fragment of the wild-type Cas9 are 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. 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% amino acid length of 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.

[0551] In some embodiments, the Cas9 fusion proteins provided herein include the full-length amino acid sequence of the Cas9 protein, for example, one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not include the full-length Cas9 sequence, but 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.

[0552] The Cas9 protein can be associated with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Examples of nucleic acid programmable DNA-binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b / C2C1, and Cas12c / C2C3.

[0553] 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):

[0554]

[0555]

[0556] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0557] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence:

[0558]

[0559]

[0560]

[0561] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0562] 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):

[0563]

[0564]

[0565]

[0566] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0567] In some embodiments, Cas9 refers to Cas9 from the following: 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.

[0568] 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.

[0569] In some embodiments, the Cas9 domain is a nuclease-inactivated 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 strand of the double-helical nucleic acid molecule. In some embodiments, the nuclease-inactivated 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-inactivated 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-inactivated Cas9 domain comprises the amino acid sequence shown in the cloning vector pPlatTET-gRNA2 (accession number BAV54124).

[0570]

[0571] (See, e.g., Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.” Cell. 2013; 152(5): 1173-83, the entire contents of which are incorporated herein by reference).

[0572] Other suitable nuclease-inactivating dCas9 domains are within the skill of 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).

[0573] In some embodiments, the Cas9 nuclease has an inactivated (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase, referred to as a "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 producing 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, the contents of which have been incorporated herein by reference in their entirety). 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 complementary strand of the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations in these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337: 816-821 (2012); Qi et al., Cell. 28; 152(5): 1173-83 (2013)).

[0574] 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 or more 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 consecutive amino acid residues compared to any one of the amino acid sequences disclosed herein.

[0575] 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 D10A and H840A mutations or corresponding mutations in another Cas9.

[0576] In some embodiments, dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A):

[0577]

[0578]

[0579] (Single bottom line: HNH domain; double bottom line: RuvC domain).

[0580] In some embodiments, the Cas9 domain comprises a D10A mutation, while 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.

[0581] In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which, for example, result in nuclease-inactive Cas9 (dCas9). 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). In some embodiments, the variants or homologs of the dCas9 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. In some embodiments, the provided dCas9 variants have shorter or longer amino acid sequences, differing by about 5 amino acids, differing by about 10 amino acids, differing by about 15 amino acids, differing by about 20 amino acids, differing by about 25 amino acids, differing by about 30 amino acids, differing by about 40 amino acids, differing by about 50 amino acids, differing by about 75 amino acids, differing by about 100 or more amino acids.

[0582] 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-helix nucleic acid molecule (e.g., a double-helix DNA molecule). In some embodiments, the Cas9 nickase cleaves the target strand of the double-helix 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 editing strand of the double-helix nucleic acid molecule, meaning that the strand cleaved by the Cas9 nickase will not be base-paired 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 within the scope of the present invention and are known to those skilled in the art.

[0583] The amino acid sequence of an example of a catalytic Cas9 nickase (nCas9) is as follows:

[0584]

[0585] In some embodiments, Cas9 refers to Cas9 from an archaea (e.g., nanoarchaea), which belongs to the unicellular prokaryotic microbial domain and the unicellular prokaryotic microbial kingdom. In some embodiments, the programmable nucleotide binding protein can be a CasX or CasY protein, which has been 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 have been incorporated herein by reference. Many CRISPR-Cas systems have been identified using genome-resolved metagenomics, including Cas9, which was first reported in the archaeal domain of life. This diverse Cas9 protein has appeared in the little-studied nanoarchaea as part of an active CRISPR-Cas system. Two previously unknown systems have been discovered in bacteria: CRISPR-CasX and CRISPR-CasY, which are the most robust systems discovered to date. In some embodiments, in the base editor systems described herein, Cas9 is replaced by CasX, or a variant of CasX. In some embodiments, in the base editor systems described herein, Cas9 is replaced by CasY, or a variant of CasY. It should be understood that other RNA-guided DNA binding proteins can be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present invention.

[0586] 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 natural CasX or CasY protein. In some embodiments, the programmable nucleotide binding protein is a natural 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 should be understood that CasX and CasY from other bacterial species may also be used according to the present invention.

[0587] 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:

[0588] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGE。

[0589] An exemplified CasX (>tr|F0NH53|F0NH53_SULIR CRISPR - associated protein, Casx OS = Sulfolobus islandicus (strain REY15A) GN = SiRe_0771 PE = 4 SV = 1) amino acid sequence is as follows:

[0590] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGE。

[0591] Deltaproteobacteria CasX

[0592] MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQ KWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0593] 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:

[0594]

[0595] The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. When Cas9 binds to its target, it 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.

[0596] The "efficacy" of non-homologous end joining (NHEJ) and / or homology-guided repair (HDR) can be calculated by 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 product 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 substrates that are 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).

[0597] In some embodiments, the 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 generated by hybridization of wild-type and mutant DNA strands (NHEJ produces 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).

[0598] The NHEJ repair pathway is the most active repair mechanism, often causing small nucleotide insertions or deletions at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical implications because populations of cells expressing Cas9 and gRNA or guide polynucleotides will produce divergent mutations. In most cases, NHEJ produces small insertions or deletions in the target DNA, resulting in amino acid deletions, insertions, or frameshift mutations, resulting in premature stop codons within the open reading frame (ORF) of the target gene. The ideal end result is a loss-of-function mutation within the target gene.

[0599] 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 adding fluorophores or tags. In order to use HDR for gene editing, a DNA repair template containing the desired sequence can be delivered to a cell type with gRNA (group) and Cas9 or Cas9 nickase. The repair template can include the desired edit and additional homology sequences (called left homology arms and right homology arms) immediately upstream and downstream of the target. The length of each homology arm can depend on the size of the change to be introduced, and the larger the block, the longer the homology arm is required. 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 exogenous repair templates, the efficacy of HDR is generally still low (<10% of modified alleles). The efficacy of HDR can be enhanced by cell synchronization because HDR occurs during the S and G2 phases of the cell cycle. Chemical or genetic inhibitors of NHEJ can also increase the frequency of HDR.

[0600] 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 produces 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 produces DNA nicks instead of DSBs. The nickase system can also be combined with HDR-mediated gene editing for specific gene editing.

[0601] In some embodiments, Cas9 is a variant Cas9 protein. The amino acid sequence of a variant Cas9 polypeptide differs from the amino acid sequence of a wild-type Cas9 protein by one amino acid (e.g., having a deletion, insertion, substitution, fusion). In some examples, the amino acid changes (e.g., deletion, insertion, or substitution) of a variant Cas9 polypeptide reduce the nuclease activity of the Cas9 polypeptide. For example, in some examples, a 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 an individual Cas9 protein is a variant Cas9 protein without substantial nuclease activity, it may be referred to as "dCas9".

[0602] 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).

[0603] 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 it can cleave the complementary strand of the double-stranded guide target sequence, 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).

[0604] In some embodiments, the variant Cas9 protein can cleave the non-complementary strand of the double-stranded guide target sequence, but the ability to cleave the complementary strand of the guide target sequence is reduced. 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 an alanine amino acid) mutation, so that the non-complementary strand of the guide target sequence can be cleaved, but the ability to cleave the complementary strand of the guide target sequence is reduced (so when the variant Cas9 protein cleaves the double-stranded guide target sequence, SSB instead of DSB is generated). The ability of these Cas9 proteins to cleave guide target sequences (e.g., single-stranded guide target sequences) is reduced, but the ability to bind to guide target sequences (e.g., single-stranded guide target sequences) is retained.

[0605] In some embodiments, the variant Cas9 protein has reduced ability to cleave both the complementary and non-complementary strands of a double-stranded target DNA. As a non-limiting example, in some embodiments, the variant Cas9 protein carries both D10A and H840A mutations, and thus the polypeptide has reduced ability to cleave both the complementary and non-complementary strands of a 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).

[0606] As another non-limiting example, in some embodiments, the variant Cas9 protein has W476A and W1126A mutations, so that the ability of the polypeptide to cleave target DNA is reduced. The ability of these Cas9 proteins to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to target DNA (e.g., single-stranded target DNA) is retained.

[0607] As another non-limiting example, in some embodiments, the variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, so that the ability of the polypeptide to cleave target DNA is reduced. The ability of these Cas9 proteins to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to target DNA (e.g., single-stranded target DNA) is retained.

