Compositions and methods for treating alpha 1-antitrypsin deficiency

Abstract

The invention features compositions and methods for editing deleterious mutations associated with alpha-1 antitrypsin (A1AT) deficiency. In certain embodiments, the invention provides methods for correcting mutations in A1AT polynucleotides using an adenosine deaminase base editor (i.e., ABE8) with unprecedented efficiency levels.

Description

[0001] Cross-references to related applications

[0002] This application is an international PCT application that claims priority to U.S. Provisional Application No. 62 / 805,238, filed February 13, 2019; U.S. Provisional Application No. 62 / 805,271, filed February 13, 2019; U.S. Provisional Application No. 62 / 852,224, filed May 23, 2019; U.S. Provisional Application No. 62 / 852,228, filed May 23, 2019; U.S. Provisional Application No. 62 / 931,722, filed November 6, 2019; U.S. Provisional Application No. 62 / 941,569, filed November 27, 2019; and U.S. Provisional Application No. 62 / 966,526, filed January 27, 2020, the contents of which are incorporated herein by reference.

[0003] Incorporated by reference

[0004] All publications, patents, and patent applications mentioned herein are incorporated herein by reference as if each individual publication, patent, and patent application were expressly and independently indicated to be incorporated by reference. Unless otherwise stated, all publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety. Technical Field

[0005] This application relates to compositions and methods for editing harmful mutations associated with α-1 antitrypsin (A1AT) deficiency, and in particular to adenosine deaminase base editors and methods for correcting mutations in A1AT polynucleotides. Background Technology

[0006] In healthy individuals, alpha-1-antitrypsin (A1AT) functions as a protease inhibitor, produced by hepatocytes in the liver and secreted into the systemic circulation. It is a particularly effective inhibitor of neutrophil elastase, thus protecting tissues and organs, such as the lungs, from elastin degradation. In patients with alpha-1-antitrypsin deficiency (A1AD), mutations in the gene encoding A1AD result in reduced protein production. Consequently, elastin in the lungs is more easily degraded by neutrophil elastase, and over time, this loss of lung elasticity develops into chronic obstructive pulmonary disease (COPD).

[0007] The most common pathogenic A1AT variant is a mutation from guanine to adenine, resulting in the substitution of lysine for glutamic acid at amino acid 342. This substitution causes misfolding and polymerization of the protein in hepatocytes, and the toxic aggregates can ultimately lead to liver damage and cirrhosis. While liver toxicity can be addressed with gene knockout (CRISPR / ZFN / TALEN) or gene attenuation (siRNA), neither approach addresses lung pathology. Although lung pathology can be addressed with protein replacement therapy, this therapy does not resolve liver toxicity. Gene therapy also cannot adequately address A1AT gene defects. Because the livers of A1AD patients already carry a significant disease burden due to endogenous A1AT, gene therapy that increases hepatic A1AT may be counterproductive.

[0008] Therefore, there is a need for a treatment method that addresses both lung pathology and liver toxicity in patients with A1AD. Summary of the Invention

[0009] As described below, the present invention is characterized by compositions and methods for editing harmful mutations associated with A1-antitrypsin deficiency (A1AD). In a particular embodiment, the present invention provides a method for treating A1AD using a modified adenosine deaminase called “ABE8”, which has unprecedented levels of efficiency and specificity (e.g., >60-70%) to correct mutations associated with A1AD.

[0010] On one hand, the present invention provides a method for editing an α1-antitrypsin polynucleotide containing a single nucleotide polymorphism (SNP) associated with α1-antitrypsin deficiency, the method involving contacting the polynucleotide with one or more guide RNAs and a base editor containing a polynucleotide programmable DNA-binding domain and at least one base editor domain, said base editor domain being an adenosine deaminase variant containing a variation at amino acid position 82 or 166 of the following sequence:

[0011] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, wherein the guide RNA targets the base editor to cause changes in SNPs associated with α1-antitrypsin deficiency.

[0012] On the other hand, the present invention provides a method for editing α1-antitrypsin polynucleotides containing a single nucleotide polymorphism (SNP) associated with α1-antitrypsin deficiency, the method involving contacting the α1-antitrypsin polynucleotide with one or more guide RNAs and a fusion protein containing a polynucleotide programmable DNA-binding domain, the domain containing the following sequence:

[0013] The bolded sequences refer to sequences derived from Cas9, the italicized sequences represent linker sequences, and the underlined sequences represent binomial nuclear localization sequences, as well as at least one base editor domain containing an adenosine deaminase variant, wherein the variant contains a variation at amino acid position 82 or 166 of the following sequence:

[0014] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD.

[0015] On the other hand, the present invention provides a base editing system comprising any of the foregoing fusion proteins and a guide RNA comprising a sequence of nucleic acids selected from the following:

[0016] 5'-ACCAUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAUAAGGCUAGUC CGUUAUCAAC UUGAAAAAAGU GGCACCGAGU CGGUGCUUUU-3'

[0017] 5'-CCAUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUCCGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU-3'

[0018] 5'-CAUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUCCGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU-3'

[0019] 5'-AUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUCCGUUAUCAAC UUGAAAAAAGU GGCACCGAGU CGGUGCUUUU-3'

[0020] 5'-UCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUCCGUUAUCAAC UUGAAAAAAGU GGCACCGAGU CGGUGCUUUU-3'

[0021] 5'-CGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUCCGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU-3'

[0022] On the other hand, a cell is produced by introducing the following into the cell or its progenitor cell:

[0023] The base editor, the polynucleotide encoding the base editor, wherein the base editor contains a polynucleotide programmable DNA-binding domain and an adenosine deaminase domain as described in any of the foregoing aspects; and one or more guide polynucleotides that target the base editor to induce an A·T to G·C change in SNPs associated with α1-antitrypsin deficiency. In one embodiment, the resulting cells are hepatocytes or their progenitor cells. In another embodiment, the cells are derived from a subject with α1-antitrypsin deficiency. In yet another embodiment, the cells are mammalian cells or human cells.

[0024] In various embodiments of the above, the gRNA further contains the nucleic acid sequence 5'-GUUUUAGAGCUAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU-3.

[0025] On the other hand, the present invention provides a method for treating α1-antitrypsin deficiency in a subject, involving the administration of any of the aforementioned cells to the subject. In one embodiment, the cells are autologous or allogeneic to the subject.

[0026] On the other hand, the present invention provides isolated cells or populations of cells that have multiplied or expanded from the cells described in the foregoing aspects and embodiments.

[0027] On another aspect, the present invention provides a method for generating hepatocytes, comprising: (a) introducing a base editor or a polynucleotide encoding the base editor into hepatocytes containing SNPs associated with α1-antitrypsin deficiency, wherein the base editor comprises a polynucleotide-programmable nucleotide-binding domain and an adenosine deaminase variant domain as described in any of the foregoing aspects and embodiments; and introducing one or more guide polynucleotides, wherein the one or more guide polynucleotides target the base editor to induce an A·T to G·C change in the SNPs associated with α1-antitrypsin deficiency.

[0028] In various implementations, the liver cells are mammalian cells or human cells.

[0029] In other embodiments of the above aspects, the adenosine deaminase variant includes variations at amino acid positions 82 and 166. In other embodiments of the above aspects, the adenosine deaminase variant includes the V82S variation. In other embodiments of the above aspects, the adenosine deaminase variant includes the T166R variation. In other embodiments of the above aspects, the adenosine deaminase variant includes both V82S and T166R variations. In other embodiments of the above aspects, the adenosine deaminase variant further includes one or more of the following variations: Y147T, Y147R, Q154S, Y123H, and Q154R. In other embodiments of the above aspects, the adenosine deaminase variants comprise the following variations: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y144 7R; 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 embodiments of the foregoing aspects, the adenosine deaminase variant comprises Y147R+Q154R+Y123H. In embodiments of the foregoing aspects, the adenosine deaminase variant comprises Y147R+Q154R+I76Y. In embodiments of the foregoing aspects, the adenosine deaminase variant comprises Y147R+Q154R+T166R. In embodiments of the foregoing aspects, the adenosine deaminase variant comprises Y147T+Q154R. In embodiments of the foregoing aspects, the adenosine deaminase variant comprises Y147T+Q154S. In embodiments of the foregoing aspects, the adenosine deaminase variant comprises Y147R+Q154S. In embodiments of the foregoing aspects, the adenosine deaminase variant comprises V82S+Q154S. In embodiments of the foregoing aspects, the adenosine deaminase variant comprises V82S+Y147R. In embodiments of the foregoing aspects, the adenosine deaminase variant comprises V82S+Q154R. In embodiments of the foregoing aspects, the adenosine deaminase variant comprises V82S+Y123H. In embodiments of the foregoing aspects, the adenosine deaminase variant comprises I76Y+V82S. In embodiments of the foregoing aspects, the adenosine deaminase variant comprises V82S+Y123H+Y147T. In embodiments of the foregoing aspects, the adenosine deaminase variant comprises V82S+Y123H+Y147R. In embodiments of the foregoing aspects, the adenosine deaminase variant comprises V82S+Y123H+Q154R.In embodiments of the foregoing aspects, the adenosine deaminase variant comprises Y123H+Y147R+Q154R+I76Y. In embodiments of the foregoing aspects, the adenosine deaminase variant comprises V82S+Y123H+Y147R+Q154R. In embodiments of the foregoing aspects, the adenosine deaminase variant comprises I76Y+V82S+Y123H+Y147R+Q154R. In other embodiments of the foregoing aspects, the adenosine deaminase variant comprises a C-terminal deletion of residues starting from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 157. In other embodiments of the foregoing aspects, the base editor domain comprises a single adenosine deaminase variant containing V82S and T166R. In other embodiments of the foregoing aspects, the base editor domain comprises a wild-type adenosine deaminase domain and an adenosine deaminase variant. In other embodiments of the above aspects, the adenosine deaminase variant further comprises variations selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R. In other embodiments of the above aspects, the base editor domain comprises a TadA7.10 domain and the adenosine deaminase variant. In other embodiments of the above aspects, the adenosine deaminase variant further comprises variations selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R. In other embodiments of the above aspects, the base editor comprises a TadA7.10 domain and an adenosine deaminase variant containing variations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T ;V82S+Y123H+Y147R;V82S+Y123H+Q154R;Y147R+Q154R+Y123H;Y147R+Q154R+I76Y;Y147R+Q154R+T166R;Y123H+Y147R+Q154R+I76Y;V82S+Y123H+Y147R+Q154R; andI76Y+V82S+Y123H+Y147R+Q154R. In other embodiments of the above aspects, the base editor is ABE8, which comprises or consists primarily of the following sequences or fragments thereof having adenosine deaminase activity:

[0030] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD. In other embodiments of the above aspects, the adenosine deaminase variant comprises a truncated ABE8, which, relative to the full-length ABE8, lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 N-terminal amino acid residues. In other embodiments of the above, the adenosine deaminase variant is a truncated ABE8, which, relative to the full-length ABE8, has 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 missing.

[0031] In other embodiments of the above aspects, the A·T to G·C change of the SNP associated with α1-antitrypsin deficiency alters glutamic acid in the α1-antitrypsin polypeptide to lysine. In other embodiments of the above aspects, the SNP associated with α1-antitrypsin deficiency results in the expression of an α1-antitrypsin polypeptide having a lysine residue at amino acid position 342. In other embodiments of the above aspects, the SNP associated with α1-antitrypsin deficiency replaces glutamic acid with lysine. In other embodiments of the above aspects, cells are selected for the A·T to G·C change of the SNP associated with α1-antitrypsin deficiency. In other embodiments of the above aspects, 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 other embodiments of the above aspects, the polynucleotide programmable DNA binding domain comprises a variant of SpCas9 having specificity for a modified protospacer adjacent motif (PAM) or specificity for non-G PAMs. In other embodiments of the above aspects, the modified PAM is specific for the nucleic acid sequence 5'-NGC-3'. In other embodiments of the above aspects, the modified SpCas9 comprises amino acid substitutions of D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R, or their corresponding amino acid substitutions. In other embodiments of the above aspects, the polynucleotide programmable DNA binding domain is a nuclease inactivating or nicking enzyme variant. In other embodiments of the above aspects, the nicking enzyme variant comprises amino acid substitution D10A or its corresponding amino acid substitution. In other embodiments of the above aspects, the base editor further comprises a zinc finger domain. In other embodiments of the above aspects, the adenosine deaminase domain is capable of deaminating adenosine in deoxyribonucleic acid (DNA). In other embodiments of the above aspects, one or more guide RNAs comprise CRISPR RNA (crRNA) and a reverse-coding small RNA (tracrRNA), wherein the crRNA comprises a nucleic acid sequence complementary to an α1-antitrypsin nucleic acid sequence containing an SNP associated with α1-antitrypsin deficiency. In other embodiments of the above aspects, the base editor and the one or more guide polynucleotides form a complex in the cell.In other embodiments of the above, the base editor is compounded with a single guide RNA (sgRNA) containing a nucleic acid sequence complementary to an α1-antitrypsin nucleic acid sequence containing an SNP associated with α1-antitrypsin deficiency.

[0032] On the other hand, a method for treating a subject with α1-antitrypsin deficiency (A1AD) is provided, wherein the method comprises administering to the subject a fusion protein comprising an adenosine deaminase variant in which a Cas9 or Cas12 polypeptide has been inserted, or a polynucleotide encoding such fusion protein; and one or more guide polynucleotides that target the fusion protein to induce an A·T to G·C change in a single nucleotide polymorphism (SNP) associated with A1AD, thereby treating the subject with A1AD.

[0033] On the other hand, a method for treating a subject with α1-antitrypsin deficiency (A1AD) is provided, wherein the method comprises administering to the subject an adenosine base editor ABE8, or a polynucleotide encoding the base editor, wherein ABE8 comprises an adenosine deaminase variant in which a Cas9 or Cas12 polypeptide has been inserted; and one or more guide polynucleotides that target ABE8 to induce an A·T to G·C change in an A1AD-associated SNP, thereby treating the subject with A1AD.

[0034] In the implementation scheme of the method described above, ABE8 is selected from ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, ABE8.5-m, ABE8.6-m, ABE8.7-m, ABE8.8-m, ABE8.9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.13-m, ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8.23-m, ABE8.24-m, ABE8.1-d, ABE8.2-d, ABE8.3-d, ABE8.4-d, ABE8.5-d, ABE8.6-d, ABE8.7-d, ABE8.8-d, ABE8.9-d, ABE8.10-d, ABE8.11-d, ABE8.12-d , ABE8.13-d, ABE8.14-d, ABE8.15-d, ABE8.16-d, ABE8.17-d, ABE8.18-d, ABE8.19-d, ABE8.20-d, ABE8.21-d, ABE8.22-d, ABE8.23-d or ABE8.24-d. In embodiments of the method described above, the adenosine deaminase variant comprises the amino acid sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD; and wherein the amino acid sequence contains at least one variation. In embodiments, the adenosine deaminase variant contains a variation at amino acid positions 82 and / or 166. In embodiments, the at least one variation comprises: V82S, T166R, Y147T, Y147R, Q154S, Y123H, and / or Q154R.

[0035] In embodiments of the method described above, the adenosine deaminase variant comprises one of the following combinations: 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;andI76Y+V82S+Y123H+Y147R+Q154R. In an embodiment of the method 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, Tad A*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 of residues originating from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 157. In another embodiment, the adenosine deaminase variant is a monomer of an adenosine deaminase comprising a TadA*8 adenosine deaminase variant domain. In yet another embodiment, the adenosine deaminase variant is a heterodimer of an adenosine deaminase comprising a wild-type adenosine deaminase domain and a TadA*8 adenosine deaminase variant domain. Finally, in yet another embodiment, the adenosine deaminase variant is a heterodimer of an adenosine deaminase comprising a TadA domain and a TadA*8 adenosine deaminase variant domain.

[0036] In an embodiment of the method described above, the A·T to G·C change of the SNP associated with A1AD alters glutamic acid at amino acid position 342 to lysine. In an embodiment of the method described above, the SNP associated with A1AD results in the expression of an α1-antitrypsin polypeptide having lysine at amino acid position 342. In an embodiment of the method described above, the SNP associated with α1-antitrypsin deficiency replaces glutamic acid with lysine.

[0037] In embodiments of the method described above, the adenosine deaminase variant is inserted into the flexible loop, α-helix region, unstructured portion, or solvent-accessible portion of the Cas9 or Cas12 polypeptide. In embodiments of the method described above, the adenosine deaminase variant is flanked by the N-terminal and C-terminal fragments of the Cas9 or Cas12 polypeptide.

[0038] In embodiments of the methods described above, the fusion protein or ABE8 comprises the structure NH2-[N-terminal fragment of Cas9 or Cas12 polypeptide]-[adenosine deaminase variant]-[C-terminal fragment of Cas9 or Cas12 polypeptide]-COOH, wherein the “]-[” in each case are optional linkers. In embodiments, 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 embodiments, the flexible loop comprises an amino acid close to the target nucleobase when the adenosine deaminase variant deaminates the target nucleobase.

[0039] In an embodiment of the method described above, the method further comprises administering a guide nucleic acid sequence to the subject to deaminate the target nucleotide bases of the SNP associated with A1AD. In an embodiment of the method described above, the deamination of the target nucleotide bases replaces the target nucleotide bases with wild-type or non-wild-type nucleotide bases, and the deamination of the target nucleotide bases alleviates the symptoms of A1AD. In an embodiment of the method described above, the deamination of the A1AD-associated SNP replaces glutamate with lysine.

[0040] In embodiments of the method described above, the target nucleotide in the target polynucleotide sequence is 1-20 nucleotides away from the PAM sequence. In embodiments, the target nucleotide is 2-12 nucleotides upstream of the PAM sequence. In embodiments of the method described above, the N-terminal or C-terminal fragment of the Cas9 or Cas12 polypeptide binds to the target polynucleotide sequence. In some embodiments, the N-terminal or C-terminal fragment contains a RuvC domain; the N-terminal or C-terminal fragment contains an HNH domain; neither the N-terminal nor the C-terminal fragment contains an HNH domain; or neither the N-terminal nor the C-terminal fragment contains a RuvC domain. In embodiments, the Cas9 or Cas12 polypeptide comprises a partial or complete deletion of one or more structural domains and a location where the deaminase has been inserted into the partial or complete deletion of the Cas9 or Cas12 polypeptide. In some embodiments, the deletion is within the RuvC domain; the deletion is within the HNH domain; or the deletion bridges the RuvC domain and the C-terminal domain.

[0041] In embodiments of the method described above, the fusion protein or ABE8 comprises a Cas9 polypeptide. In these embodiments, the Cas9 polypeptide is Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), or a variant thereof. In these embodiments, the Cas9 polypeptide comprises the following amino acid sequence (Cas9 reference sequence):

[0042]

[0043] (Single underscore: HNH domain; Double underscore: RuvC domain; Cas9 reference sequence), or its corresponding region. In some embodiments, the Cas9 polypeptide contains the deletion of amino acids 1017-1069 as numbered in the Cas9 polypeptide reference sequence or their corresponding amino acids; the Cas9 polypeptide contains the deletion of amino acids 792-872 as numbered in the Cas9 polypeptide reference sequence or their corresponding amino acids; or the Cas9 polypeptide contains the deletion of amino acids 792-906 as numbered in the Cas9 polypeptide reference sequence or their corresponding amino acids.

[0044] In an embodiment of the method described above, an adenosine deaminase variant is inserted into a flexible loop of the Cas9 polypeptide. In this embodiment, the flexible loop comprises a region of amino acid residues selected from positions such as 530-537, 569-579, 686-691, 768-793, 943-947, 1002-1040, 1052-1077, 1232-1248, and 1298-1300 in the Cas9 reference sequence, or a group of corresponding amino acid positions.

[0045] In embodiments of the method described above, the deaminase variant is inserted between amino acid positions numbered in the Cas9 reference sequence, such as 768-769, 791-792, 792-793, 1015-1016, 1022-1023, 1026-1027, 1029-1030, 1040-1041, 1052-1053, 1054-1055, 1067-1068, 1068-1069, 1247-1248, or 1248-1249, or their corresponding amino acid positions. In embodiments of the methods described above, the deaminase variant is inserted at amino acid positions 768-769, 792-793, 1022-1023, 1026-1027, 1040-1041, 1068-1069, or 1247-1248, or their corresponding amino acid positions, as numbered in the Cas9 reference sequence. In embodiments of the methods described above, the deaminase variant is inserted at amino acid positions 1016-1017, 1023-1024, 1029-1030, 1040-1041, 1069-1070, or 1247-1248, or their corresponding amino acid positions, as numbered in the Cas9 reference sequence. In embodiments of the methods described above, the adenosine deaminase variant is inserted at the locus identified in Table 13A within the Cas9 polypeptide. In the embodiments, the N-terminal fragment comprises amino acid residues 1-529, 538-568, 580-685, 692-942, 948-1001, 1026-1051, 1078-1231 and / or 1248-1297 of the Cas9 reference sequence, or their corresponding residues. In the embodiments, the C-terminal fragment comprises amino acid residues 1301-1368, 1248-1297, 1078-1231, 1026-1051, 948-1001, 692-942, 580-685 and / or 538-568 of the Cas9 reference sequence, or their corresponding residues.

[0046] In embodiments of the methods described above, the Cas9 peptide is a modified Cas9 and is specific for altered PAM or non-G PAM polymers. In embodiments of the methods described above, the Cas9 peptide is a nicking enzyme or wherein the Cas9 peptide is a nuclease inactivated. In embodiments of the methods described above, the Cas9 peptide is a modified SpCas9 peptide. In embodiments, the modified SpCas9 peptide includes amino acid substitutions of D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and is specific for altered PAM5'-NGC-3'.

[0047] In other embodiments of the method described above, the fusion protein or ABE8 comprises a Cas12 polypeptide. In some embodiments, an adenosine deaminase variant is inserted into the Cas12 polypeptide. In some embodiments, the Cas12 polypeptide is Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, or Cas12i. In some embodiments, 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 acids of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, or Cas12i. a) residues; b) 147 and 148, 248 and 249, 299 and 300, 991 and 992 or 1031 and 1032 of BvCas12b, or 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 the corresponding amino acid residues of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h or Cas12i. In an embodiment, the adenosine deaminase variant is inserted at the locus identified in Table 13B of the Cas12 polypeptide. In an embodiment, the Cas12 polypeptide is Cas12b. In the implementation scheme, the Cas12 polypeptide includes a BhCas12b domain, a BvCas12b domain, or an AACas12b domain.

[0048] In the embodiments of the methods described above, the guide RNA comprises CRISPR RNA (crRNA) and reverse-activating crRNA (tracrRNA). In the embodiments of the methods described above, the subject is a mammal or a human.

[0049] On the other hand, a pharmaceutical composition is provided comprising a base editing system and a pharmaceutically acceptable carrier, medium, or excipient of any of the methods, aspects, and embodiments described above.

[0050] On the one hand, a pharmaceutical composition is provided that comprises cells of the aspects and embodiments described above, as well as pharmaceutically acceptable carriers, mediators, or excipients.

[0051] On the other hand, a kit is provided that includes a base editing system comprising any of the methods, aspects, and embodiments described above.

[0052] On the other hand, a kit is provided comprising cells of any of the aspects and embodiments described above. In one embodiment of the kit, the kit further comprises a packaging insert with instructions for use.

[0053] On the one hand, this article provides a base editor comprising a polynucleotide programmable DNA-binding domain and at least one base-editing domain comprising an adenosine deaminase variant containing a variation at amino acid position 82 or 166 of the following sequence.

[0054] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD.

[0055] On one hand, the base editor system comprises the base editor described above and a guide RNA, wherein the guide RNA targets the base editor to induce a change in SNPs associated with α1-antitrypsin deficiency. In some embodiments, the adenosine deaminase variant comprises a V82S variant and / or a T166R variant. In some embodiments, the adenosine deaminase variant further comprises one or more of the following variants: Y147T, Y147R, Q154S, Y123H, and Q154R. In some embodiments, the base editor domain comprises an adenosine deaminase heterodimer, which comprises a wild-type adenosine deaminase domain and an adenosine deaminase variant. In some embodiments, the adenosine deaminase variant is a truncated TadA8, which, relative to the full-length TadA8, lacks 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. In some embodiments, the adenosine deaminase variant is a truncated TadA8, which, relative to the full-length TadA8, lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues. 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 variant of SpCas9 with altered protospacer adjacent motif (PAM) specificity or specificity for non-G PAMs. In some embodiments, the polynucleotide-programmable DNA-binding domain is a nuclease-inactivating Cas9. In some embodiments, the polynucleotide-programmable DNA-binding domain is a Cas9 nickase.

[0056] On the one hand, this paper provides a base editor system comprising one or more guide RNAs and a fusion protein containing a multinucleotide programmable DNA binding domain, the binding domain comprising the following sequence:

[0057]

[0058] The bolded sequence indicates a sequence derived from Cas9, the italicized sequence indicates a linker sequence, and the underlined sequence indicates a bifid nuclear localization sequence, as well as at least one base-editing domain containing an adenosine deaminase variant comprising changes at amino acid positions 82 and / or 166 of the following sequence.

[0059] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD.

[0060] On one hand, a cell comprising the base editor system described above is provided. In some embodiments, the cell is a human cell or a mammalian cell. In some embodiments, the cell is isolated, in vivo, or in vitro.

[0061] This invention provides compositions and methods for editing mutations associated with α1-antitrypsin deficiency (A1AD). The compositions and articles defined in this invention are isolated or manufactured in association with the examples provided below. Further features and advantages of this invention will become apparent from the detailed description and claims.

[0062] definition

[0063] The following definitions supplement those in the art and are applicable to this application, and should not be attributed to any related or unrelated cases, such as any jointly owned patents or applications. While any methods and materials similar to or equivalent to those described herein may be used in practice to test this disclosure, preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. The following provides general definitions for many of the terms used herein: 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).

[0065] In this application, unless otherwise specified, the use of the singular includes the plural. It must be noted that, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” used herein include the plural reference. In this application, unless otherwise specified, the use of “or” means “and / or” and is understood to mean “included.” Furthermore, the use of the term “including” and other forms such as “include,” “includes,” and “included” is not restrictive.

[0066] The terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of inclusion, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) as used in this specification and claims are inclusive or open-ended and do not exclude additional, unmentioned elements or method steps. It is contemplated that any embodiments discussed in this specification can be implemented with respect to any method or composition of this disclosure, and vice versa. Furthermore, the compositions of this disclosure can be used to implement the methods of this disclosure.

[0067] The terms "about" or "approximately" refer to a specific value within an acceptable margin of error as determined by those skilled in the art, which will depend in part on how the value was measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may mean within or more than one standard deviation. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, particularly for biological systems or processes, the term may mean within an order of magnitude, such as within 5 times or 2 times the value. Where a specific value is described in the application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable margin of error for the specific value.

[0068] The range provided in this article is understood as an abbreviation of all values ​​within the range, including the first and last values. For example, the range 1 to 50 is understood to include any number, number combination, or subrange that comes from the following groups: 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.

[0069] References to “some embodiments,” “an embodiment,” “an embodiment,” or “other embodiments” in the specification refer to specific features, structures, or characteristics described in connection with the embodiments that are included in at least some embodiments, but not necessarily in all embodiments disclosed herein.

[0070] "Adenosine deaminase" refers to a polypeptide or fragment thereof that catalyzes the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine or deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., engineered adenosine deaminases, evolved adenosine deaminases) can be derived from any organism, such as bacteria.

[0071] In some implementations, adenosine deaminase comprises changes in the following sequences:

[0072] MSEVEFSHEY WMRHALTLAK RARDEREVPVGAVLVLNNRV IGEGWNRAIG LHDPTAHAEIMALRQGGLVM QNYRLIDATL YVTFEPCVMC AGAMIHSRIG RVVFGVRNAK TGAAGSLMDV LHYPGMNHRVEITEGILADE CAALLCYFFR MPRQVFNAQK KAQSSTD

[0073] (Also named TadA*7.10).

[0074] In some embodiments, TadA*7.10 includes at least one variation. In some embodiments, TadA*7.10 includes variations of amino acids 82 and / or 166. In particular embodiments, variants of the above-mentioned reference sequence include one or more of the following variations:

[0075] Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. The variant Y123H is also referred to herein as H123H (the variant H123Y in TadA*7.10 is reversed to Y123H (wt)). In other embodiments, variants of the TadA*7.10 sequence comprise combinations of variants 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+Y166R; V166R+Y123H+Y147T; V82S+Y ... 23H+Y147R;V82S+Y123H+Q154R;Y147R+Q154R+Y123H;Y147R+Q154R+I76Y;Y147R+Q154R+T166R;Y123H+Y147R+Q154R+I76Y;V82S+Y123H+Y147R+Q154R;andI76Y+V82S+Y123H+Y147R+Q154R.

[0076] In other embodiments, the present invention provides a variant of adenosine deaminase comprising a deletion, such as TadA*8, which includes 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 variations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant is a monomer comprising a combination of variations 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+T166 R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.

[0077] In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each having one or more of the following variations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each having a combination of variations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+ Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.

[0078] 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), wherein the adenosine deaminase variant domain comprises one or more of the following variations: 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), wherein the adenosine deaminase variant domain comprises variations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V8 2S; 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.

[0079] 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), wherein the adenosine deaminase variant domain comprises one or more of the following variations: 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), wherein the adenosine deaminase variant domain comprises the following variations: 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; Y1 47R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; I76Y+V82S+Y123H+Y147R+Q154R.

[0080] In one embodiment, the adenosine deaminase is TadA*8, which comprises or is mainly composed of the following sequence or a fragment thereof having adenosine deaminase activity:

[0081] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0082] In some embodiments, TadA*8 is truncated. In some embodiments, the truncated TadA*8, relative to the full-length TadA*8, lacks 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. In some embodiments, the truncated TadA*8, relative to the full-length TadA*8, lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues. In some embodiments, the adenosine deaminase variant is the full-length TadA*8.

[0083] In a particular embodiment, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from one of the following:

[0084] Staphylococcus aureus (S. aureus) TadA:

[0085] MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN

[0086] Bacillus subtilis (B.subtilis) TadA:

[0087] MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE

[0088] Salmonella typhimurium (S. typhimurium) TadA:

[0089] MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV

[0090] Shewanella putrefaciens (S. putrefaciens) TadA:

[0091] MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE

[0092] Haemophilus influenzae F3031 (H. influenzae)

[0093] TadA:

[0094] MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTΑΗAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK

[0095] Caulobacter crescentus (C. crescentus) TadA:

[0096] MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI

[0097] Geobacter sulfurreducens (G. sulfurreducens)

[0098] TadA:

[0099] MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP

[0100] TadA*7.10

[0101] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0102] "Adenosine deaminase base editor 8 (ABE8) polypeptide" means a base editor (BE) comprising an adenosine deaminase variant as defined and / or described herein, wherein the adenosine deaminase variant comprises a variation of amino acid positions 82 and / or 166 of the reference sequence below:

[0103] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD. In some implementations, ABE8 contains further variations relative to the reference sequence.

[0104] "Adenosine deaminase base editor 8 (ABE8) polynucleotide" refers to the polynucleotide (polynucleotide sequence) that encodes the ABE8 polypeptide.

[0105] "Administration" in this document means providing a patient or subject with one or more of the compositions described herein. For example, but not limited to, administration of the composition may be by injection, such as intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im). One or more of these routes may be used. Parenteral administration may be performed, for example, by bolus injection or gradual infusion over time. Alternatively, or concurrently, it may be administered orally.

[0106] "Agent" refers to any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide or fragment thereof.

[0107] "α1-Antitrypsin (A1AT) protein" means a polypeptide or fragment thereof having at least about 95% identity with UniProt accession number P01009. In a particular embodiment, the A1AT protein contains one or more variations relative to the reference sequence below. In one particular embodiment, the A1AT protein associated with A1AD contains the E342K mutation. An exemplary A1AT amino acid sequence is provided below.

[0108] >sp|P01009|A1AT_HUMANΑ-1-antitrypsin OS=Homo sapiens OX=9606GN=SERPINA1 PE=1SV=3

[0109] MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERP FEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK

[0110] In the A1AT protein sequence described above, the first 24 amino acids form the signal peptide (underlined). Sequence position 342 is mutated in AAD (i.e., E342K), determined by setting the amino acid residue "E" after the signal sequence to amino acid "1".

[0111] "Change" means an alteration (e.g., increase or decrease) in the structure, expression level, or activity of a gene or polypeptide detected by standard methods known in the art as described herein. As used herein, change includes alterations in polynucleotides or polypeptides or changes in expression levels, such as 25%, 40%, 50%, or more.

[0112] "Pause" means to reduce, suppress, eliminate, stop, or stabilize the development or progression of a disease.

[0113] "Analogous" refers to molecules that are not identical but have similar functional or structural characteristics. For example, polynucleotide or polypeptide analogs retain the biological activity of the corresponding naturally occurring polynucleotide or polypeptide, but have certain modifications relative to the naturally occurring polynucleotide or polypeptide to enhance the function of the analog. These modifications may increase the analog's affinity for DNA, efficiency, specificity, resistance to proteases or nucleases, membrane permeability, and / or half-life without altering, for example, ligand binding. Analogs may include non-natural nucleotides or amino acids.

[0114] The term "base editor (BE)" or "nucleobase editor (NBE)" refers to an agent that binds to polynucleotides and has nucleobase modification activity. In various embodiments, the base editor comprises a nucleobase-modifying polypeptide (e.g., a deaminase) and a nucleic acid-programmable nucleotide-binding domain linking a guide polynucleotide (e.g., guide RNA). In various embodiments, the agent is a bimolecular complex comprising a protein domain having base-editing activity, i.e., a domain capable of modifying bases (e.g., A, T, C, G, or U) within a nucleic acid molecule (e.g., DNA). In some embodiments, the polynucleotide-programmable DNA-binding domain is fused to or linked to a deaminase domain. In one embodiment, the agent is a fusion protein comprising a domain having base-editing activity. In another embodiment, the protein domain having base-editing activity is linked to guide RNA (e.g., via an RNA-binding motif on the guide RNA and an RNA-binding domain fused to a deaminase). In some embodiments, the base-editing domain is capable of deaminating bases within a nucleic acid molecule. In some embodiments, the base editor is capable of deaminating one or more bases within a DNA molecule. In some implementations, the base editor is capable of deamination of adenosine (A) within DNA. In some implementations, the base editor is an adenosine base editor (ABE).

[0115] In some embodiments, the base editor is generated by cloning an adenosine deaminase variant (e.g., TadA*8) into a scaffold comprising a circular Cas9 (e.g., spCAS9 or saCAS9) and a bigenotyping nuclear localization sequence (e.g., ABE8). Circular Cas9 is well known in the art and described, for example, in Oakes et al., Cell 176, 254–267, 2019. Illustrative circular arrangements are indicated by bold sequences representing sequences derived from Cas9, italic sequences representing linker sequences, and underlined sequences representing bigenotyping nuclear localization sequences.

[0116] CP5 (with MSP "NGC = Pam variant of Cas9 with mutation such as NGG")

[0117] PID (Protein Interaction Domain and "D10A" Cleavage Enzyme):

[0118]

[0119]

[0120] In some embodiments, ABE8 is selected from the base editors from Tables 6 to 9, 13, or 14 below. In some embodiments, ABE8 contains an adenosine deaminase variant evolved from TAdA. In some embodiments, the adenosine deaminase variant of ABE8 is the TadA*8 variant described in Tables 7, 9, 13, or 14 below. In some embodiments, the adenosine deaminase variant is the TadA*7.10 variant (e.g., TadA*8), which contains one or more variations selected from the group consisting of: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In various implementations, ABE8 includes the TadA*7.10 variant (e.g., TadA*8) having a combination of variations 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; V 82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. In some embodiments, ABE8 is a monomeric construct. In some embodiments, ABE8 is a heterodimeric construct. In some embodiments, ABE8 comprises the sequence:

[0121] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0122] In some embodiments, the polynucleotide programmable DNA-binding domain is a CRISPR-associated enzyme (e.g., Cas or Cpf1). In some embodiments, the base editor is a non-catalytic Cas9 fused to the deaminase domain (dCas9). In some embodiments, the base editor is a Cas9 cleavage enzyme fused to the deaminase domain (nCas9). Details of the base editor are described in international PCT applications PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is incorporated herein by reference in its entirety. See also Komor, AC, et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in “Genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, AC, et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A baseeditors with higher efficiency and product purity” Science Advances 3: eaao4774 (2017); and Rees, HA, et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 Dec; 19(12):770-788. doi:10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0123] For example, the adenine base editor (ABE) used in the base editing compositions, systems, and methods described herein, as provided below, has a nucleic acid sequence (8877 base pairs) (Addgene, Watertown, MA.; Gaudelli NM, et al., Nature. 2017 Nov 23; 551(7681):464-471. doi:10.1038 / nature24644; Koblan LW, et al., Nat Biotechnol. 2018 Oct; 36(9):843-846. doi:10.1038 / nbt.4172). Polynucleotide sequences with at least 95% or higher identity to the ABE nucleic acid sequence are also covered.

[0124]

[0125] "Base editing activity" refers to the action within a polynucleotide to alter a base. In one embodiment, a first base is converted to a second base. In one embodiment, base editing activity is adenosine or adenine deaminase activity, for example, converting A·T to G·C. Base editing activity may also involve adenosine or adenine deaminase activity, for example, converting A·T to G·C; and cytidine deaminase activity, for example, converting target C·G to T·A. In some embodiments, base editing activity is assessed by the efficiency of the editing. Base editing efficiency can be measured by any suitable means, for example, by Sanger sequencing or next-generation sequencing. In some embodiments, base editing efficiency is measured as the percentage of total sequencing reads resulting from nucleobase conversions induced by a base editor, for example, the percentage of total sequencing reads converting target AT base pairs to GC base pairs. In some embodiments, when base editing is performed in a cell population, base editing efficiency is measured as the percentage of total cells resulting from nucleobase conversions induced by a base editor.

[0126] The term "base editor system" refers to a system used to edit the nucleobases of a target nucleotide sequence. In various embodiments, the base editor system comprises (1) a polynucleotide-programmable nucleotide-binding domain (e.g., Cas9); (2) a deaminase domain (e.g., adenosine deaminase) for deaminating the nucleobase; 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.

[0127] 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 the target nucleic acid sequence. In some embodiments, a pair of guide polynucleotides may be used to target different deaminases to the target nucleic acid sequence.

[0128] The deaminase domain and the polynucleotide programmable nucleotide binding component of the base editor can covalently or nonvalently associate with each other or any combination of their association or interaction. For example, in some embodiments, the deaminase domain can be targeted to a target nucleotide sequence via the polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused to or linked to the deaminase domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the deaminase domain to a target nucleotide sequence by nonvalently interacting with or associating with the deaminase domain. For example, in some embodiments, the deaminase domain may include an additional heterologous portion or domain capable of interacting, associating, or forming a complex with the additional heterologous portion or domain that is part of the polynucleotide programmable nucleotide binding domain. In some embodiments, the additional heterologous portion can bind to, interact with, associate with, or form a complex with a polypeptide. In some embodiments, the additional heterologous portion can bind to, interact with, associate with, or form a complex with a polynucleotide. In some embodiments, the additional heterologous portion can bind to a guide polynucleotide. In some embodiments, the additional heterologous portion can bind to a polypeptide linker. In some embodiments, the additional heterologous portion can bind to a polynucleotide linker. The additional heterologous portion can be a protein domain. In some embodiments, the additional heterologous portion can be a K homology (KH) domain, an MS2 capsid protein domain, a PP7 capsid protein domain, an SfMuCom capsid protein domain, a sterile α motif, a telomerase Ku-binding motif and Ku protein, a telomerase Sm7-binding motif and Sm7 protein, or an RNA recognition motif.

[0129] The base editor system may further include guide polynucleotide components. It should be understood that the components of the base editor system can associate with each other via covalent bonds, non-covalent interactions, or any combination of association and interaction. In some embodiments, the deaminase domain can be targeted to a target nucleotide sequence via the guide polynucleotide. For example, in some embodiments, the deaminase domain may include additional heterologous moieties or domains (e.g., polynucleotide-binding domains such as RNA or DNA-binding proteins) that can interact, associate, or form complexes with portions or segments (e.g., polynucleotide motifs) of the guide polynucleotide. In some embodiments, additional heterologous moieties or domains (e.g., polynucleotide-binding domains such as RNA or DNA-binding proteins) may be fused to or linked to the deaminase domain. In some embodiments, additional heterologous moieties may bind to, interact with, associate with, or form complexes with peptides. In some embodiments, additional heterologous moieties may bind to, interact with, associate with, or form complexes with polynucleotides. In some embodiments, additional heterologous moieties can bind to guide polynucleotides. In some embodiments, additional heterologous moieties can bind to peptide linkers. In some embodiments, the additional heterologous portion can bind to a polynucleotide linker. The additional heterologous portion can be a protein domain. In some embodiments, the additional heterologous portion can be a K homology (KH) domain, an MS2 capsid protein domain, a PP7 capsid protein domain, an SfMuCom capsid protein domain, a sterile α motif, a telomerase Ku-binding motif and Ku protein, a telomerase Sm7-binding motif and Sm7 protein, or an RNA recognition motif.

[0130] In some embodiments, the base editor system may further include a base excision repair inhibitor (BER) component. It should be understood that the components of the base editor system may associate with each other via covalent bonds, non-covalent interactions, or any combination of association and interaction. The inhibitor of the BER component may include a BER inhibitor. In some embodiments, the BER inhibitor may be a uracil DNA glycosidase inhibitor (UGI). In some embodiments, the BER inhibitor may be an inosine BER inhibitor. In some embodiments, the BER inhibitor may be targeted to a target nucleotide sequence via a polynucleotide-programmable nucleotide-binding domain. In some embodiments, the polynucleotide-programmable nucleotide-binding domain may be fused to or linked to the BER inhibitor. In some embodiments, the polynucleotide-programmable nucleotide-binding domain may be fused to or linked to a deaminase domain and the BER inhibitor. In some embodiments, the polynucleotide-programmable nucleotide-binding domain may target the BER inhibitor to a target nucleotide sequence by non-covalent interaction or association with the BER inhibitor. For example, in some embodiments, the BER inhibitor component may include an additional heterologous moiety or domain capable of interacting, associating, or forming a complex with an additional heterologous moiety or domain that is part of the polynucleotide-programmable nucleotide-binding domain.

[0131] In some embodiments, the inhibitor of BER can be targeted to a target nucleotide sequence via a guide polynucleotide. For example, in some embodiments, the inhibitor of BER may include an additional heterologous moiety or domain (e.g., a polynucleotide-binding domain such as an RNA or DNA-binding protein) that can interact, associate, or form a complex with a portion or segment (e.g., a polynucleotide motif) of the guide polynucleotide. In some embodiments, the additional heterologous moiety or domain of the guide polynucleotide (e.g., a polynucleotide-binding domain such as an RNA or DNA-binding protein) may be fused to or linked to the inhibitor of BER. In some embodiments, the additional heterologous moiety may bind to, interact with, associate with, or form a complex with the polynucleotide. In some embodiments, the additional heterologous moiety may bind to the guide polynucleotide. In some embodiments, the additional heterologous moiety may bind to a peptide linker. In some embodiments, the additional heterologous moiety may bind to a polynucleotide linker. The additional heterologous moiety may be a protein domain. In some implementations, the additional heterologous portion may be a K homology (KH) domain, an MS2 capsid protein domain, a PP7 capsid protein domain, an SfMuCom capsid protein domain, a sterile α motif, a telomerase Ku-binding motif and Ku protein, a telomerase Sm7-binding motif and Sm7 protein, or an RNA recognition motif protein or RNA recognition motif.

[0132] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease containing the Cas9 protein or fragments thereof (e.g., a protein containing the active, inactive, or partially active DNA cleavage domain of Cas9 and / or the gRNA-binding domain of Cas9). Cas9 nucleases are sometimes also called Casnl nucleases or CRISPR (clustered regularly spaced short palindromic repeats)-associated nucleases. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugating plasmids). CRISPR clusters contain spacer sequences, sequences complementary to the precursor mobile element, and target invading nucleic acids. CRISPR clusters are truncated and processed into CRISPR RNA (crRNA). In type II CRISPR systems, proper processing of pre-crRNA requires a reverse-coded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. tracrRNA acts as a guide for ribonuclease 3-assisted processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA cleaves the linear or circular dsDNA target complementary to the spacer sequence via endonucleolysis. The target strand not complementary to the crRNA is first endonucleated and then exonucleated via 3′–5′ exonucleolysis. In nature, DNA binding and cleavage typically require both proteins and two RNAs. However, a single guide RNA (“sgRNA” or simply “gNRA”) can be engineered to incorporate both crRNA and tracrRNA into a single RNA species. See, for example, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816–821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes short motifs (PAMs or protospacer adjacent motifs) in CRISPR repeat sequences to help distinguish between self and non-self.The Cas9 nuclease sequence and structure are well known to those skilled in the art (see, for example, "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., JJ, McShan W.M., Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma C.M., 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 J.A., Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 heterologs have been described in a variety of species, including, but not limited to, Streptococcus pyogenes and Staphylococcus aureus.Additional suitable Cas9 nucleases and sequences are apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci described in Chylinski, Rhun, and Charpentier, “ThetracrRNA and Cas9 families of type IICRISPR-Cas immunity systems” (2013) RNABiology 10:5,726-737, the entire contents of which are incorporated herein by reference.

[0133] An illustrative Cas9 is Streptococcus pyogenes Cas9 (spCas9), whose amino acid sequence is provided below:

[0134]

[0135]

[0136] (Single underscore: HNH domain; Double underscore: RuvC domain)

[0137] Nuclease-inactivated Cas9 proteins are interchangeably referred to as “dCas9” proteins (for nucleases, “dead” Cas9) or catalytically inactivated Cas9. Methods for generating Cas9 proteins (or fragments thereof) with inactivated DNA cleavage domains are known (see, for example, Jinek et al., Science. 337:816-821 (2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28; 152(5):1173-83, the entire contents of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to comprise 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 can silence the nuclease activity of Cas9. For example, mutants 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 inactivating (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a cleavage enzyme, referred to as the “nCas9” protein (for “cleavage enzyme” Cas9). In some embodiments, a protein containing a fragment of Cas is provided. For example, in some embodiments, the protein contains 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 containing Cas9 or a fragment thereof is referred to as a “Cas9 variant”. Cas9 variants share homology with Cas9 or a fragment thereof. For example, the Cas9 variants share at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% similarity with wild-type Cas9.In some implementations, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid alterations compared to wild-type Cas9.

[0138] In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA-binding domain or a DNA-enumeration domain) such that the fragment has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity with the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid length of the corresponding wild-type Cas9.

[0139] 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.

[0140] In some implementations, wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI reference sequence: NC_017053.1, nucleotide and amino acid sequences are as follows).

[0141]

[0142]

[0143]

[0144] (Single underscore: HNH domain; Double underscore: RuvC domain)

[0145] In some embodiments, wild-type Cas9 corresponds to or contains the following nucleotide and / or amino acid sequences:

[0146]

[0147]

[0148]

[0149] (Single underscore: HNH domain; Double underscore: RuvC domain)

[0150] In some implementations, wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI reference sequence: NC_002737.2 (nucleotide sequence below); and Uniprot reference sequence: Q99ZW2 (amino acid sequence below)).

[0151]

[0152]

[0153] (SEQ ID NO:1. Single underscore: HNH domain; Double underscore: RuvC domain)

[0154] In some implementations, Cas9 refers to Cas9 derived 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_021846.1). 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 meningitides (NCBI Ref: YP_002342100.1), or Cas9 from any other organism.

[0155] In some embodiments, Cas9 is Neisseria meningitidis Cas9 (NmeCas9) or a variant thereof. In some embodiments, NmeCas9 is specific for NNNNGAYW PAM, where Y is C or T and W is or T. In some embodiments, NmeCas9 is specific for NNNNGYTT PAM, where Y is C or T. In some embodiments, NmeCas9 is specific for NNNNGGTCT PAM. In some embodiments, NmeCas9 is Nme1 Cas9. In some embodiments, NmeCas9 is specific for NNNNGATT PAM, NNNCCTA PAM, NNNCCTC PAM, NNNCCTT PAM, NNNCCTG PAM, NNNCCGT PAM, NNNCCGGPAM, NNNCCCA PAM, NNNCCCT PAM, NNNCCCC PAM, NNNNCCAT PAM, NNNCCAG PAM, NNNNCCAT PAM, or NNNNGATT PAM. In some embodiments, Nme1Cas9 is specific for NNNGATT PAM, NNNCCTA PAM, NNNCCTC PAM, NNNNCCTTPAM, or NNNCCTG PAM. In some embodiments, NmeCas9 is specific for CAA PAM, CAAA PAM, or CCAPAM. In some embodiments, NmeCas9 is Nme2Cas9. In some embodiments, NmeCas9 is specific for NNNCC(N4CC) PAM, where N is any one of A, G, C, or T. In some embodiments, NmeCas9 is specific for NNNCCGT PAM, NNNCCGGPAM, NNNCCCA PAM, NNNCCCT PAM, NNNCCCC PAM, NNNCCATPAM, NNNCCAG PAM, NNNNCCAT PAM, or NNNNGATT PAM. In some embodiments, NmeCas9 is Nme3Cas9. In some embodiments, NmeCas9 is specific for NNNNCAAA PAM, NNNCC PAM, or NNNCNNN PAM. In some implementations, the PAM-interacting domains are N4GAT, N4CC, and N4CAAA for Nme1, Nme2, or Nme3, respectively.Additional NmeCas9 features and PAM sequences are described in Edraki et al., A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing, Mol. Cell. (2019) 73(4): 714-726, the entire contents of which are incorporated herein by reference.

[0156] The Cas9 protein of *Neisseria meningitidis*, Nme1Cas9 (NCBI reference: WP_002235162.1; type II CRISPR RNA-guided endonuclease Cas9), has the following amino acid sequence:

[0157] 1 maafkpnpinyilgldigia svgwamveid edenpiclid lgvrvferae vpktgdslam

[0158] 61 arrlarsvrr ltrrrahrll rarrllkreg vlqaadfdenglikslpntp wqlraaaldr

[0159] 121 kltplewsav llhlikhrgy lsqrknegetadkelgallkgvadnahalq tgdfrtpael

[0160] 181 alnkfekesghirnqrgdys htfsrkdlqaelillfekqkefgnphvsgg lkegietllm

[0161] 241 tqrpalsgdavqkmlghctf epaepkaakntytaerfiwl tklnnlrile qgserpltdt

[0162] 301 eratlmdepy rkskltyaqa rkllgledtaffkglrygkd naeastlmem kayhaisral

[0163] 361 ekeglkdkks plnlspelqd eigtafslfk tdeditgrlk driqpeilea llkhisfdkf

[0164] 421 vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee kiylppipad eirnpvvlra

[0165] 481 lsqarkving vvrrygspar ihietarevg ksfkdrkeie krqeenrkdr ekaaakfrey

[0166] 541 fpnfvgepks kdilklrlye qqhgkclysg keinlgrlne kgyveidhal pfsrtwddsf

[0167] 601 nnkvlvlgse nqnkgnqtpy eyfngkdnsr ewqefkarve tsrfprskkq rillqkfded

[0168] 661 gfkernlndt ryvnrflcqf vadrmrltgk gkkrvfasng qitnllrgfw glrkvraend

[0169] 721 rhhaldavvv acstvamqqk itrfvrykem nafdgktidk etgevlhqkt hfpqpweffa

[0170] 781 qevmirvfgkpdgkpefeeadtpeklrtll aeklssrpea vheyvtplfv srapnrkmsg

[0171] 841 qghmetvksakrldegvsvl rvpltqlklkdlekmvnrer epklyealka rleahkddpa

[0172] 901 kafaepfyky dkagnrtqqv kavrveqvqktgvwvrnhng iadnatmvrv dvfekgdkyy

[0173] 961 lvpiyswqvakgilpdravv qgkdeedwql iddsfnfkfs lhpndlvevi tkkarmfgyf

[0174] 102 1aschrgtgninirihdldhkigkngilegi gvktalsfqkyqidelgkei rpcrlkkrpp

[0175] 1081 VR

[0176] Another example of the demonstrative Neisseria meningitides Cas9 protein, Nme2Cas9 (NCBI reference: WP_002230835; type II CRISPR RNA-guided endonuclease Cas9), has the following amino acid sequence:

[0177] 1 maafkpnpinyilgldigiasvgwamveid eeenpirlid lgvrvferae vpktgdslam

[0178] 61 arrlarsvrr ltrrrahrllrarrllkregvlqaadfdenglikslpntp wqlraaaldr

[0179] 121 kltplewsav llhlikhrgy lsqrknegetadkelgallkgvannahalqtgdfrtpael

[0180] 181 alnkfekesghirnqrgdys htfsrkdlqa elillfekqkefgnphvsgg lkegietllm

[0181] 241 tqrpalsgdavqkmlghctf epaepkaakntytaerfiwl tklnnlrile qgserpltdt

[0182] 301 eratlmdepy rkskltyaqa rkllgledta ffkglrygkd naeastlmem kayhaisral

[0183] 361 ekeglkdkks plnlsselqd eigtafslfk tdeditgrlk drvqpeilea llkhisfdkf

[0184] 421 vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee kiylppipad eirnpvvlra

[0185] 481 lsqarkving vvrrygspar ihietarevg ksfkdrkeie krqeenrkdr ekaaakfrey

[0186] 541 fpnfvgepks kdilklrlye qqhgkclysgkeinlvrlne kgyveidhal pfsrtwddsf

[0187] 601 nnkvlvlgse nqnkgnqtpy ​​eyfngkdnsr ewqefkarve tsrfprskkq rillqkfded

[0188] 661 gfkecnlndt ryvnrflcqf vadhilltgkgkrrvfasng qitnllrgfw glrkvraend

[0189] 721 rhhaldavvv acstvamqqkitrfvrykemnafdgktidketgkvlhqkt hfpqpweffa

[0190] 781 qevmirvfgkpdgkpefeeadtpeklrtll aeklssrpea vheyvtplfv srapnrkmsg

[0191] 841

[0192] 901 gnakqafdpk dnpfykkggq lvkavrvekt qesgvllnkk naytiadngd mvrvdvfckv

[0193] 961 dkkgknqyfi vpiyawqvae nilpdidckg yriddsytfc fslhkydlia fqkdekskve

[0194] 1021 fayyincdss ngrfylawhd kgskeqqfri stqnlvliqkyqvnelgkei rpcrlkkrpp

[0195] 1081 vr

[0196] In some embodiments, dCas9 corresponds to or contains, partially or entirely, one or more mutated Cas9 amino acid sequences having inactivating Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain contains the D10A and H840A mutations or a mutation corresponding to another Cas9. In some embodiments, dCas9 contains the amino acid sequence of dCas9 (D10A and H840A):

[0197]

[0198] (Single underscore: HNH domain; Double underscore: RuvC domain).