[0608] As another non-limiting example, in some embodiments, the variant Cas9 protein carries H840A, W476A, and W1126A mutations, so that the ability of the polypeptide to cleave target DNA is reduced. The ability of these Cas9 proteins to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to target DNA (e.g., single-stranded target DNA) is retained. As another non-limiting example, in some embodiments, the variant Cas9 protein carries H840A, D10A, W476A, and W1126A mutations, so that the ability of the polypeptide to cleave target DNA is reduced. The ability of these Cas9 proteins to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to target DNA (e.g., single-stranded target DNA) is retained. In some embodiments, the variant Cas9 has restored the catalytic His residue (A840H) at position 840 of the Cas9 HNH domain.

[0609] As another non-limiting example, in some embodiments, the variant Cas9 protein carries H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, so that the ability of the polypeptide to cleave target DNA is reduced. The ability of these Cas9 proteins to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to target DNA (e.g., single-stranded target DNA) is retained. As another non-limiting example, in some embodiments, the variant Cas9 protein carries D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, so that the ability of the polypeptide to cleave target DNA is reduced. The ability of these Cas9 proteins to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to target DNA (e.g., single-stranded target DNA) is retained. In some embodiments, when the variant Cas9 protein has W476A and W1126A mutations or when the variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, the variant Cas9 protein does not effectively bind to the PAM sequence. Therefore, in some cases, when such variant Cas9 proteins are used in a method of binding, the method does not require a PAM sequence. In other words, in some embodiments, when such variant Cas9 proteins are used in a method of binding, the method may include a guide RNA, but the method can be performed without a PAM sequence (so that the binding specificity is provided by the targeting segment of the guide RNA). Other residues can be mutated to achieve the above effects (i.e., to inactivate one or another 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.

[0610] 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, for example, D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A), the variant Cas9 protein will still bind to the target DNA in a site-specific manner (because it is still guided to the target DNA sequence by the guide RNA), as long as it still retains the ability to interact with the guide RNA.

[0611] In some embodiments, the variant Cas protein may be spCas9, spCas9-VRQR, spCas9-VRER, xCas9(sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.

[0612] In some embodiments, a modified SpCas9 comprising amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and having specificity for the altered PAM 5'-NGC-3' is used.

[0613] Alternatives to S. pyogenes Cas9 may include RNA-guided endonucleases from the Cpf1 family that 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 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 the guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, which overcomes some of the limitations of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, the result of Cpf1-mediated DNA cleavage is a double-strand break with a short 3′ overhang. The staggered cleavage pattern of Cpf1 can open up the possibility of directional gene transfer, which is similar to traditional restriction enzyme selection methods and can improve gene editing efficiency. Like the above-mentioned Cas9 variants and direct homologs, 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 contains a mixed α / β domain, RuvC-I, followed by a helical region, RuvC-II, and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. In addition, 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 shows that Cpf1 has unique functionality and is classified as a class 2 type V CRISPR system. The Cas1, Cas2, and Cas4 proteins encoded by the Cpf1 locus are more similar to type I and type III systems than 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 (about 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 discriminating the protospacer adjacent motif 5'-YTN-3'. After discriminating the PAM, Cpf1 introduces a sticky-end-like DNA double-strand break containing a protruding segment of 4 or 5 nucleotides.

[0614] Cas12 domains of nucleobase editors

[0615] Generally, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while Class 2 systems have single protein effectors. For example, Cas9 and Cpf1 are Class 2 effectors, although they are of different types (Type II and Type V, respectively). In addition to Cpf1, Class 2 Type V CRISPR-Cas systems also include Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i). See, for example, Shmakov et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems" Mol. Cell, 2015 Nov. 5; 60(3): 385-397; Makarova et al., "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR Journal, 2018, 1(5): 325-336; and Yan et al., "Functionally Diverse Type VCRISPR-Cas Systems" Science, 2019 Jan. 4; 363: 88-91; the entire contents of which have been 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.

[0616] The nucleic acid programmable DNA binding proteins contemplated by the present invention include Cas proteins (Cas12 proteins) belonging to the second type V. Non-limiting examples of Cas second type V 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 used herein may also be referred to as Cas12 nuclease, Cas12 domain, or 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).

[0617] 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 a strand in a double-helix nucleic acid (e.g., a double-helix 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% consistent with 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 amino acid sequence comprised by the Cas12 domain has 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.

[0618] In some embodiments, a protein comprising a fragment of Cas12 is provided. For example, in some embodiments, the protein comprises one or two Cas12 domains: (1) a gRNA binding domain of Cas12; or (2) a DNA cleavage domain of Cas12. In some embodiments, a protein comprising Cas12 or a fragment thereof is therefore referred to as a "Cas12 variant". Cas12 variants and Cas12 or a fragment thereof have common homology. For example, Cas12 variants are 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), so that the fragment is at least about 70% identical to the corresponding fragment of wild-type Cas12, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the 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.

[0619] 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, differing by about 5 amino acids, differing by about 10 amino acids, differing by about 15 amino acids, differing by about 20 amino acids, differing by about 25 amino acids, differing by about 30 amino acids, differing by about 40 amino acids, differing by about 50 amino acids, differing by about 75 amino acids, differing by about 100 or more amino acids.

[0620] In some embodiments, the Cas12 fusion protein provided herein comprises the full-length amino acid sequence of the Cas12 protein, for example, one of the Cas12 sequences provided herein. However, in other embodiments, the fusion protein provided herein does not comprise the full-length Cas12 sequence, but only comprises one or more fragments thereof. The amino acid sequence examples of suitable Cas12 domains provided herein and other suitable Cas12 domain sequences and fragments are generally understood by those skilled in the art.

[0621] 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., deletion, insertion, substitution, fusion) when compared to the amino acid sequence of the wild-type Cas12 protein. In some cases, the amino acid changes (e.g., deletion, insertion, or substitution) 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 a nickase activity of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the corresponding wild-type Cas12b protein. In some cases, the variant Cas12b protein has no substantial nickase activity.

[0622] 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.

[0623] In some embodiments, the Cas12 protein includes 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 viral DNA and cleave it. Cpf1 is a smaller and simpler endonuclease than Cas9, which overcomes some of the limitations of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, the result of Cpf1-mediated DNA cleavage is a double-stranded break with a short 3′ overhang. The staggered cleavage pattern of Cpf1 could open up the possibility of directional gene transfer, similar to traditional restriction enzyme selection methods, which could improve gene editing efficiency. Like the above-mentioned Cas9 variants and orthologs, Cpf1 can also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM site preferred by SpCas9. The Cpf1 locus contains a mixed α / β domain followed by a helical region of RuvC-I, RuvC-II, and zinc finger-like domains. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. In addition, unlike Cas9, 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 shows that Cpf1 has unique functionality and is classified as a Class 2, Type V CRISPR system. The Cas1, Cas2, and Cas4 proteins encoded by the Cpf1 locus are more similar to type I and type III systems than 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 (about half the number of nucleotides of Cas9). In contrast to Cas9 targeting G-rich PAM, the Cpf1-crRNA complex cleaves the target DNA or RNA by discriminating the protospacer adjacent motif 5'-YTN-3' or 5'-TTTN-3'. After discriminating the PAM, Cpf1 introduces a sticky-end-like DNA double-strand break containing a protruding segment of 4 or 5 nucleotides.

[0624] In some embodiments of the present invention, a vector encoding a CRISPR enzyme that has been mutated in the corresponding wild-type enzyme can be used, so that the mutated CRISPR enzyme lacks cleavage of one or both strands of a target polynucleotide comprising a target sequence. Cas12 can refer to and exemplify a wild-type Cas12 polypeptide (e.g., Cas12 from Bacillus hisashii) that is 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. Cas12 may refer to and exemplify wild-type Cas12 polypeptides (e.g., polypeptides from Bacillus hisashii (BhCas12b), Bacillus sp. V3-13 (BvCas12b), and Alicyclobacillus acidiphilus (AaCas12b)) 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. Cas12 may refer to a wild-type or modified form of a Cas12 protein, which may include amino acid changes, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0625] Nucleic acid programmable DNA binding protein

[0626] Some embodiments of the present invention provide fusion proteins comprising domains that can be used as nucleic acid programmable DNA binding proteins, 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, type II Cas effector protein, type V Cas effector protein, 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 they 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 have been incorporated herein by reference in their entirety.