[0199] In some implementations, the Cas9 domain contains the D10A mutation, while the residue at position 840 in the amino acid sequence provided above or the corresponding position in any amino acid sequence provided herein remains histidine.

[0200] In other embodiments, dCas9 variants are provided having mutations other than D10A and H840A, for example, causing nuclease inactivation of Cas9 (dCas9). Such mutations, for example, include other amino acids at D10 and H840 or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs of dCas9 are provided that have at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity. In some implementations, the amino acid sequences of the variants of dCas9 are shorter or longer, differing by about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids or more.

[0201] In some embodiments, such as the Cas9 fusion proteins provided herein, the Cas9 fusion protein comprises the full-length amino acid sequence of the Cas9 protein, for example, one of the Cas9 sequences provided herein. However, in other embodiments, such as the fusion proteins provided herein, the fusion protein does not comprise the full-length Cas9 sequence, but only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0202] It should be understood that additional Cas9 proteins (e.g., dead Cas9 (dCas9), Cas9 cleavage enzyme (nCas9), or live Cas9), including variants and homologs thereof, are within the scope of this disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is dead Cas9 (dCas9). In some embodiments, the Cas9 protein is Cas9 cleavage enzyme (nCas9). In some embodiments, the Cas9 protein is live Cas9.

[0203] The illustrative amino acid sequence of non-catalytically active Cas9 (dCas9) is as follows:

[0204]

[0205] The amino acid sequence of an exemplary catalytic Cas9 nickase (nCas9) is as follows:

[0206]

[0207] The illustrative amino acid sequence of catalytically active Cas9 is as follows:

[0208]

[0209] In some embodiments, Cas9 refers to Cas9 derived from archaea (e.g., nanoarchaea), which constitute the domains and kingdoms of single-celled eukaryotic microorganisms. 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 Feb21. doi:10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Macrogenomics using genome resolution has identified many CRISPR-Cas systems, including Cas9, which was first reported in the archaeal domain of life. This divergent Cas9 protein has been found in rarely studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, have been discovered, representing the most compact systems discovered to date. In some embodiments, Cas9 refers to CasX or a variant of CasX. In some embodiments, Cas9 refers to CasY or a variant of CasY. It should be understood that other RNA-guided DNA-binding proteins may be used as nucleic acid programmable DNA-binding proteins (napDNAbp) and are within the scope of this disclosure.

[0210] In some implementations, Cas9 is a Cas9 variant specific to the altered PAM sequence. In some implementations, 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 / s41587-020-0412-8The entirety of this document is incorporated herein by reference. In some embodiments, the Cas9 variant does not have a specific PAM requirement. In some embodiments, the Cas9 variant, such as the SpCas9 variant, is specific for NRNH PAM, where R is A or G and H is C or T. In some embodiments, the SpCas9 variant is specific for PAM sequences AAA, TAA, CAA, GAA, TAT, GAT, or CAC. In some implementations, the SpCas9 variant contains amino acid substitutions located at positions such as 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1218, 1219, 1221, 1249, 1256, 1264, 1290, 1318, 1317, 1320, 1321, 1323, 1332, 1333, 1335, 1337 or 1339 or their corresponding positions relative to the reference sequence numbers below.

[0211] (Single underscore: HNH domain; Double underscore: RuvC domain).

[0212] In some embodiments, the SpCas9 variant contains amino acid substitutions located at positions such as 1114, 1135, 1218, 1219, 1221, 1249, 1320, 1321, 1323, 1332, 1333, 1335, or 1337, or their corresponding positions, relative to the reference sequence numbers described above. In some embodiments, the SpCas9 variant contains amino acid substitutions located at positions such as 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1219, 1221, 1256, 1264, 1290, 1318, 1317, 1320, 1323, 1333, or their corresponding positions, relative to the reference sequence numbers described above. In some embodiments, the SpCas9 variant contains amino acid substitutions located at positions such as 1114, 1131, 1135, 1150, 1156, 1180, 1191, 1218, 1219, 1221, 1227, 1249, 1253, 1286, 1293, 1320, 1321, 1332, 1335, 1339, or their corresponding positions relative to the reference sequence numbers above. In some embodiments, the SpCas9 variant contains amino acid substitutions located at positions such as 1114, 1127, 1135, 1180, 1207, 1219, 1234, 1286, 1301, 1332, 1335, 1337, 1338, 1349, relative to the reference sequence numbers above. Exemplary SpCas9 variant amino acid substitutions and PAM specificity are shown in Tables AD and [other tables not specified in the original text]. Figure 8 middle.

[0213] Table A

[0214]

[0215]

[0216] Table B

[0217]

[0218] Table C

[0219]

[0220] Table D

[0221]

[0222] In certain embodiments, the napDNAbps useful in the methods of the present invention include circular arrangements, which are well known in the art and described, for example, in Oakes et al., Cell 176, 254–267, 2019. In the illustrative circular arrangements described below, bold sequences indicate sequences derived from Cas9, italic sequences indicate linker sequences, and underlined sequences indicate binomial nuclear localization sequences.

[0223] CP5 (containing MSP "NGC = Pam variant with mutant regular Cas9 such as NGG", PID = protein-protein interaction domain and "D10A" nickase):

[0224]

[0225]

[0226] Non-limiting examples of multinucleotide programmable nucleotide-binding domains that can be incorporated into a base editor include CRISPR protein-derived domains, restriction nucleases, macronucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs).

[0227] In some embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) of any fusion protein provided herein can be a CasX or CasY protein. In some embodiments, napDNAbp is a CasX protein. In some embodiments, napDNAbp is a CasY protein. In some embodiments, napDNAbp comprises an amino acid sequence that has 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% identity to naturally occurring CasX or CasY proteins. In some embodiments, napDNAbp is a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp contains an amino acid sequence that has 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% identity with any CasX or CasY protein described herein. It should be understood that Cas12b / C2c1, CasX, and CasY from other bacterial species may also be used according to this disclosure.

[0228] Cas12b / C2c1(uniprot.org / uniprot / T0D7A2#2)

[0229] sp|T0D7A2|C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acido-terrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB13137 / GD3B) GN=c2c1 PE=1 SV=1

[0230]

[0231] CasX(uniprot.org / uniprot / F0NN87;uniprot.org / uniprot / F0NH53)

[0232] >tr|F0NN87|F0NN87_SULIH CRISPR-associated Cassx protein OS=Sulfolobus islandicus (strain HVE10 / 4)GN=

[0233] SiH_0402PE=4SV=1

[0234] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0235] >tr|F0NH53|F0NH53_SULIR CRISPR-associated protein, Casx OS = Sulfolobus islandicus (strain REY15A) GN = SiRe_0771 PE = 4 SV = 1

[0236] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTSNIILPLSGNDKNPWTETLKCYNFPTTTVALSEVFKNFSQVKECEEVSAPSFVKPFEYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0237] δ- proteobacteria (Deltaproteobacteria) CasX

[0238] MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQK WYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0239] CasY (ncbi.nlm.nih.gov / protein / APG80656.1)

[0240] APG80656.1 CRISPR-associated protein CaSY [uncultured Parcubacteria group bacterium]

[0241]

[0242] The term "conserved amino acid substitution" or "conserved mutation" refers to the replacement of one amino acid with another amino acid that shares a common property. A functional approach to defining the common property 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 these analyses, amino acid groups can be defined where the amino acids within a group preferably exchange with each other and are therefore most similar in their effect on the overall protein structure (Schulz, GE and Schirmer, RH, ibid.). Non-limiting examples of conserved mutations include amino acid substitutions, such as lysine replacing arginine and vice versa to maintain a positive charge; glutamic acid replacing aspartic acid and vice versa to maintain a negative charge; serine replacing threonine to maintain a free -OH group; and glutamine replacing asparagine to maintain a free -NH2 group.

[0243] The terms "coding sequence" or "protein-coding sequence" are used interchangeably herein to refer to a polynucleotide segment that encodes a protein. The region or sequence has a start codon near the 5' end and a stop codon near the 3' end. A coding sequence may also be called an open reading frame.

[0244] The terms "deaminase" or "deaminase domain," as used herein, refer to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., engineered adenosine deaminases, evolved adenosine deaminases) may be derived from organisms such as bacteria. In some implementations, adenosine deaminase is derived from bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Caulobacter crescentus.

[0245] 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 derived from a naturally occurring variant of a 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 has 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% identity with naturally occurring deaminases. For example, the deaminase domain is described in international PCT applications PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is incorporated herein by reference in its entirety.See also Komor, AC, et al., "Programmable base editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, NM, et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage" Nature 551, 464-471 (2017); Komor, AC, et al., "Improved base excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A baseeditors with higher efficiency and product purity" Science Advances 3:eaao4774 (2017) and Rees, HA, et al., "Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet.2018 Dec;19(12):770-788.doi:10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0246] "Detection" refers to identifying the presence, absence, or amount of an analyte to be detected. In one embodiment, sequence changes in a polynucleotide or polypeptide are detected. In another embodiment, the presence of an insertion or deletion (indel) is detected.

[0247] "Detectable label" means that when a composition is attached to a molecule of interest, the molecule can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense agents, enzymes (e.g., as commonly used in ELISA), biotin, digoxigenin, or haptens.

[0248] "Disease" refers to any condition or disorder that damages or interferes with the normal function of cells, tissues, or organs. In the implementation plan, the disease is A1AD.

[0249] The term "effective amount," as used herein, refers to an amount of bioactive agent sufficient to elicit the desired biological response. In a particular embodiment, the effective amount is an amount of a base editor system (e.g., a fusion protein comprising a programmable DNA-binding protein, a nucleobase editor, and gRNA) sufficient to alter A1AT mutations in cells to achieve therapeutic efficacy. This therapeutic efficacy does not need to be sufficient to alter A1AD in all cells of a tissue or organ, but only to alter about 1%, 5%, 10%, 25%, 50%, 75%, or more of the A1AD present in the cells of the subject, tissue, or organ. In one embodiment, the effective amount is sufficient to alleviate one or more symptoms of A1AD. The effective amount of the active agent used in the implementation of therapeutic therapies for diseases varies depending on the method of administration, the subject's age, weight, and general health. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. This amount is referred to as the "effective" amount. In one embodiment, the effective amount is the amount of the base editor of the present invention (e.g., a fusion protein comprising a programmable DNA-binding protein, a nucleobase editor, and gRNA) sufficient to introduce changes into genes of interest in cells (e.g., in vitro or in vivo). In one embodiment, the effective amount is the amount of base editor required to achieve therapeutic efficacy (e.g., reducing or modulating a disease or its symptoms or condition).

[0250] A “fragment” refers to a portion of a polypeptide or nucleic acid molecule. This portion contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0251] "Guide RNA" or "gRNA" refers to a polynucleotide that is specific to a target sequence and can form a complex with a polynucleotide programmable nucleotide-binding domain protein (e.g., Cas9 or Cpf1). In embodiments, the guide polynucleotide is guide RNA (gRNA). gRNA can exist as a complex of two or more RNAs or as a single RNA molecule. A single RNA molecule may be referred to as a single guide RNA (sgRNA), but "gRNA" can be used interchangeably to refer to guide RNA as a single molecule or as a complex of two or more molecules. Typically, gRNA as a single RNA substance contains two domains: (1) a domain that shares homology with the target nucleic acid (e.g., and guides the binding of the Cas9 complex to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and contains a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to tracrRNA, as provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNA (e.g., including domain 2) can be found in U.S. Provisional Patent Application USSN 61 / 874,682, filed September 6, 2013, entitled “Switchable Cas9 Nucleases and Uses Thereof,” and U.S. Provisional Patent Application USSN 61 / 874,746, filed September 6, 2013, entitled “Delivery System For Functional Nucleases,” the entire contents of which are incorporated herein by reference. In some embodiments, the gRNA comprises two or more domains (1) and (2) and may be referred to as “extended gRNA.” The extended gRNA will bind two or more Cas9 proteins and target nucleic acids in two or more different regions, as described herein. gRNA contains a nucleotide sequence complementary to a target site that mediates the binding of the nuclease / RNA complex to that target site, providing sequence specificity for the nuclease:RNA complex. As will be understood by those skilled in the art, RNA polynucleotide sequences, such as gRNA sequences, include the nucleobase uracil (U) (a pyrimidine derivative) rather than the nucleobase thymine (T) (which is included in the DNA polynucleotide sequence). In RNA, the uracil base pairs with adenine and substitutes for thymine during DNA transcription.

[0252] "Hybridization" refers to hydrogen bonds between complementary nucleobases, which can be Watson-Crick, Hoogsteen, or inverse Hoogsteen hydrogen bonds. For example, adenine and thymine are complementary nucleobases that pair up via the formation of hydrogen bonds.

[0253] The term "base repair inhibitor" or "IBR" refers to a protein that inhibits the activity of a nucleic acid repair enzyme, such as a base excision repair (BER) enzyme. In some embodiments, the IBR is an inosine base excision repair inhibitor. Exemplary base repair inhibitors include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4PDG, UDG, hSMUGl, and hAAG. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is a catalytically inactivated EndoV or a catalytically inactivated 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 inactivated EndoV or a catalytically inactivated hAAG.

[0254] In some embodiments, the base repair inhibitor is a uracil glycosidase inhibitor (UGI). UGI refers to a protein that inhibits the uracil-DNA glycosidase base excision repair enzyme. In some embodiments, the UGI domain comprises wild-type UGI or a fragment of wild-type UGI. In some embodiments, the UGI protein provided herein comprises a fragment of UGI and a protein homologous to UGI or a UGI fragment. In some embodiments, the base repair inhibitor is an inosine base excision repair inhibitor. In some embodiments, the base repair inhibitor is a “catalytically inactivated inosine-specific nuclease” or a “dead inosine-specific nuclease.” Not wishing to be limited to any particular theory, a catalytically inactivated inosine glycosidase (e.g., alkyladenine glycosidase (AAG)) can bind inosine but cannot create a base-free site or remove inosine, thereby stereotactically blocking the formation of new inosine moieties and protecting DNA damage / repair mechanisms. In some embodiments, the catalytically inactivated inosine-specific nuclease can be capable of binding inosine in nucleic acids but not cleaving the nucleic acids. Non-limiting exemplary catalytically inactivated inosine-specific nucleases include catalytically inactivated alkyladenine glycosidases (AAG nucleases), for example, from humans, and catalytically inactivated endonuclease V (EndoV nucleases), for example, from *Escherichia coli* (E. coli). In some embodiments, the catalytically inactivated AAG nuclease contains an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0255] "Increase" means a positive change of at least 10%, 25%, 50%, 75%, or 100%.

[0256] An "intein" is a protein fragment that, during a known protein cleavage process, cleaves itself and binds the remaining fragment (exopeptide) with peptide bonds. Inteins are also called "protein introns." The process by which an intein cleaves itself and binds the remaining protein fragment is referred to herein as "protein cleavage" or "intein-mediated protein cleavage." In some embodiments, the intein of the precursor protein (containing the intein of the protein prior to intein-mediated protein cleavage) is derived from two genes. This intein is referred to herein as a splitting intein (e.g., splitting intein-N and splitting intein-C). For example, in cyanobacteria, DnaE, ​​the catalytic subunit of DNA polymerase III, is encoded by two different 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."

[0257] Other intein systems may also be used. For example, a synthetic intein pair based on dnaE intein, Cfa-N (e.g., splitting intein-N) and Cfa-C (e.g., splitting intein-C) intein pairs, has been described (e.g., in Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5, which is incorporated herein by reference). Non-limiting examples of intein pairs that may be used according to this disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, and Cne Prp8 intein (e.g., as described in U.S. Patent 8,394,604, which is incorporated herein by reference).

[0258] Exemplary nucleotide and amino acid sequences of the intended peptide are provided.

[0259] DnaE intima-N DNA:

[0260] TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTCTGCCAATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCGATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGGGGAGAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAGGGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTTCCTAAT

[0261] DnaE intein-N protein:

[0262] CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDR

[0263] GEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN

[0264] DnaE intein-C DNA:

[0265] ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGA

[0266] TATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAGCTTCTAAT

[0267] Intein-C:

[0268] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN

[0269] Cfa-N DNA:

[0270] TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCCTATTGGAAAGATTGTCGAAGAGAGAATTGAATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGGCACAATCGCGGCGAACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACGAGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAATAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTGCCA

[0271] Cfa-N protein:

[0272] CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP

[0273] Cfa-C DNA:

[0274] ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAGGAAAGTAAAGATAATATCTCGAAAAAGTCTTGGTACCCAAAATGTCTATGATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTAGCCAGCAAC

[0275] Cfa-C protein:

[0276] MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN

[0277] Intein-N and intein-C may be fused to the N-terminal and C-terminal portions of cleavage Cas9, respectively, to engage the N-terminal and C-terminal portions of cleavage Cas9. For example, in some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of cleavage Cas9, i.e., to form the structure N--[N-terminal portion of cleavage Cas9]-[intein-N]--C. In some embodiments, intein-C is fused to the N-terminus of the C-terminal portion of cleavage Cas9, i.e., to form the structure N-[intein-C]--[C-terminal portion of cleavage Cas9]-C. The intein-mediated protein cleavage mechanism used to engage the protein fused with the intein (e.g., cleavage Cas9) is well 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 peptides are well known in the art and described in, for example, WO2014004336, WO2017132580, US20150344549 and US20180127780, each of which is incorporated herein by reference in its entirety.

[0278] The terms “isolated,” “purified,” or “biopure” refer to substances that, to varying degrees, are free from the components typically associated with their natural state. “Isolation” indicates the degree of separation from the original source or environment. “Purity” indicates a degree of separation higher than isolation. A “purified” or “biopure” protein is defined as being sufficiently free of other substances such that any impurities do not materially affect the protein’s biological properties 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 substantially free of chemical precursors or other chemicals during chemical synthesis. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” can mean that the nucleic acid or protein produces a substantially single band in the electrophoresis gel. For proteins that can be modified (e.g., phosphorylation or glycosylation), different modifications may produce different isolated proteins that can be purified individually.

[0279] "Separated polynucleotide" means a nucleic acid (e.g., DNA) that does not contain a gene, located flanking the gene in the naturally occurring genome of the organism from which the nucleic acid molecules of this invention are derived. Therefore, the term includes, for example, recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or existing as a distinct molecule independent of other sequences (e.g., cDNA or a fragment of genome or cDNA produced by PCR or restriction endonuclease digestion). Furthermore, the term includes RNA molecules transcribed from DNA molecules, and recombinant DNA as part of a heterozygous gene encoding an additional polypeptide sequence.

[0280] "Isolated polypeptide" means that the polypeptide of the present invention has been isolated from its naturally occurring components. Typically, a polypeptide is considered isolated when it contains at least 60% (by weight) of proteins and naturally occurring organic molecules. Preferably, the formulation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the polypeptide of the present invention. The isolated polypeptide of the present invention can be obtained, for example, by extraction from natural sources, by expression of recombinant nucleic acids encoding such polypeptides, or by chemical synthesis of proteins. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or analysis by HPLC.

[0281] The term "linker," as used herein, can refer to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group, or a molecule that connects two molecules or portions, such as two components of a protein complex or ribonucleoprotein complex, or two fused domain proteins, such as 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., as described in PCT / US19 / 44935). Linkers can connect different components or different portions of a base editor system. For example, in some embodiments, a linker can connect a guide polynucleotide-binding domain of a polynucleotide-programmable nucleotide-binding domain and a catalytic domain of a deaminase. In some embodiments, a linker can connect a CRISPR peptide and a deaminase. In some embodiments, a linker can connect Cas9 and a deaminase. In some embodiments, a linker can connect dCas9 and a deaminase. In some embodiments, a linker can connect nCas9 and a deaminase. In some embodiments, a linker can connect a guide polynucleotide and a deaminase. In some embodiments, the linker may connect the deamination portion of the base editor system and the polynucleotide programmable nucleotide binding portion. In some embodiments, the linker may connect the RNA-binding portion of the deamination portion of the base editor system and the polynucleotide programmable nucleotide binding portion. In some embodiments, the linker may connect the RNA-binding portion of the deamination portion of the base editor system and the RNA-binding portion of the polynucleotide programmable nucleotide binding portion. The linker may be located between or flanking two groups, molecules, or other portions and is connected to each via covalent or non-covalent interactions, thereby connecting the two. In some embodiments, the linker may be an organic molecule, group, polymer, or chemical portion. In some embodiments, the linker may be a polynucleotide. In some embodiments, the linker may be a DNA linker. In some embodiments, the linker may be an RNA linker. In some embodiments, the linker may include an aptamer capable of binding a ligand. In some embodiments, the ligand may be a carbohydrate, peptide, protein, or nucleic acid. In some embodiments, the linker may include an aptamer derived from a riboswitch. The riboswitch of the derived aptamer can be selected from theophylline riboswitch, thiamine pyrophosphate (TPP) riboswitch, adenosylcobalamin (AdoCbl) riboswitch, S-adenosylmethionine (SAM) riboswitch, SAH riboswitch, flavin mononucleotide (FMN) riboswitch, tetrahydrofolate riboswitch, lysine riboswitch, glycine riboswitch, purine riboswitch, GlmS riboswitch, or pre-queosine1 (PreQ1) riboswitch. In some embodiments, the linker may comprise an aptamer that binds to a polypeptide or protein domain, such as a polypeptide ligand.In some embodiments, the peptide ligand may be a K homology (KH) domain, an MS2 capsid protein domain, a PP7 capsid protein domain, an SfMuCom capsid protein domain, a sterile α motif, a telomerase Ku-binding motif and Ku protein, a telomerase Sm7-binding motif and Sm7 protein, or an RNA recognition motif. In some embodiments, the peptide ligand may be part of a base editor system component. For example, the nucleobase editing component may include a deaminase domain and an RNA recognition motif.

[0282] In some embodiments, the linker can be an amino acid or multiple 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 considered.

[0283] In some embodiments, the linker conjugates 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 conjugates dCas9 and the nucleic acid-editing protein. For example, the linker is located between or flanking two groups, molecules, or other parts and is covalently linked to each group, molecule, or other part, thereby linking the two groups, molecules, or other parts together. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical part. In some implementations, 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 may also be considered.

[0284] In some embodiments, the domains of the nucleobase editor are fused via linkers containing the following amino acid sequences: SGGSSGSETPGTSESATPESSGG, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS. In some embodiments, the domains of the nucleobase editor are fused via linkers containing the amino acid sequence SGSETPGTSESATPES, which may also be referred to as the XTEN linker. In some embodiments, the linker contains the amino acid sequence SGGS. In some embodiments, the linker contains (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.

[0285] In some embodiments, the linker is 24 amino acids long. In some embodiments, the linker contains the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids long. In some embodiments, the linker contains the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker is 64 amino acids long. In some embodiments, the linker contains the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS. In some embodiments, the linker is 92 amino acids long. In some embodiments, the linker contains the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0286] "Marker" refers to any protein or polynucleotide whose expression level or activity changes in relation to a disease or condition.

[0287] The term "mutation," as used herein, refers to the substitution of a residue within a sequence (e.g., a nucleic acid or amino acid sequence) by another residue, or the deletion or insertion of one or more residues in a sequence. Mutations are generally described herein by identifying the original residue, followed by its position in the sequence, and then the identity of the newly substituted residue. Various methods for performing the amino acid substitutions (mutations) provided herein are well known in the art and are provided below, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). In some embodiments, the base editor of this disclosure can efficiently generate "desired mutations," such as point mutations, in nucleic acids (e.g., nucleic acids within a subject's genome) without generating a large number of undesired mutations, such as undesired point mutations. In some embodiments, the desired mutation is generated by a specific base editor (e.g., an adenosine base editor) specifically designed to generate the desired mutation and bound to a guide polynucleotide (e.g., gRNA).

[0288] Generally, mutations created or identified in a sequence (e.g., an amino acid sequence, as described herein) are numbered relative to a reference (or wild-type) sequence, i.e., a sequence that does not contain the mutation. Those skilled in the art will readily understand how to determine the location of mutations in amino acid and nucleic acid sequences relative to a reference sequence.

[0289] The term "non-conservative mutation" refers to amino acid substitutions between different groups, such as lysine replacing tryptophan or phenylalanine replacing serine. 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, resulting in increased biological activity compared to the wild-type protein.

[0290] The terms “nuclear localization sequence,” “nuclear localization signal,” or “NLS” refer to an input amino acid sequence that facilitates protein entry into the cell nucleus. Nuclear localization sequences are well known in the art and described, for example, in Plank et al., International PCT Application, PCT / EP2000 / 011690, filed November 23, 2000, and published May 31, 2001 as WO / 2001 / 038547, the contents of which are incorporated herein by reference for exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS, described, for example, in Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4172. In some implementations, the NLS contains the amino acid sequence KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0291] The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to compounds comprising nucleobases and acidic moieties, such as nucleosides, nucleotides, or polymers of nucleotides. Typically, polymeric nucleic acids, such as nucleic acid molecules comprising three or more nucleotides, are linear molecules in which adjacent nucleotides are linked to each other via phosphodiester bonds. 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 polymers of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single-stranded nuclear and / or double-stranded DNA. Nucleic acids can be naturally occurring, for example, in the context of genomes, transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plasmids, mucosomes, chromosomes, chromatids, 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 including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, such as analogs having bonds other than phosphodiester bonds. Nucleic acids can be purified from natural sources, produced using recombinant expression systems, or optionally purified, chemically synthesized, etc. Where appropriate, for example in the case of chemically synthesized molecules, nucleic acids may contain nucleoside analogs such as analogs having chemically modified bases or sugars and modified backbones. Nucleic acid sequences are represented in the 5′ to 3′ orientation 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); or a nucleoside analogue (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine). , 2-aminoadenosine, 7-deadenosine, 7-deadenosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanosine and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose and hexose); and / or modified phosphate groups (e.g., thiophosphates and 5′-N-phosphoramide bonds).

[0292] The terms "nucleic acid programmable DNA-binding protein" or "napDNAbp" are used interchangeably with "polynucleotide programmable nucleotide-binding domain" to refer to a nucleic acid (e.g., DNA or RNA) associated protein, such as a guide nucleic acid or guide polynucleotide (e.g., gRNA), which 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 associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, napDNAbp is a Cas9 domain, such as an active nuclease Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Unrestricted 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.Unrestricted examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, and Cse. 2. Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Cs type Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, their homologs, or modified or engineered versions thereof. Other nucleic acid-programmable DNA-binding proteins are also within the scope of this disclosure, however they may not be specifically listed herein. See, for example, Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct; 1:325-336. doi:10.1089 / crispr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan 4; 363(6422):88-91. doi:10.1126 / science.aav7271, the entire contents of which are incorporated herein by reference.

[0293] The terms “nucleobase,” “nitrogenous base,” or “base” are used interchangeably throughout this document to refer to a nitrogen-containing biological compound that forms nucleosides, which in turn are components of nucleotides. The ability of nucleobases to form base pairs and stack together directly results in long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Five nucleobases—adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U)—are referred to as primitive or standard nucleobases. Adenine and guanine are derived from purines, while cytosine, uracil, and thymine are derived from pyrimidines. DNA and RNA may also contain other (non-primitive) modified bases. Non-limiting examples of modified nucleobases may include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydromethylcytosine. Both hypoxanthine and xanthine can be created via deamination (replacing an amino group with a carbonyl group) in the presence of a mutagen. Hypoxanthine can be modified by adenine. Xanthine can be modified by guanine. Uracil can be the result of deamination from cytosine. A nucleoside consists of a nucleobase and a pentose sugar (ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides with modified nucleobases include inosine (I), xanthine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). A nucleotide consists of a nucleobase, a pentose sugar (ribose or deoxyribose), and at least one phosphate ester group.