[0627] One example of a nucleic acid programmable DNA-binding protein with a different PAM specificity than Cas9 is the clustered regularly interspaced short palindromic repeat from Prevotella and Francisella 1 (Cpf1). Similar to Cas9, Cpf1 is also a class 2 CRISPR effector. Cpf1 has been shown to mediate reliable DNA interference, with features different from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA that utilizes T-rich protospacer adjacent motifs (TTN, TTTN, or YTN). In addition, Cpf1 cleaves DNA via staggered DNA double-strand breaks. Of the 16 Cpf1-f family proteins, two enzymes from Acidaminococcus and Lachnospiraceae have been shown to have efficient genome-editing activity in human cells. The Cpf1 protein is known in the art and has been described in the past, for example: Yamano et al., "Crystal structure of Cpf1 in complex with guide RNA and target DNA." Cell (165) 2016, p. 949-962; the entire contents of which are incorporated herein by reference.

[0628] Suitable for use in the present compositions and methods are nuclease-inactive Cpf1 (dCpf1) variants with programmable DNA-binding protein domains using a guide nucleotide sequence. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9, but without the HNH endonuclease domain, and the N-terminus of Cpf1 lacks the α-helical recognition lobe of Cas9. It is shown in Zetsche et al., Cell, 163, 759-771, 2015 (the contents of which have been 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 the nuclease activity of Cpf1. For example: mutations corresponding to D917A, E1006A, or D1255A in Francisella novicida Cpf1 inactivate the nuclease activity of Cpf1. In some embodiments, dCpf1 of the 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 invention.

[0629] 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.

[0630] Wild-type Francisella novicida Cpf1 (D917, E1006, and D1255 are bold and underlined):

[0631]

[0632]

[0633] Francisella novicida Cpf1 D917A (A917, E1006, and D1255 are bold and underlined):

[0634]

[0635] Francisella novicida Cpf1 E1006A (D917, A1006, and D1255 are bold and underlined):

[0636]

[0637]

[0638] Francisella novicida Cpf1 D1255A (D917, E1006, and A1255 are bold and underlined)

[0639]

[0640]

[0641] Francisella novicida Cpf1 D917A / E1006A (A917, A1006, and D1255 are bold and underlined):

[0642]

[0643]

[0644] Francisella novicida Cpf1 D917A / D1255A (A917, E1006, and A1255 are bold and underlined):

[0645]

[0646] Francisella novicida Cpf1 E1006A / D1255A (D917, A1006, and A1255 are bold and underlined):

[0647]

[0648] Francisella novicida Cpf1D917A / E1006A / D1255A (A917, A1006, and A1255 are bold and underlined):

[0649]

[0650]

[0651] In some embodiments, one of the Cas9 domains present in the fusion protein can be replaced by a guide nucleotide sequence programmable DNA-binding protein domain that does not require a PAM sequence.

[0652] 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 amino acid sequence provided herein.

[0653] 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.

[0654] SaCas9 sequence example:

[0655]

[0656] The residue N579 indicated above in underline and bold can be mutated (e.g., to form A579) to produce a SaCas9 nickase.

[0657] SaCas9n sequence example:

[0658]

[0659]

[0660] The above residue A579, which can generate SaCas9 nickase from N579 mutation, is underlined and in bold.

[0661] SaKKH Cas9 Example:

[0662]

[0663]

[0664] The above residue A579, which can generate SaCas9 nickase from N579 mutation, is underlined and in bold. The residues K781, K967, and H1014, which can generate SaKKH Cas9 from E781, N967, and R1014 mutations, are underlined and in italics.

[0665] 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.

[0666] CP5 (with MSP "NGC" PID and "D10A" nickase):

[0667]

[0668]

[0669] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a single effector of a microbial CRISPR-Cas system. Single effectors of a microbial CRISPR-Cas system include, but are not limited to, Cas9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multiple 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 two of the systems, Cas12b / C2c1 and Cas12c / C2c3, contain a RuvC-like endonuclease domain associated with Cpf1. The third system contains an effector with two predicted HEPN RNase domains. The production of mature CRISPR RNA does not rely on tracrRNA, which is different from the CRISPR RNA produced by Cas12b / C2c1. Cas12b / C2c1 relies on both CRISPR RNA and tracrRNA to cleave DNA.

[0670] The crystal structure of Alicyclobaccillus acidoterrastris Cas12b / C2c1 (AacC2c1) has been described in complex with a chimeric single molecule guide RNA (sgRNA). See, for example, Liu et al., "C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism", Mol. Cell, 2017 Jan. 19; 65(2): 310-322, the entire contents of which are incorporated herein by reference. The crystal structure was also reported in Alicyclobaccillus acidoterrastris C2c1 bound to a target DNA as a ternary complex. See, for example, Yang et al., "PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease", Cell, 2016 Dec. 15; 167(7): 1814-1828, the entire contents of which are incorporated herein by reference. The catalytic component configurations of AacC2c1, both with target and non-target DNA strands, have been independently captured and placed in a single RuvC catalytic pocket, with Cas12b / C2c1-mediated cleavage resulting in staggered seven nucleotide breaks in the target DNA. Comparison of the structures of the Cas12b / C2c1 ternary complex with the previously identified Cas9 and Cpf1 counterparts confirms the diversity of mechanisms employed by the CRISPR-Cas9 system.

[0671] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein may be Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, napDNAbp is Cas12b / C2c1 protein. In some embodiments, napDNAbp is Cas12c / C2c3 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% consistent with natural Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, napDNAbp is natural Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the napDNAbp sequences provided herein. It should be understood that Cas12b / C2c1 or Cas12c / C2c3 from other bacterial species may also be used according to the present invention.

[0672] Cas12b / C2c1 ((uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2|C2C1_ALIAGCRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acido-terrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) GN=c2c1 PE=1SV=1) amino acid sequence is as follows:

[0673]

[0674] BhCas12b (Bacillus hisashii) NCBI reference sequence: WP_095142515

[0675]

[0676] In some embodiments, Cas12b is BvCas12B. In some embodiments, Cas12b comprises amino acid substitutions S893R, K846R, and E837G, which are numbered according to the BvCas12b amino acid sequence example provided below.

[0677] BvCas12b (Bacillus sp. V3-13) NCBI reference sequence: WP_101661451.1:

[0678]

[0679] Guide polynucleotide

[0680] In one embodiment, the guide polynucleotide is a guide RNA. The RNA / Cas complex can assist in "guiding" the Cas protein to the target DNA. Cas9 / crRNA / tracrRNA cleaves linear or circular dsDN targets complementary to the spacer in an endonucleolytic manner. The target strand that is not complementary to the crRNA is first cut in an endonucleolytic manner, and then 3′-5′ is trimmed in an exonucleolytic manner. In nature, DNA-binding and cleavage generally require proteins and two RNAs. However, a single guide RNA ("sgRNA" or "gNRA" for short) can be engineered to incorporate both crRNA and tracrRNA into a single RNA species. See, for example: Jinek M. et al., Science 337:816-821 (2012), the entire contents of which have been incorporated herein by reference. Cas9 recognizes short motifs (PAM or protospacer adjacent motifs) in CRISPR repeat sequences to help distinguish between self and non-self. 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, JJ et al., Natl. Acad. Sci. USA 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E. et al., Nature 471:602-607 (2011); and “Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M. et al., Science 337:816-821 (2012), the entire contents of which have been incorporated herein by reference). Cas9 orthologs have been described in various species, including but not limited to: Streptococcus pyogenes and Streptococcus thermophilus.Other suitable Cas9 nucleases and sequences are those of ordinary skill in the art, and include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type IICRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 nuclease has an inactivated (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase.

[0681] In some embodiments, the guide polynucleotide is at least one single guide RNA ("sgRNA" or "gNRA"). In some embodiments, the guide polynucleotide is at least one tracrRNA. In some embodiments, the guide polynucleotide does not require a PAM sequence to guide the polynucleotide programmable DNA-binding domain (e.g., Cas9 or Cpf1) to the target nucleotide sequence.

[0682] The polynucleotide programmable nucleotide binding domain (e.g., CRISPR-derived domain) of the base editor disclosed herein can recognize the target polynucleotide sequence by associating with the guide polynucleotide. The guide polynucleotide (e.g., gRNA) is usually single-stranded and can be programmed to site-specifically bind to the target sequence of the polynucleotide (i.e., via complementary base pairing), thereby directing the base editor that has been linked to the guide nucleic acid to the target sequence. The guide polynucleotide may be DNA. The guide polynucleotide may be RNA. In some embodiments, the guide polynucleotide comprises a natural nucleotide (e.g., adenosine). In some embodiments, the guide polynucleotide comprises a non-natural (or non-natural) nucleotide (e.g., a peptide nucleic acid or a nucleotide analog). In some embodiments, the length of the targeting region of the guide nucleic acid sequence may be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The targeting region of the guide nucleic acid can be between 10-30 nucleotides in length, or between 15-25 nucleotides in length, or between 15-20 nucleotides in length.