[0294] The terms "nucleobase editing domain" or "nucleobase editing protein," as used herein, refer to a protein or enzyme that catalyzes nucleobase modifications in RNA or DNA, such as the deamination of adenine (or adenosine) to hypoxanthine (or inosine), and the addition and insertion of non-template nucleotides. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., adenine deaminase or adenosine deaminase). In some embodiments, the nucleobase editing domain is more than one deaminase domain (e.g., adenine deaminase or adenosine deaminase and cytidine or cytosine deaminase, e.g., as described in PCT / US19 / 44935). In some embodiments, the nucleobase editing domain can be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase editing domain can be an engineered or evolved nucleobase editing domain derived from a naturally occurring nucleobase editing domain. Nucleobase editing domains can originate from any organism, such as bacteria, humans, chimpanzees, gorillas, monkeys, cattle, dogs, rats, or mice.

[0295] As used in this article, “obtaining” in “obtaining agent” includes obtaining the agent by synthesis, purchase, production, preparation or other methods.

[0296] The term "patient" or "subject," as used herein, refers to an animal subject or individual who has been diagnosed with a disease or disorder, is at risk of having or developing a disease or disorder, or is predisposed to having or developing a disease or disorder. In some embodiments, the term "patient" refers to a mammalian subject with a higher-than-average likelihood of developing a disease or disorder. Exemplary patients may be humans, non-human primates, cats, dogs, pigs, cattle, horses, camels, vicuñas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), and other mammals that may benefit from the therapies disclosed herein. Exemplary human patients may be male and / or female.

[0297] "Patients in need" or "subjects in need" in this article refers to patients or subjects who have been diagnosed with, have, are at risk of, are likely to have, or are expected to have a disease or disorder.

[0298] The terms “pathogenic mutation,” “pathogenic variant,” “pathogenic mutation,” “pathogenic agent,” “harmful mutation,” or “predisposing mutation” refer to gene changes or mutations that increase an individual’s predisposition or expectation of a particular disease or ailment. In some embodiments, a pathogenic mutation comprises the substitution of at least one wild-type amino acid by at least one pathogenic amino acid in the protein encoded by the gene.

[0299] The terms “protein,” “peptide,” “polypeptide,” and their grammatical equivalents are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The term refers to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide is at least three amino acids long. A protein, peptide, or polypeptide may refer 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 chemical addition such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or modification, etc. Proteins, peptides, or polypeptides may also be single molecules or multi-molecule complexes. Proteins, peptides, or polypeptides may be simply fragments of naturally occurring proteins or peptides. Proteins, peptides, or polypeptides may be naturally occurring, recombinant, synthetic, or any combination thereof. The term “fusion protein,” as used herein, refers to a hybrid polypeptide containing protein domains from at least two different proteins. A protein may be located at the N-terminal (N-terminal) portion of a fusion protein or at the C-terminal (C-terminal) portion of a fusion protein, thereby forming an N-terminal fusion protein or a C-terminal fusion protein, respectively. The protein may contain various domains, such as nucleic acid-binding domains (e.g., the gRNA-binding domain of Cas9, which guides the protein to bind to a target site) and nucleic acid editing protein cleavage domains or catalytic domains. In some embodiments, the protein comprises a proteinaceous moiety, such as an amino acid sequence constituting the 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 complexed or associated with a nucleic acid (e.g., RNA or DNA). Any of the proteins provided herein can be manufactured using methods known in the art. For example, the proteins provided herein can be manufactured via recombinant protein expression and purification, which is particularly suitable for fusion proteins containing linkers. Methods for the expression and purification of recombinant proteins are well known, including 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.

[0300] The polypeptides and proteins disclosed herein (including functional portions and their functional variants) may contain synthetic amino acids in place of one or more naturally occurring amino acids. These synthetic amino acids are known in the art and include, for example, minocyclohexanecarboxylic acid, leucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, and indole-3-hydroxylamine. Doline-2-carboxylic acid, 2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoacylamine, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornene)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. Peptides and proteins can be associated with post-translational modifications of one or more amino acids in the peptide structure. Non-limiting examples of post-translational modifications include phosphorylation, acylation (including acetylation and formylation), glycosylation (including N- or C-linked), amidation, hydroxylation, alkylation (including methylation and ethylation), ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristylation, palmitoylation, isopreneation, farnesylation, geraniylation, glycosylphosphatidylinositylation, thioctylation, and iodination.

[0301] The term “recombinant,” as used herein, in the context of proteins or nucleic acids, refers to a protein or nucleic acid that is not naturally occurring but is a human-engineered product. For example, in some embodiments, recombinant protein or nucleic acid molecules contain an amino acid or nucleotide sequence that contains 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.

[0302] "Reduction" means a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0303] “Reference” means a standard or control condition. In one embodiment, the reference is wild-type or healthy cells. In other embodiments, and without limitation, the reference is untreated cells, which are control vectors that have not undergone the test conditions or have been given a placebo or saline, medium, buffer, and / or do not contain the polynucleotide of interest.

[0304] A “reference sequence” is a defined sequence used as a basis for sequence comparison. The reference sequence can be a subset or the whole of a specific sequence; for example, a full-length cDNA or gene sequence or a segment of a complete cDNA or gene sequence. For peptides, the reference peptide sequence will typically be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids in length. For nucleic acids, the reference nucleic acid sequence will typically be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides in length, or any integer close to or between these values. In some embodiments, the reference sequence is the wild-type sequence of the protein of interest. In other embodiments, the reference sequence is a polynucleotide sequence encoding a wild-type protein.

[0305] The terms “RNA-programmable nuclease” and “RNA-guided nuclease” are used in conjunction with one or more RNAs that are not cleavage targets (e.g., binding or associating). In some embodiments, an RNA-programmable nuclease, when complexed with RNA, may refer to a nuclease:RNA complex. Typically, the bound RNA is called a guide RNA (gRNA). gRNA may exist as a complex of two or more RNAs or as a single RNA molecule. gRNA existing as a single RNA molecule may be called a single guide RNA (sgRNA), but “gRNA” is used interchangeably to refer to guide RNA existing as a single molecule or as a complex of two or more molecules. Typically, gRNA existing as a single RNA substance contains two domains: (1) a domain that shares homology with the target nucleic acid (e.g., and guides the binding of the Cas9 complex to the target); and (2) a domain that binds the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and contains a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to tracrRNA, as provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNA (e.g., including domain 2) can be found in U.S. Provisional Patent Application No. 6, September 2013, entitled “Switchable Cas9 Nucleases and Uses Thereof”, and U.S. Provisional Patent Application No. 6, September 2013, entitled “Delivery System For Functional Nucleases”, the entire contents of which are incorporated herein by reference. In some embodiments, the gRNA comprises two or more domains (1) and (2) and may be referred to as “extended gRNA”. The extended gRNA will bind two or more Cas9 proteins and bind target nucleic acids in two or more different regions, as described herein. gRNA contains a nucleotide sequence complementary to a target site, which mediates the binding of the nuclease / RNA complex to the target site, providing sequence specificity for the nuclease:RNA complex.

[0306] In some implementations, the RNA-programmable nuclease is a Cas9 endonuclease (CRISPR-related system), for example, Cas9 (Casn1) from Streptococcus pyogenes (see, for example, "Completegenome 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)).

[0307] Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybrids to target target DNA cleavage sites, in principle these proteins can be targeted to any sequence specifically modified by the guide RNA. Methods using RNA-programmable nucleases such as Cas9 for site-specific cleavage (e.g., to modify the genome) are well known in the field (see, for example, Cong, L. et al., Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et ah, RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, WY et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et ah, RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, 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); their respective full contents are incorporated herein by reference.

[0308] The term "single nucleotide polymorphism (SNP)" refers to a single nucleotide variation occurring at a specific location in the genome, where each variation is present to a considerable degree (e.g., >1%) within a population. For example, at a specific base position in the human genome, the C nucleotide may be present in most individuals, but in a minority of individuals, that position is occupied by A. This means there is an SNP at that specific location, and the two possible nucleotide variations, C or A, are called the alleles at that location. SNPs can potentially contribute to differences in disease susceptibility. The severity of illness and how our bodies respond to treatment are also manifestations of genetic variation. SNPs can be located in the coding region of a gene, the non-coding region of a gene, or the intergenic region (the area between genes). In some implementations, due to the degeneracy of the genetic code, SNPs within the coding sequence do not necessarily alter the amino acid sequence of the resulting protein. There are two types of SNPs in coding regions: synonymous SNPs and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, while non-synonymous SNPs alter the amino acid sequence of the protein. There are two types of non-synonymous SNPs: missense and nonsense. SNPs not located 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 an eSNP (expressed SNP), which can be located upstream or downstream of the gene. Single nucleotide variants (SNVs) are variations of a single nucleotide, with no frequency limit, and can occur in somatic cells. Somatic single nucleotide variants are also called single nucleotide alterations.

[0309] "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 or binds to the polypeptide and / or nucleic acid molecules of the present invention in a sample, such as a biological sample, but does not substantially recognize or bind to other molecules.

[0310] Nucleic acid molecules that can be used in the methods of the present invention include any nucleic acid molecule encoding the polypeptide or a fragment thereof of the present invention. Such nucleic acid molecules do not need to be 100% identical to an endogenous nucleic acid sequence, but generally exhibit substantial identity. Polynucleotides having “substantial identity” with an endogenous sequence are generally capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. “Hybridization” refers to pairing under various stringent conditions to form a double-stranded molecule between complementary polynucleotide sequences (e.g., the genes described herein) or portions thereof. (See, for example, Wahl, GM and SLBerger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0311] For example, stringent salt concentrations are typically less than about 750 mM sodium chloride and 75 mM trisodium citrate, preferably less than about 500 mM sodium chloride and 50 mM trisodium citrate, and more preferably less than about 250 mM sodium chloride and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvents such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions typically include temperatures of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. For example, sodium dodecyl sulfate (SDS), and the inclusion or exclusion of vector DNA, are well known to those skilled in the art. Different degrees of stringency are achieved by combining these different conditions as needed. In one embodiment, hybridization will occur at 30°C in 750 mM sodium chloride, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization will occur at 37°C in 500 mM sodium chloride, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In another embodiment, hybridization will occur at 42°C in 250 mM sodium chloride, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be apparent to those skilled in the art.

[0312] For most applications, the stringency of the post-hybridization washing step will vary. The stringency of the washing is defined by the salt concentration and temperature. As mentioned above, the stringency of the washing is increased by decreasing the salt concentration or increasing the temperature. For example, the stringent salt concentration for the washing step is preferably less than about 30 mM sodium chloride and 3 mM trisodium citrate, most preferably less than about 15 mM sodium chloride and 1.5 mM trisodium citrate. The stringent temperature conditions for the washing step typically 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 occur at 25°C in 30 mM sodium chloride, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step will be carried out at 42°C in 15 mM sodium chloride, 1.5 mM trisodium citrate, and 0.1% SDS. In an even more preferred embodiment, the washing step will be carried out at 68°C in 15 mM sodium chloride, 1.5 mM trisodium citrate, and 0.1% SDS. The additional variations in these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and have been described, for example, by 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.

[0313] "Split" means to divide into two or more segments.

[0314] "Split Cas9 protein" or "split Cas9" refers to a Cas9 protein provided as an N-terminal and C-terminal fragment encoded by two distinct nucleotide sequences. The polypeptides corresponding to the N-terminal and C-terminal portions of the Cas9 protein can be cleaved to form a "reconstructed" Cas9 protein. In particular embodiments, the Cas9 protein is split into two fragments within the disordered region of the protein, as described, for example, in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014 or in Jiang et al., (2016) Science 351:867-871. PDB file: 5F9R, each of which is incorporated herein by reference. In some embodiments, the protein is split into two fragments at any C, T, A, or S position within the region of approximately amino acids A292-G364, F445-K483, or E565-T637 of SpCas9, or at the corresponding position in any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other nap DNAbp. In some embodiments, the protein is split into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of splitting the protein into two fragments is referred to as “split” the protein.

[0315] In other embodiments, the N-terminal portion of the Cas9 protein contains amino acids 1-573 or 1-637 of wild-type Streptococcus pyogenes Cas9 (SpCas9) (NCBI reference sequence: NC_002737.2, Uniprot reference sequence: Q99ZW2) or its corresponding position / mutation, and the C-terminal portion of the Cas9 protein contains amino acids 574-1368 or 638-1368 of wild-type SpCas9.

[0316] The C-terminal portion of split Cas9 can bind to the N-terminal portion of split Cas9 to form the complete Cas9 protein. In some embodiments, the C-terminal portion of the Cas9 protein originates from the end of the N-terminal portion of the Cas9 protein. Thus, in some embodiments, the C-terminal portion of split Cas9 contains the amino acid (551-651)-1368 portion of spCas9. "(551-651)-1368" means originating from amino acids between 551 and 651 (inclusive) and edited at amino acid 1368.For example, the C-terminal portion of split Cas9 may contain any of the following amino acid moieties of spCas9: 551-1368, 552-1368, 553-1368, 554-1368, 555-1368, 556-1368, 557-1368, 558-1368, 559-1368, 560-1368, 561-1368, 562-1368, 563-1368, 564-1368, 565-1368, 566-1368, 567-1368, 568-1368, 569-1368, 570-1368, 571-1368, 572-1368, 573... -1368, 574-1368, 575-1368, 576-1368, 577-1368, 578-1368, 579-1368, 580-1368, 581-1368, 582-1368, 583-1368, 584-1368, 585-1368, 586-1368, 587-1368, 588-1368, 589-1368, 590-1368, 591-1368, 592-1368, 593-1368, 594-1368, 595-1368, 596-1368, 597-1368, 598-1368, 599-1 368, 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, 625-136 8. 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 implementations, the C-terminal portion of the Cas9 protein contains amino acid 574-1368 or 638-1368 of SpCas9.

[0317] "Serpin1A polynucleotide" refers to a nucleic acid molecule encoding the A1AT protein or a fragment thereof. An exemplary Serpin1A polynucleotide sequence is available from NCBI accession number NM_000295 and is provided below:

[0318] 1 acaatgactc ctttcggtaa gtgcagtgga agctgtacac tgcccaggca aagcgtccgg

[0319] 61 gcagcgtagg cgggcgactc agatcccagc cagtggactt agcccctgtt tgctcctccg

[0320] 121 ataactgggg tgaccttggt taatattcac cagcagcctc ccccgttgcc cctctggatc

[0321] 181 cactgcttaa atacggacga ggacagggcc ctgtctcctc agcttcaggc accaccactg

[0322] 241 acctgggaca gtgaatcgac aatgccgtct tctgtctcgt ggggcatcct cctgctggca

[0323] 301 ggcctgtgct gcctggtccc tgtctccctg gctgaggatc cccagggaga tgctgcccag

[0324] 361 aagacagata catcccacca tgatcaggat cacccaacct tcaacaagat cacccccaac

[0325] 421 ctggctgagt tcgccttcag cctataccgc cagctggcac accagtccaa cagcaccaat

[0326] 481 atcttcttct ccccagtgag catcgctaca gcctttgcaa tgctctccct ggggaccaag

[0327] 541 gctgacactc acgatgaaat cctggagggc ctgaatttca acctcacgga gattccggag

[0328] 601 gctcagatcc atgaaggctt ccaggaactc ctccgtaccc tcaaccagcc agacagccag

[0329] 661 ctccagctga ccaccggcaa tggcctgttc ctcagcgagg gcctgaagct agtggataag

[0330] 721 tttttggagg atgttaaaaa gttgtaccac tcagaagcct tcactgtcaa cttcggggac

[0331] 781 accgaagagg ccaagaaaca gatcaacgat tacgtggaga agggtactca agggaaaatt

[0332] 841 gtggatttgg tcaaggagct tgacagagac acagtttttg ctctggtgaa ttacatcttc

[0333] 901 tttaaaggca aatgggagag accctttgaa gtcaaggaca ccgaggaaga ggacttccac

[0334] 961 gtggaccagg tgaccaccgt gaaggtgcct atgatgaagc gtttaggcat gtttaacatc

[0335] 1021 cagcactgta agaagctgtc cagctgggtg ctgctgatga aatacctggg aatgccacc

[0336] 1081 gccatcttct tcctgcctga tgaggggaaa ctacagcacc tggaaaatga ctcacccac

[0337] 1141 gatatcatca ccaagttcct ggaaatga gacagaaggt ctgccagctt catttaccc

[0338] 1201 aaactgtcca ttactggaac ctatgatctg aagagcgtcc tgggtcaact ggcatcact

[0339] 1261 aaggtcttca gcaatggggc tgacctctcc ggggtcacag aggaggcacc ctgaagctc

[0340] 1321 tccaaggccg tgcataaggc tgtgctgacc atcgac g aga aagggactga gc tgctggg

[0341] 1381 gccatgtttt tagaggccat acccatgtct atccccccg aggtcaagtt aacaaaccc

[0342] 1441 tttgtcttct taatgattga acaaaatacc aagtctcccc tcttcatggg aaagtggtg

[0343] 1501 aatcccaccc aaaataact gcctctcgct cctcaacccc tcccctccat cctggcccc

[0344] 1561 ctccctggat gacattaaag aagggttgag ctggtccctg cctgcatgtg ctgtaaatc

[0345] 1621 cctcccatgt tttctctgag tctccctttg cctgctgagg ctgtatgtgg ctccaggta

[0346] 1681 acagtgctgtcttcgggccc cctgaactgt gttcatggag catctggctg gtaggcaca

[0347] 1741 tgctgggctt gaatccaggg gggactgaat cctcagctta cggacctggg ccatctgtt

[0348] 1801 tctggagggc tccagtcttc cttgtcctgt cttggagtcc ccaagaagga tcacagggg

[0349] 1861 aggaaccaga taccagccat gaccccaggc tccaccaagc atcttcatgt cccctgctc

[0350] 1921 atcccccact cccccccacc cagagttgct catcctgcca gggctggctg gcccacccc

[0351] 1981 aaggctgccc tcctgggggc cccagaactg cctgatcgtg ccgtggccca ttttgtggc

[0352] 2041 atctgcagca acacaagaga gaggacaatg tcctcctctt gacccgctgt acctaacca

[0353] 2101 gactcgggcc ctgcacctct caggcacttc tggaaaatga ctgaggcaga tcttcctga

[0354] 2161 agcccattct ccatggggca acaaggacac ctattctgtc cttgtccttc atcgctgcc

[0355] 2221 ccagaaagcc tcacatatct ccgtttagaa tcaggtccct tctccccaga gaagaggag

[0356] 2281 ggtctctgct ttgttttctc tatctcctcc tcagacttga ccaggcccag aggccccag

[0357] 2341 aagaccatta ccctatatcc cttctcctcc ctagtcacat ggccataggc tgctgatgg

[0358] 2401 ctcaggaagg ccattgcaag gactcctcag ctatgggaga ggaagcacat acccattga

[0359] 2461 cccccgcaac ccctcccttt cctcctctga gtcccgactg gggccacatg agcctgact

[0360] 2521 tctttgtgcc tgttgctgtc cctgcagtct tcagagggcc accgcagctc agtgccacg

[0361] 2581 gcaggaggct gttcctgaat agcccctgtg gtaagggcca ggagagtcct ccatcctcc

[0362] 2641 aaggccctgc taaaggacac agcagccagg aagtcccctg ggcccctagc gaaggacag

[0363] 2701 cctgctccct ccgtctctac caggaatggc cttgtcctat ggaaggcact ccccatccc

[0364] 2761 aaactaatct aggaatcact gtctaaccac tcactgtcat gaatgtgtac taaaggatg

[0365] 2821 aggttgagtc ataccaaata gtgatttcga tagttcaaaa tggtgaaatt gcaattcta

[0366] 2881 catgattcag tctaatcaat ggataccgac tgtttcccac acaagtctcc gttctctta

[0367] 2941 agcttactca ctgacagcct ttcactctcc acaaatacat taaagatatg ccatcacca

[0368] 3001 agccccctag gatgacacca gacctgagag tctgaagacc tggatccaag tctgacttt

[0369] 3061 tccccctgac agctgtgtga ccttcgtgaa gtcgccaaac ctctctgagc ccagtcatt

[0370] 3121 gctagtaaga cctgcctttg agttggtatg atgttcaagt tagataacaa atgtttata

[0371] 3181 cccattagaa cagagaataa atagaactac atttcttgca

[0372] The PAM sequence is highlighted, and the correct sequence with adenine base editing is shown.

[0373] "Subject" means mammal, including but not limited to human or non-human mammals such as cattle, horses, dogs, sheep, or cats. Subjects include livestock, domesticated animals raised for productive labor and to provide goods such as food, including but not limited to cattle, goats, chickens, horses, pigs, rabbits, and sheep.

[0374] "Substantial identity" means that the polypeptide or nucleic acid molecule is identical to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). In one embodiment, such a sequence is at least 60%, 80%, 85%, 90%, 95%, or even 99% identical to the sequence used for comparison at the amino acid or nucleic acid level.

[0375] Sequence identity is typically measured using sequence analysis software (e.g., the Genetics Computing Group Sequence Analysis Suite, 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 for various substitutions, deletions, and / or other modifications. Conserved substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, where e -3 and e -100 The probability scores between them represent closely related sequences.

[0376] Use COBALT, for example, with the following parameters:

[0377] a) Alignment parameters: Vacancy penalty -11, -1 and end-vacancy penalty -5, -1

[0378] b) CDD parameters: Enable RPS BLAST; Blast E-value 0.003; Find conservative columns and enable Recompute, and

[0379] c) Query clustering parameters: Enable query clustering; word length 4; maximum clustering distance 0.8; Abet Regular.

[0380] Use the EMBOSS Needle, for example, with the following parameters:

[0381] a) Matrix: BLOSUM62;

[0382] b) GAP OPEN: 10;

[0383] c) GAP EXTEND: 0.5;

[0384] d)OUTPUT FORMAT: Yes;

[0385] e)END GAP PENALTY: Fake;

[0386] f)END GAP OPEN: 10; and

[0387] g)END GAP EXTEND: 0.5.

[0388] The term "target site" refers to a sequence within a nucleic acid molecule that has been modified by a nucleobase editor. In one embodiment, the target site undergoes deamination via a deaminase or a fusion protein containing a deaminase (e.g., adenine deaminase).

[0389] As used herein, the terms “treat,” “treating,” “treatment,” etc., refer to reducing or alleviating a disease and / or its associated symptoms or achieving a desired pharmacological and / or physiological effect. It should be understood that, although not excluded, treating a disease or condition does not require the complete elimination of the disease, condition, or its associated symptoms. In some embodiments, the effect is therapeutic, i.e., but not limited to, the effect partially or completely reduces, weakens, eliminates, alleviates, lessens, reduces in intensity, and / or cures the disease and / or undesirable symptoms attributable to the disease. In some embodiments, the effect is preventative, i.e., the effect protects against or prevents the occurrence or recurrence of the disease or condition. For this purpose, the methods of this disclosure comprise administering a therapeutically effective amount of the composition as described herein. In an embodiment, the disease is alpha-1 antitrypsin deficiency (A1AD).

[0390] "Uracil glycosidase inhibitor" or "UGI" refers to an agent that inhibits the uracil excision repair system. In one embodiment, the agent is a protein or fragment thereof that binds to a host uracil-DNA glycosylase and prevents the removal of uracil residues from DNA. In one embodiment, the UGI is a protein, fragment thereof, or domain capable of inhibiting the uracil-DNA glycosylase 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 exemplary amino acid sequence set forth below. In some embodiments, the UGI fragment comprises an amino acid sequence comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the exemplary UGI sequences provided below. In some embodiments, the UGI comprises an amino acid sequence homologous to the exemplary UGI amino acid sequence or a fragment thereof, as described below. In some embodiments, the UGI or a portion thereof has 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% identity with a wild-type UGI or a portion thereof, as described below. An exemplary UGI comprises an amino acid sequence as follows:

[0391] >splP14739IUNGI_BPPB2 Uracil-DNA glycosidase inhibitor

[0392] MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML.

[0393] The term "vector" refers to a mechanism that introduces a nucleic acid sequence into a cell, resulting in cell transformation. Vectors include plasmids, transposons, bacteriophages, viruses, liposomes, and cell-free genomes. An "expression vector" is a nucleic acid sequence containing a nucleotide sequence that will be expressed in the recipient cell. Expression vectors may include additional nucleic acid sequences to promote and / or facilitate the expression of the introduced sequence, such as initiation, termination, enhancer, promoter, and secretion sequences.

[0394] Any composition or method provided herein may be combined with any one or more other compositions or methods provided herein.

[0395] The terms “some embodiments,” “implementation,” “one embodiment,” or “other embodiments” used in reference to the specification mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of this disclosure, but not necessarily in all embodiments.

[0396] References to a list of chemical groups in any definition of a variable herein include defining that variable as any single group or combination of the listed groups. Descriptions of embodiments of a variable or aspect herein include embodiments as a single embodiment or as a combination of any other embodiment or part thereof.

[0397] DNA editing has become a viable means of altering disease states by correcting pathogenic mutations at the gene level. Until recently, all DNA editing platforms functioned by inducing DNA double-strand breaks (DSBs) at specific genomic sites and relying on endogenous DNA repair pathways to determine product outcomes in a semi-random manner, resulting in complex populations of gene products. While precise, user-defined repair outcomes can be achieved through the homology-directed repair (HDR) pathway, several challenges have prevented efficient repair using HDR in treatment-related cell types. In fact, this pathway is inefficient compared to the competitive, error-prone non-homologous end joining pathway. Furthermore, HDR is strictly limited to the G1 and S phases of the cell cycle, preventing precise repair of DSBs in post-mitotic cells. Therefore, it has proven difficult or impossible to efficiently alter genome sequences in these populations in a user-defined, programmable manner. Attached Figure Description

[0398] Figures 1A to 1C Describe the plasmid. Figure 1A This is an expression vector for encoding the TadA7.10-dCas9 base editor. Figure 1B This is a plasmid containing nucleic acid molecules encoding proteins that confer resistance to chloramphenicol (CamR) and spectinomycin (SpectR). The plasmid also contains a kanamycin resistance gene that is disabled through two point mutations. Figure 1C This is a plasmid containing nucleic acid molecules encoding proteins that confer resistance to chloramphenicol (CamR) and spectinomycin (SpectR). The plasmid also contains a kanamycin resistance gene that is disabled through two point mutations.

[0399] Figure 2 For Figure 1A The bacterial community transduced by the expression vector depicted in Figure C includes a deactivated kanamycin resistance gene. The vector contains the ABE7.10 variant, generated using error-prone PCR. Bacterial cells expressing these "evolved" ABE7.10 variants were selected against kanamycin antibodies using increased concentrations of kanamycin. Bacteria expressing the ABE7.10 variant with adenosine deaminase activity were able to correct the mutation in the introduced kanamycin resistance gene, thereby restoring kanamycin resistance. Kanamycin-resistant cells were selected for further analysis.

[0400] Figure 3 A graph to quantify the efficiency and specificity of the selected ABE8s listed in Table 6. Detection of the α-1 antitrypsin locus in HEK293T cells.

[0401] Figure 4A and 4B This section elucidates the editing efficiency and specificity of ABE8. Figure 4A and 4B A graph illustrating the base editing and specificity of the selected ABE8 enzymes listed in Table 6 for quantification. Detection of single, variant TadA deaminase domains, or wild-type TadA deaminases.

[0402] Figure 5 The presented charts show the efficiency of ABE8 in editing target adenine (A) bases and bystander A. Specifically, ABE8 caused a 5-fold increase in editing (i.e., the A·T to G·C conversion) at the A1AD site compared to the efficient TadA deaminase ABE7.10.