[0683] In some embodiments, the guide polynucleotide comprises two or more individual polynucleotides that can interact with each other via, for example, complementary base pairing (e.g., dual guide polynucleotides). For example, a guide polynucleotide can comprise a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). For example, a guide polynucleotide can comprise one or more trans-activating CRISPR RNAs (tracrRNAs).

[0684] In type II CRISPR systems, targeting of nucleic acids by CRISPR proteins (e.g., Cas9) typically requires complementary base pairing between a first RNA molecule (crRNA) containing a sequence that recognizes the target sequence and a second RNA molecule (trRNA) containing a repeating sequence that forms a backbone region to stabilize the guide RNA-CRISPR protein complex. Such dual guide RNA systems can be used as guide polynucleotides to direct the base editors disclosed herein to the target polynucleotide sequence.

[0685] In some embodiments, the base editors provided herein utilize a single guide polynucleotide (e.g., gRNA). In some embodiments, the base editors provided herein utilize dual guide polynucleotides (e.g., dual gRNAs). In some embodiments, the base editors provided herein utilize one or more guide polynucleotides (e.g., multiple gRNAs). In some embodiments, a single guide polynucleotide is used for different base editors described herein. For example, a single guide polynucleotide can be used for an adenosine base editor, or an adenosine base editor and a cytidine base editor, for example, as described in PCT / US19 / 44935.

[0686] In other embodiments, the guide polynucleotide may include both the polynucleotide targeting portion of the nucleic acid and the backbone portion of the nucleic acid in a single molecule (i.e., a single-molecule guide nucleic acid). For example, a single-molecule guide polynucleotide may be a single guide RNA (sgRNA or gRNA). The term guide polynucleotide sequence herein contemplates the use of any single, dual, or multi-molecule nucleic acid that can interact with a base editor and direct to a target polynucleotide sequence.

[0687] Typically, a guide polynucleotide (e.g., a crRNA / trRNA complex or a gRNA) comprises a "polynucleoside-targeting segment" that includes a sequence that can recognize and bind to a target polynucleotide sequence, and a "protein binding segment" that stabilizes the guide polynucleotide within the polynucleotide programmable nucleotide binding domain component of the base editor. In some embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to a DNA polynucleotide, thereby facilitating the editing of bases in the DNA. In other examples, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to an RNA polynucleotide, thereby facilitating the editing of bases in the RNA. A "segment" herein refers to a section or region of a molecule, for example, a contiguous section of nucleotides in a guide polynucleotide. A segment may also refer to a region / segment of a complex, and thus the segment may include a region of more than one molecule. For example, if the guide polynucleotide comprises multiple nucleic acid molecules, the protein binding segment may include all or a portion of multiple separate molecules, for example, hybridization along complementary regions. In some embodiments, a protein-binding segment of a DNA-targeting RNA comprising two separate molecules may comprise (i) base pairs 40-75 of a first RNA molecule of 100 base pairs in length; and (ii) base pairs 10-25 of a second RNA molecule of 50 base pairs in length. Unless otherwise expressly defined in a particular context, the definition of "segment" is not limited to a specific total number of base pairs, is not limited to any specific number of base pairs from a given RNA molecule, is not limited to a specific number of separate molecules within a complex, and may include a region of any total length in an RNA molecule, and may include a region that is complementary to other molecules.

[0688] The guide RNA or guide polynucleotide may include two or more RNAs, for example, CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The guide RNA or guide polynucleotide may sometimes include single-stranded RNA, or a single guide RNA (sgRNA) formed by the fusion of a portion (e.g., a functional portion) of crRNA and tracrRNA. The guide RNA or guide polynucleotide may also be a dual RNA comprising crRNA and tracrRNA. In addition, crRNA can hybridize with target DNA.

[0689] As discussed above, the guide RNA or guide polynucleotide can be an expression product. For example, the DNA encoding the guide RNA can be a vector comprising a sequence encoding the guide RNA. The guide RNA or guide polynucleotide can be transferred into the cell by transfecting the cell with an isolated guide RNA or a plasmid DNA comprising a sequence encoding the guide RNA and a promoter. The guide RNA or guide polynucleotide can also be transferred into the cell by other means, such as by virus-mediated gene delivery.

[0690] The guide RNA or guide polynucleotide can be isolated. For example, the guide RNA can be transfected into a cell or organism in the form of isolated RNA. The guide RNA can be prepared by in vitro transcription using any in vitro transcription system known in the art. The guide RNA can be transferred into the cell in the form of isolated RNA rather than a plasmid containing a sequence encoding the guide RNA.

[0691] The guide RNA or guide polynucleotide may comprise three regions: a first region at the 5' end that may be complementary to a target site in a chromosomal sequence; a second internal region that may form a stem-loop structure, and a third 3' region that may be single-stranded. The first region of each guide RNA may also be different, so that each guide RNA guides the fusion protein to a specific target site. In addition, the second and third regions of each guide RNA in all guide RNAs may be the same.

[0692] The first region of the guide RNA or guide polynucleotide may be complementary to the sequence of the target site in the chromosome sequence, so that the first region of the guide RNA can be base paired with the target site. In some embodiments, the first region of the guide RNA may include 10 or about 10 nucleotides to 25 nucleotides (i.e., 10 nucleotides to 25 nucleotides; or about 10 nucleotides to about 25 nucleotides; or 10 nucleotides to about 25 nucleotides; or about 10 nucleotides to 25 nucleotides) or more. For example: the length of the base pairing region between the first region of the guide RNA and the target site in the chromosome sequence may be or may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides. Sometimes, the length of the first region of the guide RNA may be or may be about 19, 20, or 21 nucleotides.

[0693] The guide RNA or guide polynucleotide can also include a second region that forms a secondary structure. For example, the secondary structure formed by the guide RNA can include a stem (or hairpin) and a loop. The lengths of the loop and stem can vary. For example, the length of the loop can range from 3 or about 3 to 10 nucleotides, and the length of the stem can range from 6 or about 6 to 20 base pairs. The stem can include one or more protrusions containing 1 to 10 or about 10 nucleotides. The total length of the second region group can range from 16 or about 16 to 60 nucleotides. For example, the length of the loop can be 4 or about 4 nucleotides and the stem can be 12 or about 12 base pairs.

[0694] The guide RNA or guide polynucleotide can also include a third region at the 3' end that is substantially single-stranded. For example, the third region is sometimes not complementary to any chromosomal sequence in the cell of interest, and sometimes is not complementary to the rest of the guide RNA. In addition, the length of the third region can vary. The length of the third region can be more than or about 4 nucleotides. For example, the length of the third region can be from 5 or about 5 to 60 nucleotides.

[0695] The guide RNA or guide polynucleotide can target any exon or intron of a gene target. In some embodiments, the guide sequence can target exon 1 or 2 of a gene; in other examples, the guide sequence can target exon 3 or 4 of a gene. The composition can include multiple guide RNAs that all target the same exon, or in some embodiments, the multiple guide RNAs can target different exons. Exons and introns of a gene can be targeted.

[0696] The guide RNA or guide polynucleotide can target a nucleic acid sequence of 20 or about 20 nucleotides. The target nucleic acid can be less than or less than about 20 nucleotides. The length of the target nucleic acid can be at least or at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or any number of nucleotides between 1-100. The length of the target nucleic acid can be at most or at most about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or any number of nucleotides between 1-100. The target nucleic acid sequence can be or can be about 20 bases immediately 5' of the first nucleotide of the PAM. The guide RNA can target a nucleic acid sequence. A target nucleic acid can be at least or at least about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, or 1-100 nucleotides.

[0697] A guide polynucleotide, such as a guide RNA, can refer to a nucleic acid that can hybridize to another nucleic acid, such as a target nucleic acid or a protospacer in the genome of a cell. A guide polynucleotide can be RNA. A guide polynucleotide can be DNA. A guide polynucleotide can be programmed or designed to bind to a nucleic acid in a sequence site-specific manner. A guide polynucleotide can comprise a polynucleotide strand and can be referred to as a single guide polynucleotide. A guide polynucleotide can comprise two polynucleotide strands and can be referred to as a dual guide polynucleotide. A guide RNA can be introduced into a cell as an RNA molecule. For example, an RNA molecule can be transcribed in vitro and / or can be chemically synthesized. RNA can be transcribed from a synthetic DNA molecule, such as: Gene fragment. The guide RNA is then introduced into the cell as an RNA molecule. The guide RNA can also be introduced into the cell as a non-RNA nucleic acid molecule, such as a DNA molecule. For example, the DNA encoding the guide RNA is operably linked to a promoter control sequence to express the guide RNA in the cell of interest. The RNA coding sequence is operably linked to a promoter sequence recognized by RNA polymerase III (Pol III). Plasmid vectors that can be used to express guide RNA include (but are not limited to): px330 vectors and px333 vectors. In some embodiments, the plasmid vector (e.g., px333 vector) may contain at least two DNA sequences encoding guide RNA.

[0698] Methods for selecting, designing, and validating guide polynucleotides, such as guide RNAs, and targeting sequences are described herein and are known to those skilled in the art. For example, to minimize the effect of promiscuity in the deaminase domain (e.g., AID domain) of a nucleobase editor system, the number of residues that may be inadvertently targeted for deamination (e.g., off-target C residues that may remain on ssDNA within the target nucleic acid locus) can be minimized. In addition, software tools can be used to optimize gRNAs corresponding to target nucleic acid sequences, such as to minimize total off-target activity throughout the genome. For example, when using S. pyogenes Cas9 to select each possible targeting domain, all off-target sequences (previously selected PAMs, such as NAG or NGG) can be identified throughout the genome, which contain at most a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base pairs. The first region of the gRNA complementary to the target site can be identified, and all first regions (e.g., crRNA) can be ranked according to their predicted total off-target scores; the top-ranked targeting domains represent those that may have the greatest on-target activity and the least off-target activity. The function of the targeting gRNA candidate can be analyzed using methods known in the art and / or described herein.

[0699] As a non-limiting example, the target DNA hybridization sequence in the crRNA of the guide RNA used by Cas9 can be identified using a DNA sequence search algorithm. gRNA design can be performed using the public tool cas-offinder custom gRNA design software described in Bae S., Park J., & Kim J.-S.Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473-1475 (2014). After calculating its whole genome off-target tendency, the software calculates the score of the guide sequence. Generally, for guide sequences with a length range of 17 to 24, the matching range is considered from perfect matching to 7 mismatches. Once the off-target site is determined by computer calculation, the total score is calculated for each guide sequence, and the web interface is used to summarize the output in a table. In addition to identifying possible target sites adjacent to the PAM sequence, the software can also identify all PAM adjacent sequences that differ from the selected target site by 1, 2, 3 or more nucleotides. The genomic DNA sequence of the target nucleic acid sequence, such as the target gene, can be obtained and screened for repetitive elements using publicly available tools such as the RepeatMasker program. RepeatMasker searches the input DNA sequence for repetitive elements and regions of low complexity. The output is a detailed annotation of the repetitive sequences present in the specified query sequence.

[0700] Following the identification, the first region of the guide RNA (e.g., crRNA) can be ranked based on its distance from the target site, its homology, and the presence of a 5' nucleotide to closely match the relevant PAM sequence (e.g., 5'G identified based on high matching in a human genome containing a relevant PAM (e.g., NGG PAM of Streptococcus pyogenes (S. pyogenes), NNGRRT or NNGRRV PAM of Staphylococcus aureus (S. aureus)). As used herein, orthog...

Claims

1. A method of editing a beta globin (HBB) polynucleotide containing a single nucleotide polymorphism (SNP) related to sickle cell disease in vitro, the method comprising contacting a beta globin polynucleotide with one or more guide RNA and a fusion protein comprising a Cas9 polynucleotide programmable DNA binding domain and adenosine deaminase domain, in, The adenosine deaminase domain consists of the following amino acid sequence or fragments thereof that lack only N-terminal methionine: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, and have a single modification composed of Y147T or Y147R or have the following single group modifications: Y123H, Y147R and Q154R; Y147R, Q154R and T166R; Y147T and Q154R; Y147T and Q154S; I76Y, Y123H, Y147R and Q154R; V82S and Q154R; or I76Y, V82S, Y123H, Y147R and Q154R; Among them, the above modification is carried out for the adenosine deaminase domain of the amino acid sequence shown in MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKAQSSTD; Wherein, the guide RNA mediates the binding of the adenosine deaminase domain and a target site in the HBB polynucleotide to cause modification of the A·T to G·C of the SNP related to sickle cell disease, wherein the A·T to G·C of the SNP related to sickle cell disease changes the valine at amino acid position 7 in the HBB polypeptide encoded by the HBB polynucleotide to alanine, and wherein the HBB polypeptide consists of the following amino acid sequences:

2. A method of editing a beta globin (HBB) polynucleotide containing a single nucleotide polymorphism (SNP) related to sickle cell disease in vitro, the method comprising contacting a beta globin polynucleotide with one or more guide RNA and a fusion protein comprising a Cas9 polynucleotide programmable DNA binding domain of the following sequence: EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKDLIIKLPKYSLF ELENGRKRMLASAKFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSGGSGG SGGSGGMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKE IFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTK VKYVTEGMRKPAFLSGEQKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEGADKRTADGSEFESPKKKRKV, and adenosine deaminase domain, in, The adenosine deaminase domain consists of the following amino acid sequence or fragments thereof that lack only N-terminal methionine: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, and have a single modification composed of Y147T or Y147R or have the following single group modifications: Y123H, Y147R and Q154R; Y147R, Q154R and T166R; Y147T and Q154R; Y147T and Q154S; I76Y, Y123H, Y147R and Q154R; V82S and Q154R; or I76Y, V82S, Y123H, Y147R and Q154R; Among them, the above modification is carried out for the adenosine deaminase domain of the amino acid sequence shown in MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKAQSSTD; wherein the guide RNA mediates the binding of the adenosine deaminase domain and the target site in the HBB polynucleotide to cause modification of the A·T to G·C of the SNP related to sickle cell disease, wherein the A·T to G·C of the SNP related to sickle cell disease changes the valine at amino acid position 7 in the HBB polypeptide encoded by the HBB polynucleotide to alanine, and wherein the HBB polypeptide consists of the following amino acid sequence: MVHLTPVEKSAVTALWGKVN VDEVGGEALG RLLVVYPWTQ RFFESFGDLS TPDAVMGNPK VKAHGKKVLG AFSDGLAHLDNLKGTFATLS ELHCDKLHVD PENFRLLGNV LVCVLAHHFG KEFTPPVQAA YQKVVAGVAN ALAHKYH.

3. The method according to claim 1 or 2, in, The programmable DNA binding domain of the Cas9 polynucleotide is Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9) or Streptococcus pyogenes Cas9 (SpCas9).

4. The method of claim 3, in, The SpCas9 has specificity for the proto-spaced adjacent motif composed of the following nucleic acid sequences: 5'-NGC-3'.

5. The method according to claim 1 or 2, in, The Cas9 polynucleotide programmable DNA binding domain lacks nuclease activity or is a nickase.

6. The method of claim 5, in, The nickase comprises D10A amino acid substitution.

7. The method of claim 1 or 2, in, The fusion protein also comprises a zinc finger domain.

8. The method of claim 7, in, The zinc finger domain comprises the amino acid sequences RNEHLEV, QSTTLKR and RTEHLAR or the amino acid sequences RGEHLRQ, QSGTLKR and RNDKLVP.

9. The method of claim 1 or 2, in, The adenosine deaminase domain can deaminate adenine in deoxyribonucleic acid.

10. The method of claim 1 or 2, in, The one or more guide RNAs include CRISPR RNA (crRNA) and trans-encoded small RNA, wherein the crRNA comprises a nucleic acid sequence complementary to the HBB nucleic acid sequence comprising the SNP involved in sickle cell disease.

11. The method of claim 1 or 2, in, The fusion protein is complexed with the one or more guide RNAs, and wherein the spacer of the one or more guide RNAs comprises a nucleic acid sequence complementary to the HBB nucleic acid sequence comprising the SNP involved in sickle cell disease.

12. A base editing system comprising a fusion protein according to any one of claims 1 to 11 and a guide RNA (gRNA) comprising a spacer having a nucleic acid sequence selected from any of the following: CUUCUCCACAGGAGUCAGAU; ACUUCUCCACAGGAGUCAGAU; GACUUCUCCACAGGAGUCAGA; or ACUUCUCCACAGGAGUCAGG, in, The guide RNA mediates the binding of the adenosine deaminase domain and a target site in the HBB polynucleotide to cause modification of the A·T to G·C of the SNP related to sickle cell disease, wherein the A·T to G·C of the SNP related to sickle cell disease changes the valine at amino acid position 7 in the HBB polypeptide encoded by the HBB polynucleotide to alanine, and wherein the HBB polypeptide consists of the following amino acid sequences:

13. The base editing system of claim 12, wherein the backbone of the gRNA comprises the following nucleic acid sequence: GUUUUUUGUACUCAAGAAUUUAAGUAACUUAAAUC UUGCAGAAGCUACAAAGAUAAGGCUUCAUCAUGCCGAAAUCAACCCUGUCAUUUAUGGCA GGGUG.