[0403] Figures 6A to 6D Nucleic acid sequences, tables, and bar charts are presented in relation to improved nucleobase correction rates in primary PiZ fibroblasts via base editor engineering. Figure 6A The DNA sequence encoding the target site of the PiZ mutation associated with A1AD is displayed. The sequence includes a 20-nucleotide protospacer sequence and a non-standard spCas9 NGC PAM. Figure 6B A table describing the TadA deaminase and Cas9 PAM variant components used to correct PiZ mutations is presented. Figure 6Cand 6D The presented bar charts depict the editing rates observed in patient-derived PiZZ fibroblasts (GM11423 Corriel Biorepository) transfected with base editing reagents using the Neon electroporation system. Each treatment consisted of 10 μl of electroporation buffer containing 70,000 fibroblasts, 100 ng of mRNA encoding the base editor, and 50 ng of A-1 correction gRNA. After 48 hours of recovery, cells were lysed and loci of interest were explored via targeted amplicon sequencing. Data were obtained from two independent experiments. These data and results demonstrate the optimization of NGC PAM recognition (variants 1–3). Figure 6B and 6C ) and optimized via the incorporation of ABE8 / 9 mutant TadA deaminases (variants 4-9, Figures 6B to 6D ).

[0404] Figures 7A to 7D Nucleic acid sequences, tables, and figures related to serum A1AT enhancement generated by lipid nanoparticle (LNP)-mediated delivery and base editor in NSG-PiZ transgenic mice. Figure 7A The sequence shows the original spacer sequence, which includes 20 nucleotides, and the target site DNA sequence of the non-standard spCas9 NGC PAM. Figure 7B Tables describing the TadA deaminase and Cas9 PAM variant components used to correct PiZ mutations are presented. Figure 7C This figure depicts the editing rate observed in whole liver gDNA from an NSG-PiZ transgenic mouse model after 7 days of treatment with 1.5 mg / kg LNP containing gRNA and mRNA encoding a base editor in a 1:1 weight ratio. Commercially available NSG-PiZ mice expressed a mutant human SERPINA1 (Glu342Lys mutation) against an immunodeficient NOD-SCIDγ (NSG) background, which provides a stable background for human hepatocytes after partial hepatectomy (The Jackson Laboratory, Mount Desert Island, ME). The results indicate that ngcABEvar9 produced a higher editing rate than the earlier variant 8. Figure 7D The results showed an editing rate associated with an increase in serum α1-antitrypsin relative to pretreated samples, as determined by an MSD sandwich immunoassay. Based on these results, base editing with the ABE8 agent can resolve α1-antitrypsin deficiency and its potential pulmonary sequelae.

[0405] Figure 8A table is provided to depict all possible PAMs within the NRNN PAM space. Only Cas9 variants requiring identification of three or fewer defining 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), ( / / d oi.org / 10.1038 / s41587- 020-0412-8 (The content of which is incorporated into this article in its entirety by way of quotation). Detailed Implementation

[0406] As described below, the present invention is characterized by compositions and methods for altering mutations associated with α-1 antitrypsin deficiency (A1AD). In some embodiments, the edit corrects the harmful mutation such that the edited polynucleotide is indistinguishable from a wild-type reference polynucleotide sequence. In another embodiment, the edit alters the harmful mutation such that the edited polynucleotide contains a benign mutation.

[0407] This invention is based, at least in part, on the discovery that a base editor containing adenosine deaminase variants can effectively and accurately edit harmful mutations associated with A1A.

[0408] A1-Antipase Deficiency (A1AD)

[0409] Alpha-1 antitrypsin (A1A) is a protease inhibitor encoded by the SERPINA1 gene on chromosome 14. In healthy adults, this glycoprotein is primarily synthesized in the liver and secreted into the bloodstream, with serum concentrations ranging from 1.5 to 3.0 g / L (20 to 52 μmol / L). It diffuses into the pulmonary interstitium and alveolar lining fluid, inactivating neutrophil elastase and thus protecting lung tissue from protease-mediated damage. Alpha-1 antitrypsin deficiency (A1AD) is inherited in an autosomal codominant manner. More than 100 genetic variants of the SERPINA1 gene have been described, but not all are associated with the disease. The letter names of these variants are based on their migration rate on gel electrophoresis. The most common variant is the M (moderate migration) allele (PiM), and the two most common defective alleles are PiS and PiZ (the latter migrating the slowest). Several mutations that do not produce measurable serum proteins have been described; these are referred to as “empty” alleles. The most common genotype is MM, which produces normal serum levels of α-1 antitrypsin. Most individuals with severe deficiency are homozygous for the Z allele (ZZ). In the United States, over 60,000 patients with A1AD have a severe ZZ phenotype. The Z protein misfolds and polymerizes during its production in the endoplasmic reticulum of hepatocytes; these abnormal polymers are trapped in the liver, significantly reducing serum levels of α-1 antitrypsin. Insufficient or unstable A1AT production causes liver and / or lung lesions in patients with A1AD. Liver disease seen in patients with α-1 antitrypsin deficiency is caused by the accumulation of abnormal α-1 antitrypsin protein in hepatocytes and the ensuing cellular responses, including autophagy, endoplasmic reticulum stress, and apoptosis. Decreased circulating levels of α-1 antitrypsin lead to increased elastase activity in pulmonary neutrophils; this imbalance between the protease and antiprotease contributes to lung disease associated with this condition.

[0410] Alpha-1 antitrypsin deficiency (“A1AD”) is most common in Caucasians and most frequently affects the lungs and liver. In the lungs, the most common presentation is early-onset (in patients in their 30s and 40s) panalveolar emphysema most prominently seen at the bases of the lungs. However, diffuse or upper lobe emphysema, as well as bronchiectasis, may occur. The most commonly described symptoms include dyspnea, wheezing, and cough. Pulmonary function tests in affected individuals show results consistent with COPD; however, bronchodilator responsiveness may be observed and may be misdiagnosed as asthma. Liver disease caused by the ZZ genotype presents in multiple forms. Affected infants may present with cholestatic jaundice in the neonatal period, sometimes accompanied by abilious stools (pale or clay-colored) and hepatomegaly. Elevated levels of conjugated bilirubin, transaminases, and gamma-glutamyl transferase in the blood are observed. Liver disease in older children and adults may incidentally reveal elevated transaminases or signs of cirrhosis, including variceal bleeding or ascites. Alpha-1 antitrypsin deficiency also makes patients more susceptible to hepatocellular carcinoma. While the homozygous ZZ genotype is essential for the development of liver disease, heterozygous Z mutations can act as genetic modifiers for other diseases by conferring a greater risk of more severe liver diseases, such as hepatitis C infection and cystic fibrosis.

[0411] The two most common clinical variants of A1AD are the E264V (PiS) and E342K (PiZ) alleles. The clinical single nucleotide variant E342K (PiZ) results in unstable and / or inactivated A1AT protein, leading to liver and lung toxicity. The inheritance pattern is autosomal codominant. More than half of A1AD patients carry at least one copy of the E342K mutation.

[0412]

[0413] In some embodiments, the disease or disorder is α1-antitrypsin deficiency (A1AD). In some embodiments, the pathogenic mutation is located in the gene SERPINA1. In some embodiments, the mutation in SERPINA1 is E342K (PiZ allele). In some embodiments, the A at position 7 is edited to G to reverse the PiZ allele to the wild-type allele.

[0414] Nucleotide base editor

[0415] This document discloses a base editor or nucleobase editor for editing, modifying, or altering target nucleotide sequences of polynucleotides. It describes a nucleobase editor or base editor comprising a polynucleotide programmable nucleotide-binding domain and a nucleobase editing domain (e.g., adenosine deaminase). The polynucleotide programmable nucleotide-binding domain, upon binding to a guide polynucleotide (e.g., gRNA), specifically binds to a target polynucleotide sequence (i.e., via a complementary base pair between the bases of the bound guide nucleic acid and the bases of the target polynucleotide sequence) and thereby positions the base editor to the target nucleic acid sequence to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid.

[0416] Multinucleotide programmable nucleotide binding domain

[0417] It should be understood that polynucleotide-programmable nucleotide-binding domains may also include nucleic acid-programmable proteins that bind RNA. For example, a polynucleotide-programmable nucleotide-binding domain can associate with a nucleic acid that guides the polynucleotide-programmable nucleotide-binding domain to RNA. Other nucleic acid-programmable DNA-binding proteins are also within the scope of this disclosure, however, they are not specifically listed herein.

[0418] The polynucleotide-programmable nucleotide-binding domain of the base editor may itself contain one or more domains. For example, the polynucleotide-programmable nucleotide-binding domain may contain one or more nuclease domains. In some embodiments, the nuclease domain of the polynucleotide-programmable nucleotide-binding domain may contain an endonuclease or an exonuclease. Hereinafter, the term "exonuclease" refers to a protein or polypeptide capable of digesting nucleic acids (e.g., RNA or DNA) from its free end, and the term "endonuclease" refers to a protein or polypeptide capable of catalyzing (e.g., cleaving) internal regions within nucleic acids (e.g., RNA or DNA). In some embodiments, the endonuclease may cleave a single strand of a double-stranded nucleic acid. In some embodiments, the endonuclease may 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.

[0419] In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain can cleave the 0, 1, or both strands of the target polynucleotide. In some embodiments, the polynucleotide programmable nucleotide binding domain may include a nicking enzyme domain. Hereinafter, the term "nicking enzyme" refers to a polynucleotide programmable nucleotide binding domain containing a nuclease domain capable of cleaving only one strand of a double-stranded nucleic acid molecule (e.g., DNA). In some embodiments, the nicking enzyme can be derived from a fully catalytically activated (e.g., native) form of the polynucleotide programmable nucleotide binding domain by introducing one or more mutations into the active polynucleotide programmable nucleotide binding domain. For example, when the polynucleotide programmable nucleotide binding domain includes a Cas9-derived nicking enzyme domain, the Cas9-derived nicking enzyme domain may include a D10A mutation and a histidine residue at position 840. In these embodiments, residue H840 retains catalytic activation and thus the ability to cleave a single strand of the nucleic acid duplex. In another embodiment, the Cas9-derived nicking enzyme 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 polynucleotide programmable nucleotide-binding domain having a fully catalytically activated (e.g., native) form by removing all or part of the nuclease domains that are not required for nickase activity. For example, in cases where the polynucleotide programmable nucleotide-binding domain contains a nickase domain derived from Cas9, the Cas9-derived nickase domain may contain all or part of the deletion of the RuvC domain or the HNH domain.

[0420] An exemplary amino acid sequence for catalytically activating Cas9 is as follows:

[0421]

[0422] Base editors containing nicking enzyme domains and polynucleotide programmable nucleotide-binding domains can thus create single-strand DNA breaks (nicks) at specific polynucleotide target sequences (e.g., determined by the complementary sequence of the bound guide nucleic acid). In some embodiments, the strand of the double-stranded target polynucleotide sequence of the nucleic acid cleaved by a base editor containing a nicking enzyme domain (e.g., a Cas9-derived nicking enzyme domain) is the strand that is not edited by the base editor (i.e., the strand cleaved by the base editor is the opposite of the strand containing the base to be edited). In other embodiments, a base editor containing a nicking enzyme domain (e.g., a Cas9-derived nicking enzyme domain) can cleave a strand of DNA molecule that is targeted for editing. In such embodiments, the non-target strand is not cleaved.

[0423] This document also provides a base editor comprising a polynucleotide programmable nucleotide-binding domain that is catalytically dead (i.e., unable to cleave the target polynucleotide sequence). In this document, the terms "catalytically dead" and "nuclease dead" are used interchangeably to refer to a polynucleotide programmable nucleotide-binding domain having one or more mutations and / or deletions that prevent it from cleaving the nucleic acid strand. In some embodiments, the catalytically dead polynucleotide programmable nucleotide-binding domain base editor may lack nuclease activity as a result of specific point mutations in one or more nuclease domains. For example, in the case where the base editor comprises a Cas9 domain, Cas9 may contain both the D10A and H840A mutations. These mutations inactivate both nuclease domains, thereby resulting in a loss of nuclease activity. In other embodiments, the catalytically dead polynucleotide programmable nucleotide-binding domain may comprise one or more deletions of all or part of a catalytic domain (e.g., the RuvC1 and / or HNH domains). In a further embodiment, the catalytic death polynucleotide programmable nucleotide-binding domain comprises all or part of a point mutation (e.g., D10A or H840A) and a deletion of the nuclease domain.

[0424] This document also considers mutations that can generate catalytic death polynucleotide programmable nucleotide binding domains from prior functional versions of polynucleotide programmable nucleotide binding domains. For example, in the case of catalytic death Cas9 (“dCas9”), variants are provided having mutations other than D10A and H840A, which result in nuclease inactivation of Cas9. Such mutations include, for example, other amino acid substitutions at D10 and H840, or other substitutions within the Cas9 nuclease domain (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). Based on this disclosure and knowledge in the art, additional suitable nuclease-inactivating dCas9 domains will be apparent to those skilled in the art and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactivating Cas9 domains include, but are not limited to, the D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, for example, Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9):833-838, the entire contents of which are incorporated herein by reference).

[0425] Non-limiting examples of polynucleotide-programmable nucleotide-binding domains that can be incorporated into a base editor include CRISPR protein-derived domains, restriction nucleases, macronucleases, TALENs (taurases), and zinc finger nucleases (ZFNs). In some embodiments, the base editor comprises a polynucleotide-programmable nucleotide-binding domain comprising a native or modified protein or a portion thereof, which is capable of binding to a nucleic acid sequence via a bound guide nucleic acid during CRISPR (i.e., clustered regularly spaced short palindromic repeats)-mediated nucleic acid modification. Such proteins are referred to herein as “CRISPR proteins.” Therefore, this document discloses a base editor comprising a polynucleotide-programmable nucleotide-binding domain comprising all or part of a CRISPR protein (i.e., a base editor comprising all or part of a CRISPR protein as a domain, also referred to as a “CRISPR protein-derived domain” of the base editor). The CRISPR protein-derived domain incorporated into the base editor may be modified relative to the wild-type or native version of the CRISPR protein. For example, CRISPR protein-derived domains, as described below, may contain one or more mutations, insertions, deletions, rearrangements, and / or recombinations relative to the wild-type or natural version of the CRISPR protein.

[0426] CRISPR provides protection for the adaptive immune system against mobile genetic elements (viruses, transposable elements, and conjugating plasmids). A CRISPR cluster contains a spacer sequence, a sequence complementary to the precursor mobile element, and a target invading nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, proper processing of pre-crRNA requires a reverse-coding small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. tracrRNA acts as a guide for ribonuclease 3-assisted processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonuclease cleaves the linear or circular dsDNA target complementary to the spacer sequence. The target strand not complementary to the crRNA is first endonucleated, followed by 3'-5' exonuclease trimming. In nature, DNA binding and cleavage typically require both proteins and two RNAs. However, a single guide RNA (“sgRNA” or simply “gNRA”) can be engineered to incorporate aspects of both crRNA and tracrRNA into a single RNA material. See, for example, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 identifies short motifs (PAM or adjacent motifs of the original spacer sequence) in CRISPR repeat sequences to help distinguish itself from non-self motifs.

[0427] In some implementations, the methods described herein may utilize engineered Cas proteins. Guide RNA (gRNA) is a short synthetic RNA consisting of a scaffold sequence required for Cas binding and a user-defined spacer sequence of approximately 20 nucleotides that defines the genomic target to be modified. Therefore, those skilled in the art can alter the genomic target specificity of the Cas protein, determined in part by how the specificity of the gRNA targeting sequence for the genomic target compares to its specificity for the rest of the genome.

[0428] In some implementations, the gRNA scaffold sequence is as follows: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAUAAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU.

[0429] In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is an endonuclease (e.g., deoxyribonuclease or ribonuclease) that binds to a target polynucleotide upon binding to the guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is a nickase that binds to a target polynucleotide upon binding to the guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is a catalytic death domain that binds to a target polynucleotide upon binding to the 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.

[0430] The Cas proteins that can be used in this paper include class 1 and class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, and Cmr6. CRISPR enzymes include 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, their homologs or modified versions thereof. Unmodified CRISPR enzymes can have DNA cleavage activity, such as Cas9, which has two functional endonuclease domains: RuvC and HNH. CRISPR enzymes can guide the cleavage of one or both strands of a target sequence, such as within the target sequence and / or within its complement. For example, a CRISPR enzyme can guide the cleavage of one or both strands of a target sequence that are approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more bases away from the first or last nucleotide.

[0431] A vector encoding a CRISPR enzyme may be used, which is mutated relative to the wild-type enzyme so that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. Cas9 may refer to a polypeptide that has at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology with a wild-type exemplary Cas9 (e.g., Cas9 from Streptococcus pyogenes). Cas9 can refer to a wild-type exemplary Cas9 (e.g., Cas9 from Streptococcus pyogenes) 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. Cas9 can refer to wild-type or modified Cas9 proteins that may contain amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or combinations thereof.

[0432] In some implementations, the CRISPR protein-derived domain of the base editor may include all or part of the following Cas9: *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_021846.1). Ref:NC_021314.1); Belliella baltica (NCBI Ref:NC_018010.1); Psychroflexus torquisI (NCBI Ref:NC_018721.1); Streptococcus thermophilus (NCBI Ref:YP_820832.1); Listeria innocua (NCBI Ref:NP_472073.1); Campylobacter jejuni (NCBI Ref:YP_002344900.1); Neisseria meningitides (NCBI Ref:YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus.

[0433] Cas9 domain of the nucleobase editor

[0434] The Cas9 nuclease sequence and structure are well known to those skilled in the art (see, for example, "Complete genome sequence of an Ml 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 F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin R.E., Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 heterologs have been described in a variety of species, including, but not limited to, Streptococcus pyogenes and Streptococcus thermophilus.Additional suitable Cas9 nuclease nuclear sequences will be readily apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences described in the following organisms and loci: 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.

[0435] In some embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) is a Cas9 domain. Non-limiting, illustrative Cas9 domains are provided herein. The Cas9 domain can be a nuclease-active Cas9 domain, a nuclease-inactivating Cas9 domain (dCas9), or a Cas9 cleavage enzyme (nCas9). In some embodiments, the Cas9 domain is a nuclease-active domain. For example, the Cas9 domain can be a Cas9 domain that cleaves the double strand of a double-stranded nucleic acid (e.g., the double strand of a double-stranded DNA molecule). In some embodiments, the Cas9 domain comprises any of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that has 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% identity with any of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any of the amino acid sequences described herein. In some embodiments, the 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 consecutive amino acid residues identical to any of the amino acid sequences described herein.

[0436] 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-cleaving domain of Cas9. In some embodiments, a protein comprising Cas9 or a fragment thereof is referred to as a “Cas9 variant”. Cas9 variants share homology with Cas9 or a fragment thereof. For example, Cas9 variants have at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity with wild-type Cas9. In some implementations, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA-binding domain or a DNA-cleavage domain) such that the fragment has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity with the corresponding fragment of wild-type Cas9. In some embodiments, the fragment corresponds to 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% identity, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid length of 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.

[0437] In some embodiments, such as the Cas9 fusion protein provided herein, the Cas9 fusion protein comprises the full-length amino acid sequence of the Cas9 protein, for example, one of the Cas9 sequences provided herein. However, in other embodiments, such as the fusion protein provided herein, the fusion protein does not comprise the full-length Cas9 sequence, but only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments are readily apparent to those skilled in the art.

[0438] The Cas9 protein can associate with a guide RNA that guides Cas9 to a specific DNA sequence complementary to the guide RNA. In some embodiments, the polynucleotide programmable nucleotide-binding domain is the Cas9 domain, such as an active nuclease Cas9, a Cas9 nickase (nCas9), or a nuclease-inactivating Cas9 (dCas9). Examples of nucleic acid programmable DNA-binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b / C2C1, and Cas12c / C2C3. In some embodiments, wild-type 9 (NCBI reference sequence: NC_017053.1, nucleotide and amino acid sequences below) corresponds to Cas9 from Streptococcus pyogenes.

[0439]

[0440]

[0441]

[0442] (Single underscore: HNH domain; Double underscore: RuvC domain)

[0443] In some embodiments, wild-type Cas9 corresponds to or contains the following nucleotide and / or amino acid sequences:

[0444]

[0445]

[0446]

[0447] (Single underscore: HNH domain; Double underscore: RuvC domain).

[0448] In some implementations, the wild-type Cas9 (NCBI reference sequence: NC_002737.2 (nucleotide sequence below); and Uniprot reference sequence: Q99ZW2 (amino acid sequence below)) corresponding to Cas9 from Streptococcus pyogenes are described below:

[0449]

[0450] (Single underscore: HNH domain; Double underscore: RuvC domain)

[0451] In some implementations, Cas9 refers to Cas9 derived 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_021846.1). 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 meningitides (NCBI Ref:YP_002342100.1), or Cas9 from any other organism.

[0452] It should be understood that additional Cas9 proteins (e.g., dead Cas9 (dCas9), Cas9 nickase (nCas9), or active Cas9), including their variants and homologs, are within the scope of this disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is dead Cas9 (dCas9). In some embodiments, the Cas9 protein is Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is active Cas9.

[0453] In some embodiments, the Cas9 domain is a nuclease-inactivating Cas9 domain (dCas9). For example, the dCas9 domain can bind to a double-stranded nucleic acid molecule (e.g., via a gRNA molecule) without cleaving either strand of the double-stranded nucleic acid molecule. In some embodiments, the nuclease-inactivating dCas9 domain contains the D10X and H840X mutations of the amino acid sequences described herein, or corresponding mutations of any amino acid sequences provided herein, where X is any amino acid change. In some embodiments, the nuclease-inactivating dCas9 domain contains the D10X and H840X mutations of the amino acid sequences described herein, or corresponding mutations of any amino acid sequences provided herein. As an example, the nuclease-inactivating Cas9 domain contains the amino acid sequence detailed in the cloning vector pPlatTET-gRNA2 (Accession No. BAV54124).

[0454] An exemplary amino acid sequence of catalytically inactivated Cas9 (dCas9) is as follows:

[0455]

[0456] The additional suitable nuclease-inactivating dCas9 domain is based on the knowledge of this disclosure and the art to which it pertains, and will be readily apparent to those skilled in the art, and is within the scope of this disclosure. This additional exemplary suitable nuclease-inactivating dCas9 domain includes, but is not limited to, the D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, for example, Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9):833-838, the entire contents of which are incorporated herein by reference).

[0457] In some implementations, the Cas9 nuclease has an inactivating (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nicking enzyme, referred to as the “nCas9” protein (for “nicking enzyme” Cas9). The nuclease-inactivating Cas9 protein may be interchangeably referred to as the “dCas9” protein (for nuclease “dead” Cas9) or catalytically inactivating Cas9. Methods for generating Cas9 proteins (or fragments thereof) with an inactivating DNA cleavage domain are known (see, for example, Jinek et al., Science. 337:816-821 (2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28; 152(5): 1173-83, the entire contents of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337:816-821 (2012); Qi et al., Cell. 28; 152(5):1173-83 (2013)).

[0458] In some embodiments, the dCas9 domain contains an amino acid sequence that has 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% identity with any of the dCas9 domains provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any of the amino acid sequences described 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 consecutive amino acid residues identical to any of the amino acid sequences described herein.

[0459] In some embodiments, dCas9 corresponds to, or comprises, part or all of, a Cas9 amino acid sequence with one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain contains the D10A and H840A mutations or corresponding mutations in another Cas9.

[0460] In some implementations, dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A):

[0461] (Single underscore: HNH domain; Double underscore: RuvC domain).

[0462] In some implementations, the Cas9 domain contains the D10A mutation, while the residue at position 840 remains histidine at the corresponding position in the amino acid sequence provided herein or in any of the amino acid sequences provided herein.

[0463] In other embodiments, dCas9 variants are provided having mutations other than D10A and H840A, which, for example, cause nuclease inactivation in Cas9 (dCas9). Such mutations, for example, include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions within the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs of Cas9 are provided having at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity. In some implementations, variants of dCas9 are provided that have shorter or longer amino acid sequences, differing by about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids or more.

[0464] In some embodiments, the Cas9 domain is a Cas9 cleavage enzyme. The Cas9 cleavage enzyme can be a Cas9 protein that cleaves one strand of a double-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, the Cas9 cleavage enzyme cleaves the target strand of the double-stranded nucleic acid molecule, meaning the Cas9 cleavage enzyme cleaves the strand that is base-paired (complementary) to the gRNA (e.g., sgRNA) that binds to Cas9. In some embodiments, the Cas9 cleavage enzyme contains a D10A mutation and has a histidine residue at position 840. In some embodiments, the Cas9 cleavage enzyme cleaves the non-target, non-base-edited strand of the double-stranded nucleic acid molecule, meaning the Cas9 cleavage enzyme cleaves the strand that is not base-paired to the gRNA (e.g., sgRNA) that binds to Cas9. In some embodiments, the Cas9 cleavage enzyme contains 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 has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with any Cas9 nickase provided herein. Additional suitable Cas9 nickases will be readily apparent to those skilled in the art based on this disclosure and the knowledge thereof, and are within the scope of this disclosure.

[0465] The amino acid sequence of an exemplary catalytic Cas9 nickase (nCas9) is as follows:

[0466]

[0467] In some embodiments, Cas9 refers to Cas9 derived from archaea (e.g., nanoarchaea), which constitute domains and kingdoms of single-celled eukaryotes and microbes. In some embodiments, the programmable nucleotide-binding protein can be a CasX or CasY protein, described, for example, in Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi:10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Macrogenomics using genome resolution has identified many CRISPR-Cas systems, including Cas9, first reported in the archaeal domain of life. This divergent Cas9 protein has been found in rarely studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, have been discovered; they are the most compact systems discovered to date. In some embodiments, Cas9 in the base editor systems described herein 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 are available as nucleic acid-programmable DNA-binding proteins (napDNAbp) and are within the scope of this disclosure.

[0468] 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, napDNAbp is a CasX protein. In some embodiments, napDNAbp is a CasY protein. In some embodiments, napDNAbp contains an amino acid sequence that has 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% identity with naturally occurring CasX or CasY proteins. In some embodiments, the programmable nucleotide-binding protein is a naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide-binding protein contains an amino acid sequence that has 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% identity with any CasX or CasY protein described herein. It should be understood that CasX and CasY from other bacterial species may also be used in accordance with this disclosure.

[0469] An exemplary CasX((uniprot.org / uniprot / F0NN87;uniprot.org / uniprot / F0NH53)tr|F0NN87|F0NN87_SULIHCRISPR-Associated Casx protein OS=Sulfolobus islandicus (strain HVE10 / 4)GN=SiH_0402PE=4SV=1) amino acid sequence is as follows:

[0470] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0471] An exemplary CasX (>tr|F0NH53|F0NH53_SULIR CRISPR-associated protein, Casx OS = Sulfolobus islandicus (strain REY15A) GN = SiRe_0771 PE = 4 SV = 1) amino acid sequence is as follows:

[0472] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTSNIILPLSGNDKNPWTETLKCYNFPTTTVALSEVFKNFSQVKECEEVSAPSFVKPFEYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0473] δ- bacteriumCasX

[0474] MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQ KWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0475] An example CasY ((ncbi.nlm.nih.gov / protein / APG80656.1)>APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria]) amino acid sequence is as follows:

[0476]

[0477] The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Upon target binding, Cas9 undergoes a conformational change, repositioning itself within the nuclease domain to cleave the opposite strand of the target DNA. The final result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (approximately 3-4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired via one of two general repair pathways: (1) the efficient but error-prone non-homologous end joining (NHEJ) pathway; or (2) the less efficient but high-fidelity homologous directed repair (HDR) pathway.