14. The base editing system of claim 12, in, The gRNA comprises CUUCUCCACAGGAGUCAGAUGUUUUUGUACUCAAGAUUUAAGUAACUGUACAACGAAACUUACUUACUUAAAUCUUUGCAGAAGCUACAAAGAUAAAUCUUUGCAGAAGCUACAAAGAUAAGGCUUCAUCAUGCCGAAAUCAACCCUGUCAUUUUAUGGCAGGUG; ACUUCUCCACAGGAGUCAGAUGUUUUUGUACUCAAGAUUUAAGUAACUGUACAA CGAAACUUUACUUAAAUCUUUGCAGAAGCUACAAAGAUAAAGGCUUCAUCAAAAGAUAAGGCUUCAUCAUGCCGAA AUCAACCCCUGUCAUUUUAUGGCAGGGUG; GACUUCUCCACAGGAGUCAGAUGUUUUUGUACUCAAGAUUUAAGUAACUGUACAACUUAAAUCUUUAAAUCUUCAGAAGCUACAAAGAUAAAUCUUUGCAGAAGCUACAAAGAUAAGGCUUCAUCAUCAAAGAUAAGGCUUCAUCAUCCGAAAAUCAACCCCUGUCAUUUAUGGCAGGGUG; gsascsUUCUCCACAGGAGUCAGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAAAAAA GGCUAGUCCGUUAUCAACUUGAAAAAAAAAAGUCCGUUAUCAACUUGAAAAAAAAGUGCACCGAGUCGGUGCUsususu; ascsusUCUCCACAGGAGUCAGGGUUUAGAGCUAGAAAUAGCAAGUUAAAAAAG GCUAGUCCGUUAUCAACUUGAAAAAAAAGUGCACCGAGUCGGUGCUsususu; and csususCUCCACAGGAGUCAGGGUUUAGAGCUAGAAAUAGCAAGUUAAAAAAGG CUAGUCCGUUAUCAACUUGAAAAAAAGUGCACCGAGUCGGUGCUsususu nucleic acid sequence, where "s" represents a phosphorothioate, and "g", "a", "c" and "u" represent G, A, C or U nucleotides methylated by 2'-O- .

15. A cell whose production is introduced in hematopoietic stem cells, hematopoietic progenitor cells, pro-red blood cells or red blood cell blasts: base editor or polynucleotide encoding the base editor, in, The base editor comprises a Cas9 polynucleotide programmable DNA binding domain and an adenosine deaminase domain according to any one of claims 1 to 4; and One or more guide RNAs mediate the binding of the base editor and the target site in the HBB polynucleotide to perform modifications of the A·T to G·C of the SNP related to sickle cell disease, wherein the modifications of the A·T to G·C of the SNP related to sickle cell disease change the valine at amino acid position 7 in the HBB polypeptide encoded by the HBB polynucleotide to alanine to produce the cells, and wherein the HBB polypeptide consists of the following amino acid sequences:

16. The cell of claim 15, in, The cells produced are hematopoietic stem cells, hematopoietic progenitor cells, pro-erythrocytes, red blood cells, reticulocytes or red blood cells.

17. The cell of claim 15 or 16, in, The hematopoietic stem cells are CD34 + cell.

18. The cell of claim 15 or 16, in, The cells are from an individual suffering from sickle cell disease.

19. The cell of claim 15 or 16, in, The cells are mammalian cells.

20. The cell of claim 15 or 16, in, The programmable DNA binding domain of the Cas9 polynucleotide is Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9) or Streptococcus pyogenes Cas9 (SpCas9).

21. The cell of claim 15 or 16, in, The programmable DNA binding domain of the Cas9 polynucleotide is SpCas9.

22. The cell of claim 21, in, The SpCas9 has specificity for the proto-spaced adjacent motif composed of the following nucleic acid sequences: 5'-NGC-3'.

23. The cell of claim 15 or 16, in, The Cas9 polynucleotide programmable DNA binding domain lacks nuclease activity or is a nickase.

24. The cell of claim 23, in, The nickase comprises D10A amino acid substitution.

25. The cell of claim 15 or 16, in, The base editor also includes a zinc finger domain.

26. The cell of claim 25, in, The zinc finger domain comprises the amino acid sequence RNEHLEV, QSTTLKR, and RTEHLAR or the amino acid sequence RGEHLRQ, QSGTLKR and RNDKLVP.

27. The cell of claim 15 or 16, in, The adenosine deaminase domain can deaminate adenine in deoxyribonucleic acid.

28. The cell of claim 15 or 16, in, The one or more guide RNAs include CRISPR RNA (crRNA) and trans-encoded small RNA, wherein the crRNA comprises a nucleic acid sequence complementary to the HBB nucleic acid sequence comprising the SNP involved in sickle cell disease.

29. The cell of claim 15 or 16, in, The base editor and the one or more guide RNAs form a complex in the cell.

30. The cell of claim 29, in, The base editor is complex with the one or more guide RNAs, and wherein the spacer of the one or more guide RNAs comprises a nucleic acid sequence complementary to the HBB nucleic acid sequence comprising the SNP involved in sickle cell disease.

31. Use of a cell according to any one of claims 15 to 30 for the preparation of a medicament for the treatment of sickle cell disease in an individual.

32. The use according to claim 31, in, The cell is an autologous cell of the individual.

33. The use of claim 31, in, The cell is an allogenic cell of the individual.

34. A method of making red blood cells, include: (a) Introduction in red blood cell progenitor cells containing SNPs related to sickle cell disease, a base editor, or a polynucleotide encoding the base editor, wherein the base editor comprises a programmable DNA binding domain of Cas9 polynucleotide and an adenosine deaminase domain according to any one of claims 1 to 11; and One or more guide RNAs, wherein the one or more guide RNAs mediate the binding of the base editor and the target site in the HBB polynucleotide to cause modification of A·T to G·C of SNPs related to sickle cell disease, wherein the modification of A·T to G·C of the SNPs related to sickle cell disease changes valine at amino acid position 7 in the HBB polypeptide encoded by the HBB polynucleotide to alanine, and wherein the HBB polypeptide consists of the following amino acid sequences: MVHLTPVEKS AVTALWGKVN VDEVGGEALG RLLVVYPWTQ RFFESFGDLS TPDAVMGNPKVKAHGKKVLG AFSDGLAHLD NLKGTFATLS ELHCDKLHVD PENFRLLGNV LVCVLAHHFG KEFTPPVQAAYQKVVAGVAN ALAHKYH; and (b) Differentiation into red blood cells from the red blood cell group.

35. The method of claim 34, in, The red blood cell progenitor cells are CD34 + cell.

36. The method of claim 34, in, The erythrocytic progenitor cells are obtained from individuals suffering from sickle cell disease.

37. The method of claim 34, in, The red blood cell progenitor cells are mammalian cells.

38. The method of claim 34, in, The programmable DNA binding domain of the Cas9 polynucleotide is Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9) or Streptococcus pyogenes Cas9 (SpCas9).

39. The method of claim 38, in, The SpCas9 has specificity for the proto-spaced adjacent motif composed of the following nucleic acid sequences: 5'-NGC-3'.

40. The method of claim 34, in, The Cas9 polynucleotide programmable DNA binding domain lacks nuclease activity or is a nickase.

41. The method of claim 40, in, The nickase comprises D10A amino acid substitution.

42. The method of claim 34, in, The base editor also includes a zinc finger domain.

43. The method of claim 42, in, The zinc finger domain comprises the amino acid sequence RNEHLEV, QSTTLKR, and RTEHLAR or the amino acid sequence RGEHLRQ, QSGTLKR and RNDKLVP.

44. The method of claim 34, in, The one or more guide RNAs include CRISPR RNA (crRNA) and trans-encoded small RNA, wherein the crRNA comprises a nucleic acid sequence complementary to the HBB nucleic acid sequence comprising the SNP related to sickle cell disease.

45. The method of claim 34, in, The base editor and the one or more guide RNAs form a complex in the cell.

46. ​​The method of claim 45, in, The base editor is complex with one or more guide RNAs, and wherein the spacer of the one or more guide RNAs comprises a nucleic acid sequence complementary to the HBB nucleic acid sequence comprising the SNP involved in sickle cell disease.

47. Use of a base editor for the preparation of a drug for the treatment of sickle cell disease (SCD) in an individual, in, The base editor includes an adenosine deaminase domain inserted into the Cas9 polypeptide or a polynucleotide encoding the base editor; and one or more guide RNAs mediating the binding of the base editor and the target site in the HBB polynucleotide, resulting in modifications of A·T to G·C related to single nucleotide polymorphism (SNP) of SCD, wherein the modifications of A·T to G·C for the SNP related to SCD change valine at amino acid position 7 in the HBB polypeptide encoded by the HBB polynucleotide to alanine, and wherein the HBB polypeptide consists of the following amino acid sequences: MVHLTPVEKS AVTALWGKVN VDEVGGEALG RLLVVYPWTQ RFFESFGDLS TPDAVMGNPKVKAHGKKVLG AFSDGLAHLD NLKGTFATLS ELHCDKLHVD PENFRLLGNV LVCVLAHHFG KEFTPPVQAAYQKVVAGVAN ALAHKYH to treat SCD of the individual; Among them, the adenosine deaminase domain consists of the following amino acid sequence or fragments thereof that lack only N-terminal methionine: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, and have a single modification composed of Y147T or Y147R or have the following single group modifications: Y123H, Y147R and Q154R; Y147R, Q154R and T166R; Y147T and Q154R; Y147T and Q154S; I76Y, Y123H, Y147R and Q154R; V82S and Q154R; or I76Y, V82S, Y123H, Y147R and Q154R; Among them, the above modification is carried out against the adenosine deaminase domain of the amino acid sequence shown in MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD.

48. Use of a base editor or polynucleotide encoding the base editor in the preparation of a drug for the treatment of sickle cell disease (SCD) in an individual, in, The base editor comprises an adenosine deaminase domain inserted into the Cas9 polypeptide; and one or more guide RNAs mediating the binding of the base editor to the target site in the HBB polynucleotide, resulting in modifications of A·T to G·C of SNPs of SCD, wherein modifications of A·T to G·C of SNPs of sickle cell disease change valine at amino acid position 7 in the HBB polypeptide encoded by the HBB polynucleotide to alanine, and wherein the HBB polypeptide consists of the following amino acid sequences: MVHLTPVEKS AVTALWGKVN VDEVGGEALG RLLVVYPWTQ RFFESFGDLS TPDAVMGNPKVKAHGKKVLG AFSDGLAHLD NLKGTFATLS ELHCDKLHVD PENFRLLGNV LVCVLAHHFG KEFTPPVQAAYQKVVAGVAN ALAHKYH to treat SCD of the individual; Among them, the adenosine deaminase domain consists of the following amino acid sequence or fragments thereof that lack only N-terminal methionine: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, and have a single modification composed of Y147T or Y147R or have the following single group modifications: Y123H, Y147R and Q154R; Y147R, Q154R and T166R; Y147T and Q154R; Y147T and Q154S; I76Y, Y123H, Y147R and Q154R; V82S and Q154R; or I76Y, V82S, Y123H, Y147R and Q154R; Among them, the above modification is carried out for the adenosine deaminase domain of the amino acid sequence shown in MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKAQSSTD.

49. Use according to claim 47 or 48, in, The adenosine deaminase domain contained in the base editor is a single adenosine deaminase domain.

50. Use according to claim 47 or 48, in, The adenosine deaminase domain is flanked by the N-terminal fragment and the C-terminal fragment of the Cas9 polypeptide.

51. The use of claim 50, in, The base editor contains the structural formula NH 2 -[N-terminal fragment of the Cas9 polypeptide]-[adenosine deaminase domain]-[C-terminal fragment of the Cas9 polypeptide]-COOH, wherein the "]-[" in each example is a linker as desired.

52. Use according to claim 47 or 48, in, The deamylation of SNPs related to SCD relieves symptoms of sickle cell disease.

53. Use according to claim 47 or 48, in, The modification of A·T to G·C is 1 to 20 nucleobases away from the original spaced motif.

54. The use of claim 53, in, The SNP is 2 to 12 nucleobases upstream of the PAM sequence.

55. The use of claim 50, in, The N-terminal fragment or the C-terminal fragment of the Cas9 polypeptide binds to the HBB polynucleotide.

56. The use of claim 55, in, 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.

57. Use according to claim 47 or 48, in, The Cas9 polypeptide comprises partial or complete deletion within the RuvC domain or within the HNH domain, and wherein the deaminase is inserted in a partial or complete deletion position of the Cas9 polypeptide.

58. Use according to claim 47 or 48, in, The Cas9 polypeptide is Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9) or Streptococcus thermophilus 1Cas9 (St1Cas9).

59. Use according to claim 47 or 48, in, The Cas9 polypeptide comprises the following Cas9 reference sequence: MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQ LPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSE QEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKDLIIK LPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD.

60. The use of claim 59, in, The Cas9 polypeptide comprises the deletion of amino acids 1017 to 1069 numbered in the Cas9 polypeptide reference sequence; The Cas9 polypeptide comprises a deletion of amino acids 792 to 872 numbered in the Cas9 polypeptide reference sequence; or The Cas9 polypeptide comprises a deletion of amino acids 792 to 906 numbered in the Cas9 polypeptide reference sequence.

61. Use according to claim 47 or 48, in, The adenosine deaminase domain is inserted into the flexible loop of the Cas9 polypeptide.

62. The use of claim 61, in, The flexible loop comprises a region selected from the group consisting of amino acid residues numbered positions 530 to 537, 569 to 579, 686 to 691, 768 to 793, 943 to 947, 1002 to 1040, 1052 to 1077, 1232 to 1248 and 1298 to 1300 in the Cas9 polypeptide reference sequence.

63. The use of claim 59, in, The deaminase domain is inserted between the numbered amino acid positions 768 to 769, 791 to 792, 792 to 793, 1015 to 1016, 1022 to 1023, 1026 to 1027, 1029 to 1030, 1040 to 1041, 1052 to 1053, 1054 to 1055, 1067 to 1068, 1068 to 1069, 1247 to 1248 or 1248 to 1249 in the Cas9 polypeptide reference sequence.

64. The use of claim 59, in, The deaminase domain is inserted between the numbered amino acid positions 768 to 769, 792 to 793, 1022 to 1023, 1026 to 1027, 1040 to 1041, 1068 to 1069, or 1247 to 1248 in the Cas9 polypeptide reference sequence.

65. The use of claim 59, in, The deaminase domain is inserted between the numbered amino acid positions 1016 to 1017, 1023 to 1024, 1029 to 1030, 1040 to 1041, 1069 to 1070, or 1247 to 1248 in the Cas9 polypeptide reference sequence.

66. The use of claim 59, in, The adenosine deaminase domain is flanked by an N-terminal fragment and a C-terminal fragment of the Cas9 polypeptide, and wherein the N-terminal fragment comprises amino acid residues 1 to 529, 538 to 568, 580 to 685, 692 to 942, 948 to 1001, 1026 to 1051, 1078 to 1231 and / or 1248 to 1297 of the Cas9 reference sequence.

67. The use of claim 59, in, The adenosine deaminase domain is flanked by an N-terminal fragment and a C-terminal fragment of the Cas9 polypeptide, and wherein the C-terminal fragment comprises amino acid residues 1301 to 1368, 1248 to 1297, 1078 to 1231, 1026 to 1051, 948 to 1001, 692 to 942, 580 to 685 and / or 538 to 568 of the Cas9 reference sequence.

68. Use according to claim 47 or 48, in, The Cas9 polypeptide is a nick enzyme, or wherein the Cas9 polypeptide lacks nuclease activity.

69. Use according to claim 47 or 48, in, The Cas9 polypeptide is a SpCas9 polypeptide.

70. Use according to claim 47 or 48, in, The guide RNA comprises CRISPR RNA (crRNA) and transactivated crRNA.

71. Use according to claim 47 or 48, in, The individual is a mammal.

72. A pharmaceutical composition comprising a base editing system comprising a fusion protein according to any one of claims 1 to 11 and the one or more guide RNAs, and a pharmaceutically acceptable carrier, vehicle or excipient, in, The one or more guide RNA mediates the binding of the base editor and the target site in the HBB polynucleotide, resulting in the modification of A·T to G·C of SNPs related to sickle cell disease, wherein the modification of A·T to G·C of the SNPs related to sickle cell disease changes valine at amino acid position 7 in the HBB polypeptide encoded by the HBB polynucleotide to alanine, and wherein the HBB polypeptide consists of the following amino acid sequences:

73. The pharmaceutical composition of claim 72, in, The spacer of the one or more guide RNAs (gRNAs) has a nucleic acid sequence selected from any of the following: CUUCUCCACAGGAGUCAGAU; ACUUCUCCACAGGAGUCAGAU; GACUUCUCCACAGGAGUCAGAU; or ACUUCUCCACAGGAGUCAGG.