[0478] The “efficiency” of non-homologous end joining (NHEJ) and / or homologous directed repair (HDR) can be calculated by any convenient method. For example, in some implementations, efficiency can be expressed as a percentage of successful HDR. For example, a Surveyor nuclease assay can be used to generate cleavage products, and the product-to-substrate ratio can be used to calculate the percentage. For example, a Surveyor nuclease can be used to directly cleave DNA containing a newly integrated restriction sequence as a result of successful HDR. More cleaved substrate indicates a higher HDR percentage (higher HDR efficiency). As an illustrative example, the fraction (percentage) of HDR can be calculated using the following equation [(cleavage products) / (substrate plus cleavage products)] (e.g., (b+c) / (a+b+c), where “a” is the band intensity of the DNA substrate, and “b” and “c” are the cleavage products).

[0479] In some implementations, efficiency can be expressed as a percentage of successful NHEJs. For example, a T7 endonuclease I assay can be used to generate cleavage products, and the product-to-substrate ratio can be used to calculate the percentage of NHEJs. T7 endonuclease I cleaves mismatched heteroduplex DNA resulting from hybridization of wild-type and mutant DNA strands (NHEJ produces small random insertions or deletions at the original break site). More cleavages indicate a higher percentage of NHEJs (higher NHEJ efficiency). As an illustrative example, the fraction (percentage) of NHEJs can be calculated using the following equation: (1 - (1 - (b+c) / (a+b+c))) 1 / 2 )×100, where “a” is the band intensity of the DNA substrate, and “b” and “c” are the cleavage products (Ran et al., Cell. 2013 Sep. 12; 154(6):1380-9; and Ran et al., Nat Protoc. 2013 Nov.; 8(11):2281–2308).

[0480] The NHEJ repair pathway is the most active repair mechanism, frequently resulting in small nucleotide insertions or deletions at DSB sites. The stochastic nature of NHEJ-mediated DSB repair has significant practical implications because cell populations expressing Cas9 and gRNA or guide polynucleotides lead to a variety of mutations. In some implementations, NHEJ induces small insertions or deletions in the target DNA, causing amino acid deletions, insertions, or frameshift mutations, leading to premature stop codons within the open reading frame (ORF) of the target gene. The ideal end result is a loss-of-function mutation within the target gene.

[0481] While NHEJ-mediated DSB repair frequently disrupts open reading frames of genes, homology-directed repair (HDR) can be used to generate specific nucleotide changes, ranging from single nucleotide changes to large 2k7 insertions, such as the addition of fluorophores or markers. To utilize HDR in gene editing, a DNA repair template containing the desired sequence is delivered to the cell type of interest along with gRNA and Cas9 or Cas9 nickase. The repair template can contain the desired edit along with additional homologous sequences upstream and downstream of the target (called left and right homologous arms). The length of each homologous arm can vary depending on the size of the introduced change; larger insertions require longer homologous arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. Even in cells expressing Cas9, gRNA, and exogenous repair templates, HDR efficiency is typically low (<10% of modified alleles). HDR efficiency can be enhanced by synchronizing cells, as HDR occurs during the S and G2 phases of the cell cycle. Chemically or genetically repressive genes involved in NHEJ can also increase HDR frequency.

[0482] In some implementations, Cas9 is a modified Cas9. A given gRNA target sequence can have additional sites throughout the genome where partial homology exists. These sites are called off-targets and need to be considered when designing gRNAs. In addition to optimizing gRNA design, CRISPR specificity can be increased by modifying Cas9. Cas9 generates double-strand breaks (DSBs) via the combined activity of two nuclease domains, RuvC and HNH. The Cas9 nickase is a D10A mutant of SpCas9 that retains one nuclease domain and generates DNA nicks instead of DSBs. Nickase systems can also be combined with HDR-mediated gene editing for specific gene editing.

[0483] In some embodiments, Cas9 is a variant Cas9 protein. The variant Cas9 polypeptide has an amino acid sequence that differs from the wild-type Cas9 protein by one amino acid (e.g., deletion, insertion, substitution, fusion). In some cases, the variant Cas9 polypeptide has amino acid alterations (e.g., deletion, insertion, or substitution) that reduce the nuclease activity of the Cas9 polypeptide. For example, in some cases, the variant Cas9 polypeptide has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 protein. In some embodiments, the variant Cas9 protein does not have substantial nuclease activity. When the primary Cas9 protein is a variant Cas9 protein that does not have substantial nuclease activity, it may be referred to as "dCas9".

[0484] In some embodiments, the variant Cas9 protein has reduced nuclease activity. For example, the variant Cas9 protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the endonuclease activity of the wild-type Cas9 protein, for example, the wild-type Cas9 protein.

[0485] In some embodiments, the variant Cas9 peptide chain can cleave the complementary strand of the guide target sequence, but has a reduced ability to cleave the non-complementary strand of the double-stranded guide target sequence. For example, the variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, the Cas9 protein has D10A (aspartic acid at amino acid position 10 is replaced with alanine), and therefore can cleave the complementary strand of the double-stranded guide target sequence, but has a reduced ability to cleave the non-complementary strand of the double-stranded guide target sequence (thus causing single-strand breaks (SSBs) instead of double-strand breaks (DSBs) when the variant Cas9 protein cleaves the double-stranded target nucleic acid) (see, for example, Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21).

[0486] In some embodiments, the variant Cas9 protein can cleave the non-complementary strand of a double-stranded guide target sequence, but has a reduced ability to cleave the complementary strand of the guide target sequence. For example, the variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif). As a non-limiting example, in some embodiments, the variant Cas9 protein has an H840A mutation (histidine at amino acid position 840 is replaced with alanine), and because it can cleave the non-complementary strand of the guide target sequence but has a reduced ability to cleave the complementary strand of the guide target sequence (thus causing SSB instead of DSB when the variant Cas9 protein cleaves the double-stranded target nucleic acid). This Cas9 protein has a reduced ability to cleave guide target sequences (e.g., single-stranded guide target sequences) but retains the ability to bind guide target sequences (e.g., single-stranded guide target sequences).

[0487] In some embodiments, the variant Cas9 protein has a reduced ability to cleave both the complementary and non-complementary strands of double-stranded target DNA. As a non-limiting example, in some embodiments, the variant Cas9 protein carries two mutations, D10A and H840A, such that the peptide has a reduced ability to cleave both the complementary and non-complementary strands of double-stranded target DNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0488] As another non-limiting example, in some embodiments, the variant Cas9 protein carries mutations of W476A and W1126A, resulting in a reduced ability of the peptide to cleave target DNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0489] As another non-limiting example, in some embodiments, the variant Cas9 protein carries mutations of P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, resulting in a reduced ability of the peptide to cleave target DNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0490] As another non-limiting example, in some embodiments, the variant Cas9 protein carries mutations of H840A, W476A, and W1126A, resulting in a reduced ability of the peptide to cleave target DNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, the variant Cas9 protein carries mutations of H840A, D10A, W476A, and W1126A, resulting in a reduced ability of the peptide to cleave target DNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind target DNA (e.g., single-stranded target DNA). In some embodiments, the variant Cas9 has a repair-catalyzing His residue at position 840 in the Cas9 HNH domain (A840H).

[0491] As another non-limiting example, in some embodiments, the variant Cas9 protein carries mutations of H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, resulting in a reduced ability of the peptide to cleave target DNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, the variant Cas9 protein carries mutations of D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, resulting in a reduced ability of the peptide to cleave target DNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind target DNA (e.g., single-stranded target DNA). In some embodiments, when the variant Cas9 protein carries mutations of W476A and W1126A, or when the variant Cas9 protein carries mutations of P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, the variant Cas9 protein does not effectively bind to the PAM sequence. Therefore, in such embodiments, when the variant Cas9 protein is used in a binding method, the method does not require PAM. In other words, in some embodiments, when the variant Cas9 protein is used in a binding method, the method may include guide RNA, but the method may be performed without the PAM sequence (therefore, the specificity of binding is provided by the target strand of the guide RNA). Other residues can be mutated to achieve the above effect (i.e., inactivate one or another nuclease moiety). As a non-limiting example, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 can be altered (i.e., substituted). Furthermore, mutations other than alanine substitution are also suitable.

[0492] In some implementations, the variant Cas9 protein has reduced catalytic activity (e.g., when the Cas9 protein has mutations such as D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or 987, such as D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A). The variant Cas9 protein can still bind to the target DNA in a site-specific manner (because it is still guided to the target DNA via guide RNA), as long as it retains the ability to interact with guide RNA.

[0493] In some implementations, the variant Cas protein can be spCas9, spCas9-VRQR, spCas9-VRER, xCas9(sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.

[0494] In some embodiments, a modified SpCas9 is used, which includes amino acid substitutions of D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER), and is specific for the modified PAM5'-NGC-3'.

[0495] As an alternative to Cas9 in *S. pyogenes*, RNA-guided endonucleases from the Cpf1 family can be included, exhibiting cleavage activity in mammalian cells. CRISPR (CRISPR / Cpf1) from *Prevotella* and *Francisella 1* 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 is an acquired immune mechanism found in *Prevotella* and *Francisella* bacteria. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses guide RNA to locate and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some limitations of the CRISPR / Cas9 system. Unlike Cas9 nucleases, Cpf1-mediated DNA cleavage results in double-strand breaks with short 3' protrusions. The staggered cutting pattern of Cpf1 opens up possibilities for targeted gene transfer, similar to traditional restriction enzyme cloning, which can improve the efficiency of gene editing. Like the aforementioned Cas9 variants and orthologs, Cpf1 can also extend the number of CRISPR-targetable sites to AT-rich regions or AT-rich genomes lacking the NGG PAM sites favored by SpCas9. The Cpf1 locus contains a hybrid α / β domain, a RuvC-I followed by a helical region, a RuvC-II, and a zinc finger-like domain. The Cpf1 protein possesses a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, Cpf1 lacks an HNH endonuclease domain, and the N-terminus of Cpf1 does not possess the α-helical recognition leaf of Cas9. The Cpf1 CRISPR-Cas domain architecture reveals Cpf1 as functionally unique and classifies it as a type 2 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 trans-activated CRISPR RNA (tracrRNA), therefore, only CRISPR (crRNA) is needed. This is advantageous for genome editing because Cpf1 is not only smaller than Cas9, but it also has a smaller sgRNA molecule (approximately half the nucleotide size of Cas9). Compared to the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex cleaves target DNA or RNA by identifying the 5'-YTN-3' motif adjacent to the protospacer sequence. Upon PAM identification, Cpf1 introduces a 4 or 5-nucleotide overhang resembling a sticky-terminal DNA double-strand break.

[0496] The Cas12 domain of the nucleobase editor

[0497] Typically, microbial CRISPR-Cas systems are classified 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 each are incorporated herein by reference. Type V Cas proteins contain RuvC (or RuvC-like) endonuclease domains. However, the production of mature CRISPR RNA (crRNA) generally does not depend on tracrRNA; for example, Cas12b / C2c1 requires tracrRNA for crRNA production. Cas12b / C2c1 depends on both crRNA and tracrRNA for RNA cleavage.

[0498] The nucleic acid programmable DNA-binding proteins considered in this invention include Cas proteins, which are classified as type 2V (Cas12 proteins). Non-limiting examples of type 2V Cas proteins include Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i, their homologs, or modified versions thereof. As used herein, Cas12 protein may also be referred to as Cas12 nuclease, Cas12 domain, or Cas12 protein domain. In some embodiments, the Cas12 protein of this invention comprises an amino acid sequence interrupted by an internal fusion protein domain (such as a deaminase domain).

[0499] In some embodiments, the Cas12 domain is a nuclease-inactivating Cas12 domain or a Cas12 cleavage enzyme. In some embodiments, the Cas12 domain is a nuclease-activating domain. For example, the Cas12 domain can be a Cas12 domain of one strand of a cleavage double-stranded nucleic acid (e.g., a double-stranded DNA molecule). In some embodiments, the Cas12 domain comprises any of the amino acid sequences described herein. In some embodiments, the Cas12 domain comprises an amino acid sequence that has 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% identity with any of the amino acid sequences described herein. In some embodiments, the Cas12 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations relative to any of the amino acid sequences described herein. In some embodiments, the Cas12 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 consecutive amino acid residues identical to any of the amino acid sequences described herein.

[0500] In some embodiments, a protein comprising a Cas12 fragment is provided. For example, in some embodiments, the protein comprises one of two Cas12 domains: (1) a Cas12 gRNA-binding domain; or (2) a Cas12 DNA-cleaving domain. In some embodiments, a protein comprising Cas12 or a fragment thereof is referred to as a “Cas12 variant”. Cas12 variants share homology with Cas12 or a fragment thereof. For example, Cas12 variants have at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity with wild-type Cas12. In some implementations, the Cas12 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas12. In some embodiments, the Cas12 variant comprises a fragment of Cas12 (e.g., a gRNA-binding domain or a DNA cleavage domain) such that the fragment has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity with the corresponding fragment of wild-type Cas12. In some embodiments, the fragment has 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% identity, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the corresponding fragment of wild-type Cas12. In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0501] In some embodiments, Cas12 partially or wholly corresponds to or comprises a Cas12 amino acid sequence having one or more mutations that alter the activity of the Cas12 nuclease. This mutation, for example, includes an amino acid substitution within the RuvC nuclease domain of Cas12. In some embodiments, variants or homologs of Cas12 are provided that have at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identity with wild-type Cas12. In some embodiments, variants of Cas12 are provided that have shorter or longer amino acid sequences, differing by about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, or more amino acids.

[0502] In some embodiments, such as the Cas12 fusion proteins provided herein, the Cas12 protein comprises the full-length amino acid sequence of a Cas12 protein, for example, one of the Cas12 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas12 sequence, but only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas12 domains are provided herein, and additional suitable sequences of Cas12 domains and fragments will be readily apparent to those skilled in the art.

[0503] Generally, type V Cas proteins possess a single-function 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 Cas12b protein (see Strecker et al., Nature Communications, 2019, 10(1):Art.No.:212). In one embodiment, the variant Cas12 peptide has an amino acid sequence that differs from the wild-type Cas12 protein in that it contains 1, 2, 3, 4, 5, or more amino acids (e.g., deletions, insertions, substitutions, or fusions). In some cases, the variant Cas12 peptide has amino acid alterations (e.g., deletions, insertions, or substitutions) that reduce the activity of the Cas12 peptide. For example, in some cases, the variant Cas12 is the Cas12b polypeptide, which 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 cleavage enzyme activity of the corresponding wild-type Cas12b protein. In some cases, the variant Cas12b protein does not have substantial cleavage enzyme activity.

[0504] In some cases, variant Cas12b proteins exhibit reduced cleavage enzyme activity. For example, variant Cas12b proteins exhibit 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 cleavage enzyme activity of wild-type Cas12b proteins.

[0505] In some implementations, the Cas12 protein includes an RNA-guided endonuclease of the Cas12a / Cpf1 family that exhibits activity in mammalian cells. CRISPR (CRISPR / Cpf1) from *Prevotella* and *Francisella* is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of the class II CRISPR / Cas system. This acquired immune mechanism is found in *Prevotella* and *Francisella* bacteria. The Cpf1 gene is associated with a CRISPR locus and encodes an endonuclease that uses guide RNA to discover and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some limitations of the CRISPR / Cas9 system. Unlike Cas9 nucleases, Cpf1-mediated DNA cleavage results in double-strand breaks with short 3' protrusions. The staggered cleavage pattern of Cpf1 opens up possibilities for targeted gene transfer, similar to traditional restriction enzyme cloning, which can improve gene editing efficiency. Like the aforementioned Cas9 variants and orthologs, Cpf1 can also extend the number of CRISPR-targetable sites to AT-rich regions or AT-rich genomes lacking the NGG PAM sites favored by SpCas9. The Cpf1 locus contains a hybrid α / β domain, a RuvC-I followed by a helical region, a RuvC-II, and a zinc finger-like domain. The Cpf1 protein possesses a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, unlike Cas9, Cpf1 lacks an HNH endonuclease domain, and the N-terminus of Cpf1 lacks the α-helical recognition leaflet of Cas9. The Cpf1 CRISPR-Cas domain architecture indicates that Cpf1 is functionally unique and is classified as a type 2 V CRISPR system. The Cpf1 locus encodes the Cas1, Cas2, and Cas4 proteins, which are more similar to type I and III systems than type II systems. Functional Cpf1 does not require reverse activation of CRISPR RNA (tracrRNA), therefore, only CRISPR (crRNA) is needed. This is advantageous for genome editing because Cpf1 is not only smaller than Cas9, but its sgRNA molecule is also smaller (approximately half the nucleotide size of Cas9). Compared to the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex cleaves target DNA or RNA by recognizing the protospacer sequence adjacent to the motif 5'-YTN-3' or 5'-TTTN-3'. Upon PAM recognition, Cpf1 introduces a sticky-terminal DNA double-strand break with a 4 or 5 nucleotide protrusion.

[0506] In some aspects of the invention, a vector encoding a CRISPR enzyme may be used, the enzyme having been mutated relative to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. Cas12 may refer to wild-type exemplary Cas12 having at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology (e.g., Cas12 from Bacillus jutelidis, Bacillus V3-13 (BvCas12b), and Cyclocarya acidophilus (AaCas12b)). Cas12 can refer to a polypeptide having up to or about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology with the wild-type Cas12 polypeptide. Cas12 can refer to the wild-type or modified form of the Cas12 protein, which may include amino acid alterations such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0507] Nucleic acid programmable DNA-binding protein

[0508] Some aspects of this disclosure provide fusion proteins comprising domains that function 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. Unrestricted examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, and 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, their homologs or modified or engineered versions thereof. Other nucleic acid programmable DNA-binding proteins are within the scope of this disclosure, however they may not be specifically listed herein.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 full contents of which are incorporated herein by reference.

[0509] An example of a nucleic acid-programmable DNA-binding protein with PAM specificity distinct from Cas9 is the clustered, regularly spaced short palindromic repeats from *Prevotella* and *Francisella* 1 (Cpf1). Similar to Cas9, Cpf1 is also a class 2 CRISPR effector. Cpf1 has been shown to mediate robust DNA interference, characterized differently from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA and utilizing adjacent motifs (TTN, TTTN, or YTN) rich in T-rich protospacer sequences. Furthermore, Cpf1 cleaves DNA through staggered double-strand breaks. Among the 16 Cpf1 family proteins, two enzymes from the genera *Acidaminococcus* and *Lachnospiraceae* have been shown to possess potent genome editing activity in human cells. The Cpf1 protein is known in the field of science and has been previously described, for example, Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, pp. 949-962; the entire contents of which are incorporated herein by reference.

[0510] Useful in this composition and method is a nuclease-inactivating Cpf1 (dCpf1) variant, which can serve as a guide nucleotide sequence-programmable DNA-binding protein domain. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9 but lacks the HNH endonuclease domain, and the N-terminus of Cpf1 does not possess the α-helical recognition leaf of Cas9. As shown in Zetsche et al., Cell, 163, 759-771, 2015 (incorporated herein by reference), the RuvC-like domain of Cpf1 is responsible for cleaving both DNA strands, while the RuvC-like domain is responsible for inactivating the Cpf1 nuclease activity. For example, mutations corresponding to D917A, E1006A, or D1255A in Francisella novicida Cpf1 inactivate the Cpf1 nuclease activity. In some embodiments, the dCpf1 of this disclosure includes mutations corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It should be understood that any mutation, such as a substitution mutation, deletion, or insertion that inactivates the RuvC domain of Cpf1, may be used according to this disclosure.

[0511] In some embodiments, the nucleic acid-programmable DNA-binding protein (napDNAbp) of any fusion protein provided herein may be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 cleavage enzyme (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 has 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% identity with the Cpf1 sequence disclosed herein. In some embodiments, dCpf1 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the Cpf1 sequence disclosed herein, and contains mutations corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It should be understood that Cpf1 from other bacterial species may also be used according to this disclosure.

[0512] Wild-type Francisella novicida Cpf1 (D917, E1006, and D1255 are in bold and underlined)

[0513]

[0514]

[0515] Francisella novicida Cpf1 D917A (A917, E1006 and D1255 are in bold and underlined)

[0516]

[0517]

[0518] Francisella novicida Cpf1 E1006A (D917, A1006 and D1255 are in bold and underlined)

[0519]

[0520]

[0521] Francisella novicida Cpf1 D1255A (D917, E1006 and A1255 are in bold and underlined)

[0522]

[0523]

[0524] Francisella novicida Cpf1 D917A / E1006A (A917, A1006, and D1255 are in bold and underlined)

[0525]

[0526]

[0527] Francisella novicida Cpf1 D917A / D1255A (A917, E1006 and A1255 are in bold and underlined)

[0528]

[0529] Francisella novicida Cpf1 E1006A / D1255A (D917, A1006, and A1255 are in bold and underlined)

[0530]

[0531] Francisella novicida Cpf1 D917A / E1006A / D1255A (A917, A1006, and A1255 are in bold and underlined)

[0532]

[0533]

[0534] In some implementations, a Cas9 domain present in the fusion protein can be replaced with a guide nucleotide sequence-programmable DNA-binding protein domain that is not required to have a PAM sequence.

[0535] In some embodiments, the Cas9 domain is the Cas9 domain from Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domain is a nuclease-activated SaCas9, a nuclease-inactivated SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, SaCas9 contains an N579A mutation or a corresponding mutation of any amino acid sequence provided herein.

[0536] In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain may bind to a nucleic acid sequence having a non-standard PAM. In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain may bind to a nucleic acid sequence having an NNGRRT or NNGRRT PAM sequence. In some embodiments, the SaCas9 domain contains one or more of the E781X, N967X, and R1014X mutations or a corresponding mutation of any amino acid sequence provided herein, where X is any amino acid. In some embodiments, the SaCas9 domain contains one or more of the E781K, N967K, and R1014H mutations or one or more corresponding mutations of any amino acid sequence provided herein. In some embodiments, the SaCas9 domain contains the E781K, N967K, or R1014H mutation or a corresponding mutation of any amino acid sequence provided herein.

[0537] Exemplary SaCas9 sequence

[0538]

[0539] The aforementioned residue N579, which is underlined and bold, can be mutated (e.g., to A579) to produce the SaCas9 nickase.

[0540] Exemplary SaCas9n sequence

[0541]

[0542] The above residue A579 can be mutated from N579 to produce the SaCas9 nickase, which is underlined and in bold.

[0543] Example SaKKH Cas9

[0544]

[0545]

[0546] The residue A579 mentioned above can be mutated from N579 to produce the SaCas9 nickase, and is shown as underlined and bold. The residues K781, K967, and H1014 mentioned above can be mutated from E781, N967, and R1014 to produce SaKKH Cas9, and are shown as underlined and italicized text.

[0547] In some implementations, the napDNAbp is arranged in a circular pattern. In the following sequences, plain text indicates adenosine deaminase sequences, bold sequences indicate sequences derived from Cas9, italic sequences indicate linker sequences, and underlined sequences indicate binomial nuclear localization sequences.

[0548] CP5 (with MSP "NGC" PID and "D10A" nickase):

[0549]

[0550]

[0551] In some implementations, the nucleic acid programmable DNA-binding protein (napDNAbp) is the single effector of the microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, but are not limited to, Cas9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Typically, microbial CRISPR-Cas systems are classified into Class 1 and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while Class 2 systems have single protein effectors. For example, Cas9 and Cpf1 are Class 2 effectors. In addition to Cas9 and Cpf1, three different Class 2 CRISPR-Cas systems (Cas12b / C2c1 and Cas12c / C2c3) have been described in Shmakov et al., “Discovery and Functional Characterization of DiverseClass 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov. 5; 60(3):385-397, the entire contents of which are incorporated herein by reference. The effectors of these two systems, Cas12b / C2c1 and Cas12c / C2c3, contain a Cpf1-associated RuvC-like endonuclease domain. The third system contains an effector with two predicted HEPN RNase domains. The production of mature CRISPR RNA is independent of tracrRNA, unlike the CRISPR RNA produced by Cas12b / C2c1, which depends on DNA cleavage involving both CRISPR RNA and tracrRNA.

[0552] The crystal structure of Alicyclobaccillus acidoterrastris Cas12b / C2c1 (AacC2c1) has been reported in a complex with a chimeric single-molecule guide RNA (sgRNA). See, for example, Liu et al., “C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism”, Mol. Cell, 2017 Jan. 19; 65(2):310-322, the entire contents of which are incorporated herein by reference. The crystal structure has also been reported in Alicyclobacillus acidoterrastris C2c1 bound to 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 configuration of AacC2c1, containing both target and non-target DNA strands, has been independently captured within a single RuvC catalytic pocket, resulting in a staggered heptanucleotide break of the target DNA via Cas12b / C2c1-mediated cleavage. Structural comparisons of the Cas12b / C2c1 ternary complex with previously identified Cas9 and Cpf1 counterparts demonstrate the diversity of mechanisms employed by the CRISPR-Cas9 system.

[0553] In some embodiments, the nucleic acid-programmable DNA-binding protein (napDNAbp) of any fusion protein provided herein can be a Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, napDNAbp is a Cas12b / C2c1 protein. In some embodiments, napDNAbp is a Cas12c / C2c3 protein. In some embodiments, napDNAbp comprises an amino acid sequence that has 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% identity with naturally occurring Cas12b / C2c1 or Cas12c / C2c3 proteins. In some embodiments, napDNAbp is a naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp contains an amino acid sequence that has 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% identity with 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 this disclosure.

[0554] The amino acid sequence of Cas12b / C2c1((uniprot.org / uniprot / T0D7A2#2)sp|T0D7A2|C2C1_ALIAGCRISPR-related endonuclease C2c1 OS=Alicyclobacillus acido-terrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B)GN=c2c1 PE=1SV=1) is as follows:

[0555]

[0556] BhCas12b (Bacillus hisashii) NCBI reference sequence: WP_095142515

[0557]

[0558] In some embodiments, Cas12b is BvCas12B. In some embodiments, Cas12b contains amino acid substitutions S893R, K846R, and E837G, as numbered in the exemplary BvCas12b amino acid sequence below.

[0559] BvCas12b (Bacillus sp.) V3-13 NCBI reference sequence: WP_101661451.1

[0560]

[0561] Guide polynucleotides

[0562] In the implementation, the guide polynucleotide is a guide RNA. The RNA / Cas complex helps to "guide" the Cas protein to the target DNA. Cas9 / crRNA / tracrRNA endonuclease cleaves linear or circular dsDNA targets complementary to the spacer sequence. The target strand not complementary to the crRNA is first endonucleated and then subjected to 3'-5' exonuclease trimming. In nature, DNA binding and cleavage typically require both proteins and two RNAs. However, single guide RNAs ("sgRNA" or simply "gNRA") can be engineered to incorporate aspects of both crRNA and tracrRNA into a single RNA material. See, for example, Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes short motifs in CRISPR repeat sequences (PAM or protospacer adjacent motifs) to help distinguish between self and non-self. The Cas9 nuclease sequence and structure are well known to those skilled in the art (see, for example, “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti, JJ et al., Natl. Acad. Sci. USA 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E. et al., Nature 471:602-607 (2011); and “Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference). Cas9 heterologs have been described in various species, including, but not limited to, Streptococcus pyogenes and Streptococcus thermophilus.Additional suitable Cas9 nucleases and sequences are readily apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci described below: Chylinski, Rhun, and Charpentier, “ThetracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNABiology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 nuclease has an inactivating (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nicking enzyme.

[0563] 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.

[0564] The multinucleotide programmable nucleotide-binding domain (e.g., a CRISPR-derived domain) of the base editor disclosed herein can recognize target polynucleotide sequences by associating with a guide polynucleotide. The guide polynucleotide (e.g., gRNA) is typically single-stranded and can be programmed to bind site-specifically (i.e., via complementary base pairing) to the target sequence of the polynucleotide, thereby guiding the base editor, which is bound to the guide nucleic acid, to the target sequence. The guide polynucleotide can be DNA. The guide polynucleotide can be RNA. In some embodiments, the guide polynucleotide comprises a natural nucleotide (e.g., adenosine). In some embodiments, the guide polynucleotide comprises a non-natural (or unnatural) nucleotide (e.g., a peptide nucleic acid or nucleotide analogue). In some embodiments, the target region of the guide nucleic acid sequence can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The target region of a guide nucleic acid can be between 10 and 30 nucleotides in length, or between 15 and 25 nucleotides in length, or between 15 and 20 nucleotides in length.

[0565] In some embodiments, the guide polynucleotide comprises two or more different polynucleotides that can interact with each other via, for example, complementary base pairing (e.g., dual guide polynucleotide). For example, the guide polynucleotide may comprise CRISPR RNA (crRNA) and trans-activated CRISPR RNA (tracrRNA). For example, the guide polynucleotide may comprise one or more trans-activated CRISPR RNAs (tracrRNA).

[0566] In type II CRISPR systems, targeting nucleic acids via CRISPR proteins (e.g., Cas9) typically requires complementary base pairing between a first RNA molecule (crRNA) containing the target sequence and a second RNA molecule (trRNA) containing the repetitive sequence. This complementary base pairing forms a scaffold region that stabilizes the guide RNA-CRISPR protein complex. This dual guide RNA system can be used as a guide polynucleotide to guide the base editor disclosed herein to the target polynucleotide sequence.

[0567] In some embodiments, the base editor provided herein utilizes a single-guide polynucleotide (e.g., gRNA). In some embodiments, the base editor provided herein utilizes a double-guide polynucleotide (e.g., dual gRNA). In some embodiments, the base editor provided herein utilizes one or more guide polynucleotides (e.g., multiple gRNAs). In some embodiments, a single-guide polynucleotide is used in different base editors described herein. For example, a single-guide polynucleotide can be used in an adenosine base editor or an adenosine base editor and a cytidine base editor, as described, for example, in PCT / US19 / 44935.

[0568] In other embodiments, the guide polynucleotide may contain both the polynucleotide targeting portion and the scaffold portion of the nucleic acid within 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). Throughout this document, the term guide polynucleotide sequence encompasses any single-molecule, bimolecule, or multimolecule nucleic acid capable of interacting with a base editor and guiding the base editor to a target polynucleotide sequence.

[0569] Typically, a guide polynucleotide (e.g., a crRNA / trRNA complex or gRNA) comprises a "polynucleotide targeting segment" which includes a sequence capable of recognizing and binding to a target polynucleotide sequence; and a "protein-binding segment" which is a guide polynucleotide sequence within a polynucleotide programmable nucleotide-binding domain component that stabilizes the base editor. In some embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to DNA polynucleotides, thereby facilitating base editing in DNA. In other embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to RNA polynucleotides, thereby facilitating base editing in RNA. Hereinafter, "segment" refers to a segment or region of a molecule, such as a continuous extension of nucleotides in a guide polynucleotide. A segment may also refer to a region / segment of a complex, such that a segment may contain a region of more than one molecule. For example, when the guide polynucleotide contains multiple nucleic acid molecules, the protein-binding segment may include all or part of multiple separate molecules, for example, hybridizing along complementary regions. In some embodiments, a protein-binding segment of DNA-targeting RNA comprising two separate molecules may comprise (i) 40-75 base pairs of a first RNA molecule of length 100 base pairs; and (ii) 10-25 base pairs of a second RNA molecule of length 50 base pairs. Unless otherwise explicitly defined in a particular context, the definition of "segment" is not limited to a specific total number of base pairs, not limited to any specific number of base pairs from a given RNA molecule, not limited to a specific number of separate molecules within the complex, and may include a region of RNA molecules of any total length, and may include regions complementary to other molecules.

[0570] Guide RNA or guide polynucleotides may contain two or more RNAs, such as CRISPR RNA (crRNA) and trans-activated crRNA (tracrRNA). Guide RNA or guide polynucleotides may sometimes contain single-stranded RNA or single guide RNA (sgRNA), formed by fusing crRNA and a portion of tracrRNA (e.g., the functional portion). Guide RNA or guide polynucleotides can also be dual RNAs containing crRNA and tracrRNA. Furthermore, crRNA can hybridize with target DNA.

[0571] As discussed above, guide RNA or guide polynucleotides can be expression products. For example, DNA encoding guide RNA can be a vector containing a sequence encoding guide RNA. Guide RNA or guide polynucleotides can be transferred into cells by transfecting cells with isolated guide RNA or plasmid DNA containing sequences encoding guide RNA and a promoter. Guide RNA or guide polynucleotides can also be transferred into cells by other means, such as using virus-mediated gene delivery.

[0572] Guide RNA or guide polynucleotides can be isolated. For example, guide RNA can be transfected into cells or organisms in the form of isolated RNA. Guide RNA can be prepared by in vitro transcription using any in vitro transcription system known in the art. Guide RNA can be transferred to cells in the form of isolated RNA rather than in the form of a plasmid containing the coding sequence of the guide RNA.

[0573] Guide RNAs or guide polynucleotides may contain three regions: a first region located at the 5' end, which is complementary to a target site in the chromosomal sequence; a second inner region, which can form a stem-loop structure; and a third 3' region, which can be single-stranded. The first region of each guide RNA can also be different, allowing each guide RNA to guide the fusion protein to a specific target site. Furthermore, the second and third regions of each guide RNA are identical in all guide RNAs.

[0574] The first region of the guide RNA or guide polynucleotide can be complementary to a target site in the chromosomal sequence, allowing the first region of the guide RNA to pair with the bases of the target site. In some embodiments, the first region of the guide RNA may contain 10 to 25 nucleotides (i.e., 10 to 25 nucleotides; or about 10 to about 25 nucleotides; or 10 to about 25 nucleotides; or about 10 to 25 nucleotides) or more. For example, the base-pairing region between the first region of the guide RNA and the target site in the chromosomal sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25 or more nucleotides in length. Sometimes, the first region of the guide RNA can be about 19, 20 or 21 nucleotides in length.

[0575] Guide RNA or guide polynucleotides may also contain a second region that forms a secondary structure. For example, the secondary structure formed by guide RNA may contain a stem (or hairpin) and a loop. The lengths of the loop and the stem can vary. For example, the loop may range from about 3 to 10 nucleotides in length, and the stem may range from about 6 to 20 base pairs in length. The stem may contain one or more protrusions of 1 to 10 or about 10 nucleotides. The total length of the second region may range from about 16 to 60 nucleotides in length. For example, the loop may be (about) 4 nucleotides in length, and the stem may be (about) 12 base pairs in length.

[0576] Guide RNA or guide polynucleotides may also contain a third region located at the 3' end, which can be substantially single-stranded. For example, the third region is sometimes not complementary to any chromosomal sequence in the cell of interest and sometimes not complementary to the rest of the guide RNA. Furthermore, the length of the third region can vary. The third region can be more than (about) 4 nucleotides long. For example, the length of the third region can range from about 5 to 60 nucleotides.

[0577] Guide RNA or guide polynucleotides can target any exon or intron of a gene target. In some embodiments, the guide RNA can target exon 1 or 2 of the gene; in other embodiments, the guide RNA can target exon 3 or 4 of the gene. The composition may comprise multiple guide RNAs that all target the same exon, or in some embodiments, it may comprise multiple guide RNAs that target different exons. The guide RNA can target both exons and introns of the gene.

[0578] Guide RNA or guide polynucleotides can target nucleic acid sequences of approximately 20 nucleotides. Target nucleic acids can be fewer than approximately 20 nucleotides. Target nucleic acids can be at least approximately 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or any length between 1 and 100 nucleotides. Target nucleic acids can be up to approximately 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or any length between 1 and 100 nucleotides. The target nucleic acid sequence can be the 5' (approximately 20 bases) of the first nucleotide immediately adjacent to the PAM. Guide RNA can target nucleic acid sequences. The target nucleic acid can be 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.

[0579] Guide polynucleotides, such as guide RNA, can refer to a nucleic acid that can hybridize to another nucleic acid, such as a target nucleic acid or protospacer sequence in the cellular genome. Guide polynucleotides can be RNA. Guide polynucleotides can be DNA. Guide polynucleotides can be sequences programmed or designed to bind specifically to nucleic acid sites. Guide polynucleotides can contain a polynucleotide chain and can be called monoguide polynucleotides. Guide polynucleotides can contain two polynucleotide chains and can be called biguide polynucleotides. Guide RNA is introduced into the cell as an RNA molecule. For example, RNA molecules can be transcribed in vitro and / or can be chemically synthesized. RNA can be synthesized from DNA molecules, such as... The gene fragment is transcribed. The guide RNA can then be introduced into the cell as an RNA molecule. Guide RNA can also be introduced into the cell in the form of a non-RNA nucleic acid molecule (e.g., a DNA molecule). For example, the DNA encoding the guide RNA can be operatively linked to a promoter regulatory sequence for expression of the guide RNA in the cells of interest. The RNA coding sequence can be operatively 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, the px330 vector and the px333 vector. In some embodiments, the plasmid vector (e.g., the px333 vector) may contain at least two DNA sequences encoding guide RNA.

[0580] Methods for selecting, designing, and validating guide polynucleotides (e.g., guide RNAs and target sequences) are described herein and are well known to those skilled in the art. For example, to minimize the impact of potential substrate contamination from deaminase domains (e.g., AID domains) in a nucleobase editor system, the number of residues that may be unintentionally targeted for deamination (e.g., ssDNA that may potentially reside within the target nucleic acid locus) can be minimized. Furthermore, software tools can be used to optimize gRNAs corresponding to target nucleic acid sequences, e.g., to minimize overall off-target activity across the entire genome. For example, for each possible target domain selection using *Streptococcus pyogenes* Cas9, all off-target sequences (preceding the selected PAM, e.g., NAG or NGG) can be identified throughout the genome, containing up to a specific number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mismatched base pairs. The first region of gRNA complementary to the target site can be identified, and all first regions (e.g., crRNAs) can be ranked according to their predicted total off-target score; the top-ranked target domains represent those that are likely to have the greatest on-target activity and the least off-target activity. Functional evaluation of candidate target gRNAs can be performed using methods known in the art and / or as described herein.

[0581] As a non-restricted example, DNA sequence search algorithms can be used to identify target DNA hybridization sequences in the crRNA of guide RNAs used with Cas9. gRNA design can be performed using custom gRNA design software based on the public tool cas-offinder, documented 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-guidedendonucleases. Bioinformatics 30, 1473-1475 (2014). This software scores guides after calculating genome-wide off-target probabilities. For guides ranging in length from 17 to 24, matches from perfect matches to 7 non-matches are typically considered. Once off-target sites are determined through calculation, a total score is calculated for each guide and summarized in a tabular output using a web interface. In addition to identifying potential target sites adjacent to PAM sequences, the software also identifies all PAM-adjacent sequences that differ from the selected target site by 1, 2, 3, or more than 3 nucleotides. Target nucleic acid sequences, such as the genomic DNA sequence of a target gene, can be obtained, and repetitive elements can be screened using publicly available tools such as the RepeatMasker program. RepeatMasker searches for repetitive elements and low-complexity regions in the input DNA sequence. The output is a detailed annotation of the repetitions present in the given query sequence.

[0582] Following identification, the first region of the guide RNA, such as crRNA, can be sorted according to its distance from the target site, its orthogonality, and the presence of a 5' nucleotide to match the associated PAM sequence (e.g., 5'G based on the identification of close matches of associated PAMs in the human genome, such as NGG PAM for Streptococcus pyogenes, NNGRRT or NNGRRV PAM for Staphylococcus aureus). As used herein, orthogonality refers to the minimum number of sequences in the human genome that mismatch with the target sequence. For example, "high level of orthogonality" or "good orthogonality" can mean that there are no identical 20-mer target domains in the human genome other than the intended target, nor any sequences containing one or two mismatches in the target sequence. Target domains with good orthogonality can be selected to minimize off-target DNA fragmentation.

[0583] In some embodiments, the reporter system can be used to detect base editing activity and test candidate guide polynucleotides. In some embodiments, the reporter system may include a reporter gene-based assay, wherein base editing activity results in the expression of the reporter gene. For example, the reporter system may include a reporter gene containing an inactivated start codon, such as a mutation from 3'-TAC-5' to 3'-CAC-5' in the template strand. Upon successful deamination of the target C, the corresponding mRNA will be transcribed to 5'-AUG-3' instead of 5'-GUG-3', thereby achieving the translation of the reporter gene. Suitable reporter genes are readily apparent to those skilled in the art. Non-limiting examples of reporter genes include genes encoding green fluorescent protein (GFP), red fluorescent protein (RFP), luciferase, secretory alkaline phosphatase (SEAP), or any other gene whose expression is detectable and readily apparent to those skilled in the art. The reporter system can be used to test many different gRNAs, for example, to determine which residues of the target DNA sequence the corresponding deaminase will target. sgRNAs targeting non-template strands can also be tested to assess off-target effects on proteins that edit specific bases, such as Cas9 deaminase fusion proteins. In some embodiments, such gRNAs can be programmed so that the start codon of the mutation does not pair with the gRNA bases. Guide polynucleotides may include standard ribonucleotides, modified ribonucleotides (e.g., pseudouridine), ribonucleotide isomers, and / or ribonucleotide analogs. In some embodiments, guide polynucleotides may contain at least one detectable label. The detectable label may be a fluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluors, Halo tags, or suitable fluorescent dyes), a detection tag (e.g., biotin, digoxigenin, etc.), quantum dots, or gold particles.

[0584] Guide polynucleotides can be chemically synthesized, enzymatically synthesized, or a combination thereof. For example, guide RNA can be synthesized using standard solid-phase synthesis methods based on phosphoramide. Alternatively, guide RNA can be synthesized in vitro by operatively linking DNA encoding the guide RNA to a promoter regulatory sequence recognized by a phage RNA polymerase. Examples of suitable phage promoter sequences include the T7, T3, SP6 promoter sequences, or variants thereof. In embodiments where the guide RNA comprises two separate molecules (e.g., crRNA and tracrRNA), the crRNA can be chemically synthesized and the tracrRNA can be enzymatically synthesized.

[0585] In some implementations, the base editor system may comprise multiple guide polynucleotides, such as gRNAs. For example, the gRNAs may target one or more target loci contained in the base editor system (e.g., at least 1 gRNA, at least 2 gRNAs, at least 5 gRNAs, at least 10 gRNAs, at least 20 gRNAs, at least 30 gRNAs, at least 50 gRNAs). The multiple gRNA sequences may be tandemly arranged and are preferably separated by direct repeat sequences.

[0586] The DNA sequence encoding guide RNA or guide polynucleotides can be part of a vector. Furthermore, the vector may contain additional expression regulatory sequences (e.g., enhancer sequences, Kozak sequences, polyadenosine sequences, transcription termination sequences, etc.), selectable marker sequences (e.g., GFP or antibiotic resistance genes such as puromycin), origin of replication, etc. The DNA molecule encoding guide RNA can also be linear. The DNA molecule encoding guide RNA or guide polynucleotides can also be circular.

[0587] In some implementations, one or more components of the base editor system may be encoded by DNA sequences. Such DNA sequences may be introduced into the expression system (e.g., a cell) together or separately. For example, DNA sequences encoding a polynucleotide programmable nucleotide binding domain and a guide RNA may be introduced into the cell, and the DNA sequences may be part of separate molecules (e.g., a vector containing a sequence encoding a polynucleotide programmable nucleotide binding domain and a second vector containing a sequence encoding a guide RNA) or both may be part of the same molecule (e.g., a vector containing sequences encoding (and regulating) a polynucleotide programmable nucleotide binding domain and a guide RNA).

[0588] Guided polynucleotides may contain one or more modifications to provide nucleic acids with novel or enhanced characteristics. Guided polynucleotides may contain nucleic acid affinity tags. Guided polynucleotides may contain synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides.

[0589] In some embodiments, the gRNA or guide polynucleotide may contain modifications. Modifications may be made at any site on the gRNA or guide polynucleotide. More than one modification may be made to a single gRNA or guide polynucleotide. The gRNA or guide polynucleotide may be subjected to quality control after modification. In some embodiments, quality control may include PAGE, HPLC, MS, or any combination thereof.

[0590] Modifications to gRNA or guide polynucleotides can include substitution, insertion, deletion, chemical modification, physical modification, stabilization, purification, or any combination thereof.

[0591] gRNA or guide polynucleotides can also be modified by the following: 5' adenosine, 5' guanosine triphosphate capping, triphosphate capping, 5' N7-methylguanosine triphosphate capping, 5' triphosphate capping, 3' phosphate, 3' thiophosphate, 5' phosphate, 5' thiophosphate, Cis-Syn thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9, 3'-3' modification, 5'-5' modification, no base, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesterol-based TEG, desulfurized biotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, Dibiotin, PC-Biotin, Psoralen C2, Psoralen C6, TINA, 3'DABCYL, Black Hole Quencher 1, Black Hole Quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thio linker, 2'-deoxyribonucleoside analog purine, 2'-deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-O-methylribonucleoside analog, sugar-modified analog, wobble / universal base, fluorescent dye label, 2'-fluoroRNA, 2'-O-methylRNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, thiophosphate DNA, thiophosphate RNA, UNA, pseudouridine-5'-triphosphate, 5'-methylcytidine-5'-triphosphate, or any combination thereof.

[0592] In some embodiments, the modification is permanent. In other embodiments, the modification is temporary. In some embodiments, multiple modifications are made to the gRNA or guide polynucleotide. Modifications to the gRNA or guide polynucleotide can alter the physicochemical properties of the nucleotide, such as its conformation, polarity, hydrophobicity, chemical activity, base pairing interactions, or any combination thereof.

[0593] The PAM sequence can be any PAM sequence known in the art. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NGN, NGT, NGCG, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNNGATT, NNAGAAW, or NAAAAAC. Y is pyrimidine; N is any nucleotide base; W is A or T.

[0594] Modifications can also involve phosphate thioester substitution. In some embodiments, native phosphodiester bonds tend to be rapidly degraded by cellular nucleases; modifications using phosphate thioester (PS) bonds to replace the nucleotide inner linkages can provide greater stability towards hydrolysis by cellular nucleases. Modifications can increase the stability of gRNA or guide polynucleotides. Modifications can also enhance biological activity. In some embodiments, phosphate thioester-enhanced gRNA can inhibit RNase A, RNase T1, bovine serum nuclease, or any combination thereof. These properties allow PS-RNA gRNA to be used in applications where there is a high probability of exposure to nucleases, either in vivo or in vitro. For example, a phosphate thioester (PS) bond can be introduced between the last 3-5 nucleotides at the 5'- or "-" end of the gRNA, which can inhibit exonuclease degradation. In some embodiments, the phosphate thioester bond can be added throughout the gRNA to reduce endonuclease attack.

[0595] Original spacer sequence adjacent motif

[0596] The term "protospacer adjacent motif (PAM)" or a PAM-like motif refers to a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. In some embodiments, the PAM may be a 5' PAM (i.e., located upstream of the 5' end of the protospacer sequence). In other embodiments, the PAM may be a 3' PAM (i.e., located downstream of the 5' end of the protospacer).

[0597] The PAM sequence is important for target binding, but the exact sequence depends on the type of Cas protein.

[0598] The base editors provided herein may contain CRISPR protein-derived domains that bind nucleotide sequences containing standard or non-standard protospacer adjacent motif (PAM) sequences. A PAM site is a nucleotide sequence adjacent to a target polynucleotide sequence. Some aspects of this disclosure provide base editors that contain all or part of a CRISPR protein with different PAM specificities. For example, Cas9 proteins, such as Cas9 (spCas9) from Streptococcus pyogenes, typically require a standard NGG PAM sequence to bind a specific nucleic acid region, where "N" in "NGG" stands for adenosine (A), thymine (T), guanine (G), or cytosine (C), and G stands for guanine. The PAM can be CRISPR protein-specific, and differences may exist between different base editors containing different CRISPR protein-derived domains. The PAM can be 5' or 3' of the target sequence. The PAM can be upstream or downstream of the target sequence. The PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides in length. Typically, PAMs are between 2 and 6 nucleotides in length. Several PAM variants are described in Table 1 below.

[0599] Table 1. Cas9 protein and corresponding PAM sequence

[0600]

[0601]

[0602] In some embodiments, the PAM is NGC. In some embodiments, the NGC PAM is recognized by a Cas9 variant. In some embodiments, the NGC PAM variant includes one or more amino acid substitutions selected from the following: D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (collectively, "MQKFRAER").

[0603] In some embodiments, the PAM is NGT. In some embodiments, the NGT PAM is recognized by a Cas9 variant. In some embodiments, the NGT PAM variant is generated via a targeted mutation at one or more residues 1335, 1337, 1135, 1136, 1218, and / or 1219. In some embodiments, the NGT PAM variant is created via a targeted mutation at one or more residues 1219, 1335, 1337, 1218. In some embodiments, the NGT PAM variant is created via a targeted mutation at one or more residues 1135, 1136, 1218, 1219, and 1335. In some embodiments, the NGT PAM variant is selected from the targeted mutation groups provided in Tables 2 and 3 below.

[0604] Table 2: NGT PAM variants with mutations at residues 1219, 1335, 1337, and 1218

[0605] variants E1219V R1335Q T1337 G1218 1 F V T 2 F V R 3 F V Q 4 F V L 5 F V T R 6 F V R R 7 F V Q R 8 F V L R 9 L L T 10 L L R 11 L L Q 12 L L L 13 F I T 14 F I R 15 F I Q 16 F I L 17 F G C 18 H L N 19 F G C A 20 H L N V 21 L A W 22 L A F 23 L A Y 24 I A W 25 I A F 26 I A Y

[0606] Table 3: NGT PAM variants with mutations at residues 1135, 1136, 1218, 1219, and 1335

[0607]

[0608]

[0609] In some implementations, the NGT PAM variants are selected from variants 5, 7, 28, 31, or 36 in Tables 2 and 3. In some implementations, the variants have improved NGT PAM recognition.

[0610] In some embodiments, the NGT PAM variant has mutations at residues 1219, 1335, 1337, and / or 1218. In some embodiments, the NGT PAM variant is selected to have mutations from the variants provided in Table 4 below that are designed to improve recognition.

[0611] Table 4: NGT PAM variants with mutations at residues 1219, 1335, 1337, and 1218

[0612] variants E1219V R1335Q T1337 G1218 1 F V T 2 F V R 3 F V Q 4 F V L 5 F V T R 6 F V R R 7 F V Q R 8 F V L R

[0613] In some implementations, a base editor specific to NGT PAM can be generated as provided in Table 5A below.

[0614] Table 5A. NGT PAM variants

[0615]

[0616]

[0617] In some embodiments, the NGTN variant is variant 1. In some embodiments, the NGTN variant is variant 2. In some embodiments, the NGTN variant is variant 3. In some embodiments, the NGTN variant is variant 4. In some embodiments, the NGTN variant is variant 5. In some embodiments, the NGTN variant is variant 6.

[0618] In some embodiments, the Cas9 domain is the Cas9 domain (SpCas9) derived from *Streptococcus pyogenes*. In some embodiments, the SpCas9 domain is a nuclease-activated SpCas9, a nuclease-inactivated SpCas9 (SpCas9d), or a SpCas9 cleavage enzyme (SpCas9n). In some embodiments, SpCas9 contains a D10X mutation or a corresponding mutation of any amino acid sequence provided herein, where X is any amino acid other than D. In some embodiments, SpCas9 contains a D10A mutation or a corresponding mutation of any amino acid sequence provided herein. In some embodiments, the SpCas9 domain, SpCas9d domain, or SpCas9n domain may bind to a nucleic acid sequence having a non-standard PAM. In some embodiments, the SpCas9 domain, SpCas9d domain, or SpCas9n domain may bind to a nucleic acid sequence having an NGG, NGA, or NGCG PAM sequence. In some embodiments, the SpCas9 domain comprises one or more D1135X, R1335X, and T1337X mutations or corresponding mutations of any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more D1135E, R1335Q, and T1337R mutations or corresponding mutations of any amino acid sequence provided herein. In some embodiments, the SpCas9 domain comprises one or more D1135X, R1335X, and T1337X mutations or corresponding mutations of any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more D1135V, R1335Q, and T1337R mutations or corresponding mutations of any amino acid sequence provided herein. In some embodiments, the SpCas9 domain contains the D1135V, R1335Q, and T1337R mutations or corresponding mutations of any amino acid sequence provided herein. In some embodiments, the SpCas9 domain contains one or more D1135X, G1218X, R1335X, and T1337X mutations or corresponding mutations of any amino acid sequence provided herein, where X is any amino acid. In some embodiments, the SpCas9 domain contains one or more D1135V, G1218R, R1335Q, and T1337R mutations or corresponding mutations of any amino acid sequence provided herein. In some embodiments, the SpCas9 domain contains the D1135V, G1218R, R1335Q, and T1337R mutations or corresponding mutations of any amino acid sequence provided herein.

[0619] In some implementations, Cas9 is a Cas9 variant specific to the altered PAM sequence. In some implementations, 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). https: / / doi.org / 10.1038 / s41587-020-0412-8The full text of this document is incorporated herein by reference. In some embodiments, the Cas9 variant does not have specific PAM requirements. In some embodiments, the Cas9 variant, for example, the SpCas9 variant, is specific for NRNHPAM, where R is A or G and H is A, C, or T. In some embodiments, the SpCas9 variant is specific for PAM sequences AAA, TAA, CAA, GAA, TAT, GAT, or CAC. In some embodiments, the SpCas9 variant comprises amino acid substitutions located at positions 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1218, 1219, 1221, 1249, 1256, 1264, 1290, 1318, 1317, 1320, 1321, 1323, 1332, 1333, 1335, 1337, or 1339, or their corresponding positions, as specified in SEQ ID NO:1. In some embodiments, the SpCas9 variant comprises amino acid substitutions located at positions 1114, 1135, 1218, 1219, 1221, 1249, 1320, 1321, 1323, 1332, 1333, 1335, or 1337, or their corresponding positions, as specified in SEQ ID NO:1. In some embodiments, the SpCas9 variant contains amino acid substitutions at positions 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1219, 1221, 1256, 1264, 1290, 1318, 1317, 1320, 1323, 1333, or their corresponding positions as shown in SEQ ID NO:1. In some embodiments, the SpCas9 variant contains amino acid substitutions at positions 1114, 1131, 1135, 1150, 1156, 1180, 1191, 1218, 1219, 1221, 1227, 1249, 1253, 1286, 1293, 1320, 1321, 1332, 1335, 1339, or their corresponding positions as shown in SEQ ID NO:1. In some embodiments, the SpCas9 variants contain amino acid substitutions located at positions 1114, 1127, 1135, 1180, 1207, 1219, 1234, 1286, 1301, 1332, 1335, 1337, 1338, 1349, or their corresponding positions as numbered in SEQ ID NO:1. Exemplary amino acid substitutions and PAM specificities of the SpCas9 variants are shown in Tables 5B, 5C, 5D, and 5E below.

[0620] Table 5B.

[0621]

[0622] Table 5C.