74. The pharmaceutical composition of claim 73, in, The backbone of the one or more gRNAs comprises the following nucleic acid sequence GUUUUUGUACUGUACAACGAAACUUUACUUAAAUCUUUGCAGAAGCUACAAAGAUAAGGCUUCAUCAUGCCGAAAUCAACCCUGUCAUUUAUGGCAGGGUG.

75. The pharmaceutical composition of claim 73, in, The one or more gRNAs include CUUCUCCACAGGAGUCAGAUGUUUUUGUACUCAAGAUUUAAGUAACUGUACAACGAAACUUACUUACUUAAAUCUUUGCAGAAGCUACAAAGAUAAAUCUUUGCAGAAGCUACAAAGAUAAGGCUUCAUCAUGCCGAAAUCAACCCUGUCAUUUUAUGGCAGGUG; ACUUCUCCACAGGAGUCAGAUGUUUUUGUACUCAAGAUUUAAGUAACUGUACAA CGAAACUUUACUUAAAUCUUUGCAGAAGCUACAAAGAUAAAGGCUUCAUCAAAAGAUAAGGCUUCAUCAUGCCGAA AUCAACCCCUGUCAUUUUAUGGCAGGGUG; GACUUCUCCACAGGAGUCAGAUGUUUUUGUACUCAAGAUUUAAGUAACUGUACAACUUAAAUCUUUAAAUCUUCAGAAGCUACAAAGAUAAAUCUUUGCAGAAGCUACAAAGAUAAGGCUUCAUCAUCAAAGAUAAGGCUUCAUCAUCCGAAAAUCAACCCCUGUCAUUUAUGGCAGGGUG; gsascsUUCUCCACAGGAGUCAGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAAAAAA GGCUAGUCCGUUAUCAACUUGAAAAAAAAAAGUCCGUUAUCAACUUGAAAAAAAAGUGCACCGAGUCGGUGCUsususu; ascsusUCUCCACAGGAGUCAGGGUUUAGAGCUAGAAAUAGCAAGUUAAAAAAG GCUAGUCCGUUAUCAACUUGAAAAAAAAGUGCACCGAGUCGGUGCUsususu; and csususCUCCACAGGAGUCAGGGUUUAGAGCUAGAAAUAGCAAGUUAAAAAAGG CUAGUCCGUUAUCAACUUGAAAAAAAGUGCACCGAGUCGGUGCUsususu nucleic acid sequence, where "s" represents a phosphorothioate, and "g", "a", "c" and "u" represent G, A, C or U nucleotides methylated by 2'-O- .

76. A pharmaceutical composition comprising a base editor or a polynucleotide encoding a base editor, in, The base editor comprises a programmable DNA binding domain of Cas9 polynucleotide and an adenosine deaminase domain according to any one of claims 1 to 4; and one or more guide RNAs mediating the binding of the base editor and a target site in the HBB polynucleotide to perform the A·T to G·C modification of the SNP related to sickle cell disease, and a pharmaceutically acceptable vector, vehicle or excipient, wherein the A·T to G·C modification of the SNP related to sickle cell disease changes the valine at amino acid position 7 in the HBB polypeptide encoded by the HBB polynucleotide to alanine, and wherein the HBB polypeptide consists of the following amino acid sequences:

77. A pharmaceutical composition comprising a cell according to any one of claims 15 to 30, and a pharmaceutically acceptable carrier, vehicle or excipient.

78. A set comprising a base editing system comprising a fusion protein according to any one of claims 1 to 11 and the one or more guide RNAs.

79. The set of claim 78, wherein the spacers of the one or more guide RNAs have a nucleic acid sequence selected from any of the following: CUUCUCCACAGGAGUCAGAU; ACUUCUCCACAGGAGUCAGAU; GACUUCUCCACAGGAGUCAGAU; or ACUUCUCCACAGGAGUCAGG.

80. A set comprising a base editor or a polynucleotide encoding the base editor, in, The base editor comprises a programmable DNA binding domain of Cas9 polynucleotide and an adenosine deaminase domain according to any one of claims 1 to 4; and one or more guide RNAs mediating the binding of the base editor and a target site in the HBB polynucleotide to cause modification of the A·T to G·C of the SNP related to sickle cell disease, wherein the A·T to G·C of the SNP related to sickle cell disease changes the valine at amino acid position 7 in the HBB polypeptide encoded by the HBB polynucleotide to alanine, and wherein the HBB polypeptide consists of the following amino acid sequences: MVHLTPVEKS AVTALWGKVN VDEVGGEALG RLLVVYPWTQ RFFESFGDLS TPDAVMGNPKVKAHGKKVLG AFSDGLAHLD NLKGTFATLS ELHCDKLHVD PENFRLLGNV LVCVLAHHFG KEFTPPVQAAYQKVVAGVAN ALAHKYH.

81. A set comprising the cells according to any one of claims 15 to 30.

82. The set according to any of claims 78 to 81, further comprising a packaging insert with instructions for use.

83. A base editor system comprising a base editor comprising a Cas9 polynucleotide programmable DNA binding domain and at least one adenosine deaminase domain, the adenosine deaminase domain consisting of the following amino acid sequence or fragments thereof lacking only N-terminal methionine Place Composition: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, and have a single modification composed of Y147T or Y147R or have the following single group modifications: Y123H, Y147R and Q154R; Y147R, Q154R and T166R; Y147T and Q154R; Y147T and Q154S; I76Y, Y123H, Y147R and Q154R; V82S and Q154R; or I76Y, V82S, Y123H, Y147R and Q154R; in, The above modification is performed for adenosine deaminase of the amino acid sequence shown in MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKAQSSTD; and guide RNA, wherein the guide RNA mediates the binding of the base editor and the target site in the HBB polynucleotide to cause modification of A·T to G·C of SNPs related to sickle cell disease (SCD), wherein modification of A·T to G·C of the SNPs related to sickle cell disease changes valine at amino acid position 7 in the HBB polypeptide encoded by the HBB polynucleotide to alanine, and wherein the HBB polypeptide is from the following amino acid sequence composition:

84. The base editor system of claim 83, in, The programmable DNA binding domain of the Cas9 polynucleotide is Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9) or Streptococcus pyogenes Cas9 (SpCas9).

85. The base editor system of claim 84, in, The programmable DNA binding domain of the Cas9 polynucleotide is Cas9 which lacks nuclease activity.

86. The base editor system of claim 84, in, The programmable DNA binding domain of the Cas9 polynucleotide is Cas9 nickase.

87. A base editor system comprising one or more guide RNAs and a fusion protein comprising a Cas9 polynucleotide programmable DNA binding domain comprising the following sequences: EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKDLIIKLPKYSLFELEN GRKRMLASAKFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSGGSGGSGGSGGMD KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAHMIKFRGHFLIEGDLNPDNSDVDKL FIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGS PAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEGADKRTADGSEFESPKKKRKV, and the adenosine deaminase domain, which is composed of the following amino acid sequence or fragments thereof that lack only N-terminal methionine. composition: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, and have a single modification composed of Y147T or Y147R or have the following single group modifications: Y123H, Y147R and Q154R; Y147R, Q154R and T166R; Y147T and Q154R; Y147T and Q154S; I76Y, Y123H, Y147R and Q154R; V82S and Q154R; or I76Y, V82S, Y123H, Y147R and Q154R; Among them, the above modification is carried out for the adenosine deaminase domain of the amino acid sequence shown in MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD; wherein the one or more guide RNA mediates the binding of the base editor and the target site in the HBB polynucleotide to cause modification of A·T to G·C of SNP related to sickle cell disease, wherein the modification of A·T to G·C of the SNP related to sickle cell disease changes valine at amino acid position 7 in the HBB polypeptide encoded by the HBB polynucleotide to alanine, and wherein the HBB polypeptide consists of the following amino acid sequences:

88. A cell comprising the base editor system according to claim 83 or 87.

89. The cell of claim 88, in, The cells are human cells.

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