[0623]

[0624]

[0625] Table 5D.

[0626]

[0627] Table 5E.

[0628]

[0629] In some embodiments, the Cas9 domain of any fusion protein provided herein comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the Cas9 peptide described herein. In some embodiments, the Cas9 domain of any fusion protein provided herein comprises the amino acid sequence of any peptide described herein. In some embodiments, the Cas9 domain of any fusion protein provided herein consists of the amino acid sequence of any Cas9 peptide described herein.

[0630] In some embodiments, the PAM recognized by a CRISPR protein-derived domain of the base editor disclosed herein may be provided to the cell on an oligonucleotide separate from the insert encoding the base editor (e.g., an AAV insert). In such embodiments, providing the PAM on a separate oligonucleotide allows for the cleavage of the target sequence, which would otherwise not be cleaved due to the absence of an adjacent PAM on the same polynucleotide as the target sequence.

[0631] In some embodiments, *S. pyogenes* Cas9 (SpCas9) can be used as a CRISPR endonuclease for genome engineering. However, others can be used. In some embodiments, different endonucleases can be used to target certain genomic targets. In some embodiments, synthetic SpCas9-derived variants with non-NGG PAM sequences can be used. Furthermore, other Cas9 heterologs from various species have been identified, and these “non-SpCas9s” can bind to various PAM sequences and can also be used in this disclosure. For example, the relatively large size of SpCas9 (approximately 4 kb coding sequence) can result in plasmids carrying SpCas9 cDNA not being efficiently expressed in cells. Conversely, the coding sequence of *Staphylococcus aureus* Cas9 (SaCas9) is approximately 1 kbp shorter than SpCas9, potentially allowing for efficient intracellular expression. Similar to SpCas9, the SaCas9 endonuclease can modify target genes in vivo in mammalian cells and in vivo in mice. In some embodiments, the Cas protein can target different PAM sequences. In some implementations, the target gene may be adjacent to the Cas9 PAM (e.g., 5'-NGG). In other implementations, other Cas9 heterologs may have different PAM requirements. For example, other PAMs such as those for *Streptococcus thermophilus* (5'-NNAGAA for CRISPR1; and 5'-NGGNG for CRISPR3) and *Neisseria meningiditis* (5'-NNNNGATT) may also be observed to be adjacent to the target gene.

[0632] In some embodiments, for the *S. pyogenes* system, the target gene sequence may be preceding the 5'-NGG PAM (i.e., located at 5'), and the 20-nt guide RNA sequence may pair with the opposite strand to mediate Cas9 cleavage adjacent to the PAM. In some embodiments, the adjacent cleavage may be approximately 3 base pairs upstream of the PAM. In some embodiments, the adjacent cleavage may be approximately 10 base pairs upstream of the PAM. In some embodiments, the adjacent cleavage may be approximately 0-20 base pairs upstream of the PAM. For example, the adjacent cleavage may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs upstream of the PAM. The adjacent cleavage may also be 1 to 30 base pairs downstream of the PAM. The sequence of an exemplary SpCas9 protein that can bind to PAM is as follows:

[0633] An example amino acid sequence of PAM binding to SpCas9 is as follows:

[0634]

[0635] An example amino acid sequence of PAM binding to SpCas9n is as follows:

[0636]

[0637] An example amino acid sequence of PAM binding to SpEQR Cas9 is as follows:

[0638]

[0639] In the above sequence, residues E1134, Q1334, and R1336 can be mutated from D1134, R1335, and T1336 to produce SpEQR Cas9, which are underlined and bold.

[0640] An example amino acid sequence of PAM binding to SpVQR Cas9 is as follows:

[0641]

[0642] In the above sequence, residues V1134, Q1334, and R1336 can be mutated from D1134, R1335, and T1336 to produce SpVQR Cas9, which are underlined and bold.

[0643] An example amino acid sequence of PAM binding SpVRER Cas9 is as follows:

[0644]

[0645] In the above sequence, residues V1134, R1217, Q1334, and R1336 can be mutated from D1134, G1217, R1335, and T1336 to produce SpVRER Cas9, which are underlined and bold.

[0646] In some implementations, engineered SpCas9 variants can recognize protospacer adjacent motif (PAM) sequences flanked by 3′H (non-G PAM) sequences (see [link to implementation]). Figure 8 In some embodiments, SpCas9 variants recognize NRNH PAMs (where R is A or G and H is A, C, or T). In some embodiments, non-G PAMs are NRRH, NRTH, or NRCH. These variants evolve via phage-assisted discontinuous evolution (PANCE), for example, as described in Miller, SM, et al., Continuous evolution of SpCas9 variants compatible with non-G PAMs, Nat. Biotechnol. (2020), ( / / d oi.org / 10.1038 / s41587-020-0412-8 The content of which is incorporated into this article in its entirety by way of quotation.

[0647] In some embodiments, the Cas9 domain is a recombinant Cas9 domain. In some embodiments, the recombinant Cas9 domain is a SpyMacCas9 domain. In some embodiments, the SpyMacCas9 domain is a nuclease-activated SpyMacCas9, a nuclease-inactivated SpyMacCas9 (SpyMacCas9d), or a SpyMacCas9 nickase (SpyMacCas9n). In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain may bind to a nucleic acid sequence having a non-standard PAM. In some embodiments, the SpyMacCas9 domain, SpCas9d domain, or SpCas9n domain may bind to a nucleic acid sequence having an NAA PAM sequence.

[0648] The sequence of an exemplary Cas9 A homolog of SpyCas9 with natural 5'-NAAN-3' PAM specificity in Streptococcus macacae is well known in the art and has been described, for example, by Jakimo et al. www.biorxiv.org / content / biorxiv / early / 2018 / 09 / 27 / 429654.full.pdf ), and is provided below.

[0649] SpyMacCas9

[0650] MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGALLFGSGETAE

[0651] ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG

[0652] NIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSD

[0653] VDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGN

[0654] LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI

[0655] LLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA

[0656] GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH

[0657] AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE

[0658] VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL

[0659] SGEQKKAIVDLLFKTTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKI

[0660] IKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQLKRRRYTGWG

[0661] RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGHSL

[0662] HEQIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERM

[0663] KRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI

[0664] VPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT

[0665] KAERGGGLSELDKAGFIKRKQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKITLKSK

[0666] LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM

[0667] IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFA

[0668] TVRKVLSMPQVNIVKKTEIQTVGQNGGLFDDNPKSPLEVTPSKLVPLKKELNPKKYGGYQ

[0669] KPTTAYPVLLITDTKQLIPISVMNKKQFEQNPVKFLRDRGYQQVGKNDFIKLPKYTLVDI

[0670] GDGIKRLWASSKEIHKGNQLVVSKKSQILLYHAHHLDSDLSNDYLQNHNQQFDVLFNEII

[0671] SFSKKCKLGKEHIQKIENVYSNKKNSASIEELAESFIKLLGFTQLGATSPFNFLFLGVKLNQ

[0672] KQYKGKKDYILPCTEGTLIRQSITGLYETRVDLSKIGED。

[0673] In some embodiments, the variant Cas9 protein carries mutations of H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1218A, resulting in a reduced ability of the peptide to cleave target DNA or RNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, the variant Cas9 protein carries mutations of D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1218A, resulting in a reduced ability of the peptide to cleave target DNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind target DNA (e.g., single-stranded target DNA). In some embodiments, when the variant Cas9 protein carries the W476A and W1126A mutations or when the variant Cas9 protein carries the P475A, W476A, N477A, ​​D1125A, W1126A, and D1218A mutations, the variant Cas9 protein does not effectively bind to the PAM sequence. Therefore, in some such cases, when the variant Cas9 protein is used in a binding method, the method does not require the PAM sequence. In other words, in some embodiments, when the variant Cas9 protein is used in a binding method, the method may include a guide RNA, but the method can be performed in the absence of the PAM sequence (and the binding specificity is thus provided by the target strand of the guide RNA). Other residues can be mutated to achieve the above-mentioned effect (i.e., inactivating one or other nuclease portions). As non-restrictive examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 may be altered (i.e., substituted). Furthermore, mutations other than alanine substitution are acceptable.

[0674] In some embodiments, the CRISPR protein-derived domain of the base editor may contain all or part of a Cas9 protein having a standard PAM sequence (NGG). In other embodiments, the Cas9-derived domain of the base editor may employ a non-standard PAM sequence. This sequence is described in the well-known art and is readily apparent to those skilled in the art. For example, Cas9 domains binding non-standard PAM sequences are described in Kleinstiver, BP, et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015) and Kleinstiver, BP, et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are incorporated herein by reference.

[0675] Cas9 domain with reduced PAM exclusivity

[0676] Typically, Cas9 proteins, such as Cas9 (spCas9) from *S. pyogenes*, require a standard NGG PAM sequence to bind to a specific nucleic acid region, where the "N" in "NGG" stands for adenosine (A), thymidine (T), or cytosine (C), and the "G" for guanosine. This can limit the ability to edit desired bases within the genome. In some embodiments, the base-editing fusion proteins provided herein may require substitutions at precise locations, such as regions containing target bases upstream of the PAM. See, for example, Komor, AC, et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage," *Nature* 533, 420-424 (2016), the entire contents of which are incorporated herein by reference. Therefore, in some embodiments, any fusion protein provided herein may contain a Cas9 domain capable of binding a nucleotide sequence that does not contain a standard (e.g., NGG) PAM sequence. Cas9 domains that bind to non-standard PAM sequences are described in the known art and are readily apparent to those skilled in the art. For example, Cas9 domains that bind to non-standard PAM sequences are described in Kleinstiver, BP, et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015) and Kleinstiver, BP, et al., “Broadening the targeting range of Staphylococcusaureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are incorporated herein by reference.

[0677] High-fidelity Cas9 domain

[0678] Certain aspects of this disclosure provide high-fidelity Cas9 domains. In some embodiments, the high-fidelity Cas9 domain is a domain of engineered Cas9 containing one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA compared to the corresponding wild-type Cas9 domain. Not wishing to be limited to any particular theory, a high-fidelity domain with reduced electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA may have less off-target efficacy. In some embodiments, the Cas9 domain (e.g., the wild-type Cas9 domain) contains one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA. In some embodiments, the Cas9 domain contains one or more mutations that reduce the association between the Cas9 domain and the sugar-phosphate backbone of DNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70%.

[0679] In some embodiments, any Cas9 fusion protein provided herein contains one or more of the N497X, R661X, Q695X, and / or Q926X mutations or a corresponding mutation of any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, any Cas9 fusion protein provided herein contains one or more of the N497A, R661A, Q695A, and / or Q926A mutations or a corresponding mutation of any amino acid sequence provided herein. In some embodiments, the Cas9 domain contains the D10A mutation or a corresponding mutation of any amino acid sequence provided herein. High-fidelity Cas9 domains are described in the known art and are readily apparent to those skilled in the art. For example, high-fidelity Cas9 domains have been described in Kleinstiver, BP, et al., “High-fidelity CRISPR-Cas9 nucleases with nodetectable genome-wide off-target effects.” Nature 529, 490-495 (2016) and Slaymaker, IM, et al., “Rationally engineered Cas9 nucleases with improved specificity.” Science 351, 84-88 (2015); the full contents of each are incorporated herein by reference.

[0680] In some embodiments, the modified Cas9 is a high-fidelity Cas9 enzyme. In some embodiments, the high-fidelity Cas9 enzyme is SpCas9 (K855A), eSpCas9 (1.1), SpCas9-HF1, or a highly accurate Cas9 variant (HypaCas9). The modified Cas9 eSpCas9 (1.1) contains an alanine substitution that weakens the interaction between the HNH / RuvC groove and the non-target DNA strand, preventing strand segregation and cleavage at the target site. Similarly, SpCas9-HF1 reduces off-target editing via an alanine substitution that interrupts Cas9 interaction with the DNA phosphate backbone. HypaCas9 contains mutations located in the REC3 domain (SpCas9 N692A / M694A / Q695A / H698A), which increase Cas9 proofreading and target discrimination. All three high-fidelity enzymes produce lower off-target editing than wild-type Cas9.

[0681] An example high-fidelity Cas9 is provided below.

[0682] Compared to the high fidelity of Cas9, Cas9 domain mutations are shown in bold and underline.

[0683]

[0684]

[0685] Fusion proteins containing nuclear localization sequences (NLS)

[0686] In some embodiments, the fusion proteins provided herein further comprise one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, such as nuclear localization sequences (NLS). In one embodiment, a bityped NLS is used. In some embodiments, the NLS comprises an amino acid sequence that facilitates protein delivery to the nucleus (e.g., via nuclear transport), and the protein contains the NLS. In some embodiments, any fusion protein provided herein further comprises a nuclear localization sequence (NLS). In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of a Cas9 domain. In some embodiments, the NLS is fused to the N-terminus of an nCas9 domain or a dCas9 domain. In some embodiments, the NLS is fused to the N-terminus of a deaminase. In some embodiments, the NLS is fused to the C-terminus of a deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises the amino acid sequence of any NLS sequence provided or referenced herein. Additional nuclear localization sequences have been described in the known art and are readily apparent to those skilled in the art. For example, the NLS sequence is described in Plank et al., PCT / EP2000 / 011690, the contents of which, with respect to the disclosed exemplary nuclear localization sequences, are incorporated herein by reference. In some embodiments, the NLS comprises the amino acid sequences PKKKRKVEGADKRTADGSEFESPKKKRKV, KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRKPKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0687] In some embodiments, the NLS is present in a linker or flanked by a linker, such as the linker described herein. In some embodiments, the N-terminal or C-terminal NLS is a bityped NLS. A bityped NLS contains two basic amino acid clusters separated by a relatively short spacer sequence (therefore, a bityped NLS is two parts, u...

Claims

1. A base editor for the preparation of a medicament for editing an α1-antitrypsin polynucleotide comprising a single nucleotide polymorphism (SNP) associated with α1-antitrypsin deficiency, wherein the use comprises contacting the polynucleotide with one or more guide RNAs and the base editor, wherein the base editor comprises a Streptococcus pyogenes Cas9 polynucleotide programmable DNA binding domain specific for a protospacer adjacent motif having a nucleic acid sequence of 5'-NGC-3' and an adenosine deaminase variant consisting of the following amino acid sequence having an alteration or a fragment thereof in which only the N-terminal methionine is missing: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD(SEQ ID NO:3), Wherein the alteration consists of one of the following changes with reference to SEQ ID NO:3: Y147T; Y147R; V82S; Q154S; V82S and Y147T; V82S and Y147R; V82S and Q154R; V82S and Q154S; V82S, Y123H and Y147T; V82S and Y123H; I76Y and V82S; V82T; I76Y and V82T; or I76Y, V82T, Y147T, and Q154S; and wherein the guide RNA mediates the base editor to bind to the target site in the α1-antitrypsin polynucleotide to cause an A·T to G·C change in the SNP associated with α1-antitrypsin deficiency, wherein the SNP associated with α1-antitrypsin deficiency causes the expression of an α1-antitrypsin polypeptide having a lysine at amino acid position 342, and the A·T to G·C change changes the lysine at position 342 to glutamate, wherein the amino acid position 342 refers to the following amino acid sequence, and the amino acid residue "E" after the underlined sequence is the amino acid position 1: MPSSVSWGILLLAGLCCLVPVSLA EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFE VKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK(SEQ ID NO:21), and wherein the guide RNA comprises a spacer sequence selected from the group of nucleic acid sequences consisting of: a)ACCAUCGACAAGAAAGGGACUGA; b)CCAUCGACAAGAAAGGGACUGA; c)CAUCGACAAGAAAGGGACUGA; d) AUCGACAAGAAAGGGACUGA; e)UCGACAAGAAAGGGACUGA; and f)CGACAAGAAAGGGACUGA.

2. The use according to claim 1, wherein the alteration consists of a V82S change.

3. The use according to claim 1, wherein the base editor further comprises a wild-type adenosine deaminase domain.

4. The use according to claim 1, wherein the base editor further comprises an adenosine deaminase domain consisting of SEQ ID NO: 3 or a fragment thereof lacking only the N-terminal methionine.

5. The use according to any one of claims 1 to 4, wherein the polynucleotide is within a cell in vivo or ex vivo.

6. The use according to claim 1, wherein the change consists of the following combination of changes: V82S+Q154S.

7. The use according to claim 1, wherein the change consists of the following combination of changes: V82S+Y147R.

8. The use according to claim 1, wherein the change consists of the following combination of changes: V82S+Q154R.

9. The use according to claim 1, wherein the change consists of the following combination of changes: V82S+Y123H.

10. The use according to claim 1, wherein the change consists of the following combination of changes: I76Y+V82S.

11. The use according to claim 1, wherein the change consists of the following combination of changes: V82S+Y123H+Y147T.

12. The use according to any one of claims 1 to 4, wherein the polynucleotide programmable DNA binding domain is nuclease inactive or is a nickase.

13. The use according to any one of claims 1 to 4, wherein the base editor further comprises a zinc finger domain.

14. The use according to any one of claims 1 to 4, wherein the adenosine deaminase variant does not occur in nature.

15. A base editor system, comprising a base editor according to any one of claims 1 to 14 and a guide RNA, wherein the guide RNA is selected from a nucleic acid sequence consisting of: 5′-ACCAUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUCCGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU-3′; 5′-CCAUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUCCGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU-3′; 5′-CAUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUCCGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU-3′; 5′-AUCGACAAGAAAGGGACUGAGUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGUCCGGUGCUUUU-3′; 5′-UCGACAAGAAAGGGACUGAGUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGUCGGUGCUUUU-3′; and / or 5′-CGACAAGAAAGGGACUGAGUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGUCCGGUGCUUUU-3′; wherein the guide RNA mediates the base editor to bind to a target site in an α1-antitrypsin polynucleotide to cause an A·T to G·C change in a SNP associated with α1-antitrypsin deficiency, wherein the SNP associated with α1-antitrypsin deficiency causes the expression of an α1-antitrypsin polypeptide having a lysine at amino acid position 342, and the A·T to G·C change changes the lysine at position 342 to glutamate, wherein the amino acid position 342 refers to the following amino acid sequence, and the amino acid residue "E" after the underlined sequence is amino acid position 1: MPSSVSWGILLLAGLCCLVPVSLA EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK(SEQ ID NO:21)。 16. A cell produced by introducing into said cell or a progenitor cell thereof: a base editor or a polynucleotide encoding the base editor to the cell, wherein the base editor comprises a Streptococcus pyogenes Cas9 polynucleotide programmable DNA binding domain and an adenosine deaminase variant specific for a protospacer adjacent motif having a nucleic acid sequence of 5'-NGC-3' as described in any one of claims 1 to 15; and One or more guide RNAs that mediate the binding of the base editor to the target site in the α1-antitrypsin polynucleotide to cause an A·T to G·C change in a SNP associated with α1-antitrypsin deficiency, wherein the SNP associated with α1-antitrypsin deficiency causes the expression of an α1-antitrypsin polypeptide having a lysine at amino acid position 342, and the A·T to G·C change changes the lysine at position 342 to glutamic acid, wherein the amino acid position 342 refers to the following amino acid sequence, and the amino acid residue "E" after the underlined sequence is the amino acid position 1: MPSSVSWGILLLAGLCCLVPVSLA EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFE VKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK(SEQ ID NO:21), and wherein the guide RNA comprises a spacer sequence selected from the group of nucleic acid sequences consisting of: a)ACCAUCGACAAGAAAGGGACUGA; b)CCAUCGACAAGAAAGGGACUGA; c)CAUCGACAAGAAAGGGACUGA; d) AUCGACAAGAAAGGGACUGA; e)UCGACAAGAAAGGGACUGA; and f)CGACAAGAAAGGGACUGA.

17. The cell according to claim 16, wherein the produced cell is a hepatocyte or a progenitor cell thereof.

18. The cell of claim 16 or claim 17, wherein the cell is from a subject suffering from alpha 1 -antitrypsin deficiency.

19. The cell according to claim 16 or 17, wherein the cell is a mammalian cell.

20. The cell of claim 16 or 17, wherein the cell is selected for an A·T to G·C change of a SNP associated with alpha 1 -antitrypsin deficiency.

21. The cell of claim 16 or 17, wherein the polynucleotide programmable DNA binding domain is nuclease inactive or is a nickase.

22. The cell of claim 16 or 17, wherein the base editor further comprises a zinc finger domain.

23. The cell of claim 16 or 17, wherein the base editor and the one or more guide polynucleotides form a complex in the cell.

24. Use of a cell according to any one of claims 21 to 23 for the preparation of a medicament for treating alpha 1 -antitrypsin deficiency in a subject, wherein the medicament is administered to the subject.

25. The use according to claim 24, wherein the cells are autologous to the subject.

26. The use of claim 24, wherein the cells are allogeneic to the subject.

27. An isolated cell or cell population propagated or expanded from the cell of any one of claims 21 to 26.

28. Use of a base editor or a polynucleotide encoding the base editor for preparing a medicament for treating α1-antitrypsin deficiency, wherein the use comprises: (a) introducing the base editor or a polynucleotide encoding the base editor and one or more guide RNAs into a hepatocyte progenitor cell comprising a SNP associated with α1-antitrypsin deficiency, wherein the base editor comprises a Streptococcus pyogenes Cas9 polynucleotide programmable DNA binding domain and an adenosine deaminase variant specific to a protospacer adjacent motif having a nucleic acid sequence of 5'-NGC-3' as described in any one of claims 1 to 15; and wherein the one or more guide RNAs mediate the binding of the base editor to a target site in an α1-antitrypsin polynucleotide to cause an A·T to G·C change in a SNP associated with α1-antitrypsin deficiency, and wherein the SNP associated with α1-antitrypsin deficiency causes the expression of an α1-antitrypsin polypeptide having a lysine at amino acid position 342, and the A·T to G·C change changes the lysine at position 342 to glutamate, wherein the amino acid position 342 refers to the following amino acid sequence, wherein the amino acid residue "E" after the underlined sequence is amino acid position 1: MPSSVSWGILLLAGLCCLVPVSLA EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFE VKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK(SEQ ID NO:21), and wherein the guide RNA comprises a spacer sequence selected from the group of nucleic acid sequences consisting of: a)ACCAUCGACAAGAAAGGGACUGA; b)CCAUCGACAAGAAAGGGACUGA; c)CAUCGACAAGAAAGGGACUGA; d) AUCGACAAGAAAGGGACUGA; e)UCGACAAGAAAGGGACUGA; and f)CGACAAGAAAGGGACUGA; and (b) differentiating the hepatocyte progenitor cells into hepatocytes.

29. Use of a base editor or a polynucleotide encoding the base editor for preparing a medicament for treating α1-antitrypsin deficiency, wherein the use comprises: introducing the base editor or a polynucleotide encoding the base editor and one or more guide RNAs into a hepatocyte or hepatocyte progenitor cell comprising a SNP associated with α1-antitrypsin deficiency, wherein the base editor comprises a Streptococcus pyogenes Cas9 polynucleotide programmable DNA binding domain and an adenosine deaminase variant specific to a protospacer adjacent motif having a nucleic acid sequence of 5'-NGC-3' as described in any one of claims 1 to 15; and wherein the one or more guide RNAs mediate the binding of the base editor to a target site in an α1-antitrypsin polynucleotide to cause an A·T to G·C change in a SNP associated with α1-antitrypsin deficiency, wherein the SNP associated with α1-antitrypsin deficiency causes the expression of an α1-antitrypsin polypeptide having a lysine at amino acid position 342, and the A·T to G·C change changes the lysine at position 342 to glutamate, wherein the amino acid position 342 refers to the following amino acid sequence, and the amino acid residue "E" after the underlined sequence is amino acid position 1: MPSSVSWGILLLAGLCCLVPVSLA EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFE VKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK(SEQ ID NO:21), and wherein the guide RNA comprises a spacer sequence selected from the group of nucleic acid sequences consisting of: a)ACCAUCGACAAGAAAGGGACUGA; b)CCAUCGACAAGAAAGGGACUGA; c)CAUCGACAAGAAAGGGACUGA; d) AUCGACAAGAAAGGGACUGA; e)UCGACAAGAAAGGGACUGA; and f)CGACAAGAAAGGGACUGA.

30. The use according to claim 28 or 29, wherein the hepatocytes or hepatocyte progenitor cells are mammalian cells.

31. The use of claim 28 or 29, wherein the cell is selected for an A·T to G·C change of a SNP associated with alpha 1 -antitrypsin deficiency.

32. The use according to claim 28 or 29, wherein the polynucleotide programmable DNA binding domain is a nuclease inactivating or nicking enzyme.

33. The use according to claim 28 or 29, wherein the base editor further comprises a zinc finger domain.

34. The use according to claim 28 or 29, wherein the base editor and the one or more guide polynucleotides form a complex in a cell.

35. A pharmaceutical composition comprising the base editor system according to claim 15, and a pharmaceutically acceptable carrier, vehicle or excipient.

36. A pharmaceutical composition comprising the cell according to any one of claims 16 to 23, and a pharmaceutically acceptable carrier, vehicle or excipient.

37. A kit comprising the base editor system according to claim 15.

38. A kit comprising the cell according to any one of claims 16 to 23.

39. The kit of claim 37 or claim 38, further comprising a package insert with instructions for use.

40. A base editor system comprising a Streptococcus pyogenes Cas9 polynucleotide programmable DNA binding domain specific for a protospacer adjacent motif having a nucleic acid sequence of 5'-NGC-3' and an adenosine deaminase variant consisting of the following amino acid sequence having an alteration or a fragment thereof lacking only the N-terminal methionine: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD(SEQ ID NO:3), Wherein the alteration consists of one of the following changes with reference to SEQ ID NO:3: Y147T; Y147R; V82S; Q154S; V82S and Y147T; V82S and Y147R; V82S and Q154R; V82S and Q154S; V82S, Y123H and Y147T; V82S and Y123H; I76Y and V82S; V82T; I76Y and V82T; or I76Y, V82T, Y147T, and Q154S; and A guide RNA, wherein the guide RNA mediates the base editor to bind to a target site in an α1-antitrypsin polynucleotide to cause an A·T to G·C change in a SNP associated with α1-antitrypsin deficiency, wherein the SNP associated with α1-antitrypsin deficiency causes the expression of an α1-antitrypsin polypeptide having a lysine at amino acid position 342, and the A·T to G·C change changes the lysine at position 342 to glutamic acid, wherein the amino acid position 342 refers to the following amino acid sequence, and the amino acid residue "E" after the underlined sequence is the amino acid position 1: MPSSVSWGILLLAGLCCLVPVSLA EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFE VKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK(SEQ ID NO:21), and wherein the guide RNA comprises a spacer sequence selected from the group of nucleic acid sequences consisting of: a)ACCAUCGACAAGAAAGGGACUGA; b)CCAUCGACAAGAAAGGGACUGA; c)CAUCGACAAGAAAGGGACUGA; d) AUCGACAAGAAAGGGACUGA; e)UCGACAAGAAAGGGACUGA; and f)CGACAAGAAAGGGACUGA.

41. The base editor system of claim 40, wherein the alteration consists of a V82S change.

42. The base editor system of claim 41, wherein the base editor further comprises a wild-type adenosine deaminase domain.

43. The base editor system of claim 40 or 41, wherein the polynucleotide programmable DNA binding domain is a nuclease-inactivating Cas9.

44. The base editor system of claim 40 or 41, wherein the polynucleotide programmable DNA binding domain is a Cas9 nickase.

45. A cell comprising the base editor system according to any one of claims 40 to 44.

46. ​​The cell of claim 45, wherein the cell is a mammalian cell.

47. The cell of claim 45, wherein the cell is in vivo or in vitro.

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