Methods for editing single nucleotide polymorphisms using a programmable base editor system

Abstract

The present disclosure describes compositions and methods for altering mutations associated with Rett syndrome (RETT). Provided herein are compositions and methods for using a base editor (e.g., ABE8) that works in concert with a guide polynucleotide, the base editor comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain. Also provided herein are base editor systems for editing the nucleobases of a target nucleotide sequence.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This International PCT Application claims the benefit of and priority to Provisional Patent Application No. 62 / 850,919, filed on May 21, 2019, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the use of an adenosine base editor and a guide polynucleotide to generate a nucleobase change in the methyl CpG binding protein 2 gene for the treatment of Rett syndrome. Background Art

[0004] Rett syndrome (RTT or RETT) is a heterogeneous population of mutations in the methyl-CpG-binding protein 2 (Mecp2) gene that damage or abrogate the ability of the encoded protein to modify chromatin and transcriptional states in the central nervous system (CNS). Gene therapy that either renders functional Mecp2 or uses RNA editing to repair endogenous Mecp2 (MECP2) mRNA transcripts is a promising approach when delivered broadly throughout the CNS. However, both approaches must overcome significant challenges to achieve therapeutic efficacy. Mecp2 gene therapy must strictly control the dose of gene delivered to a single-cell matrix or risk mimicking the Mecp2 duplication syndrome phenotype. RNA editing platforms cannot accurately correct the most common Mecp2 mutations that are responsible for more than 45% of RTT diagnosed cases, and also induce potent non-guided off-target editing.

[0005] Genetic mutations in Mecp2 that cause Rett syndrome (RTT) are highly heterogeneous. Therefore, a popular therapeutic strategy is to deliver Mecp2 carried by a recombinant adeno-associated virus (rAAV). Since this strategy is agnostic to the causal mutation of each individual, successful gene therapy will provide a therapeutic option for a large part of the RTT patient population. However, to date, this strategy has achieved limited success. RTT patients are almost all heterozygous females, with characteristic wild-type and mutant X-linked MeCP2 mosaic expression in the central nervous system (CNS) due to random X chromosome inactivation. Therefore, the delivery of wild-type MeCP2 and its expression in neurons that already express wild-type MeCP2 appear to partially mimic the phenotype of MeCP2 duplication syndrome. Consistent with this, the high transduction efficiency in the CNS of RTT model mice produces approximately 2 times higher MeCP2 expression than that found in wild-type mice.

[0006] Therefore, there is a need for novel compositions and methods for treating patients suffering from Rett syndrome.

[0007] Incorporated by Reference

[0008] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication, patent or patent application was specifically and individually indicated to be incorporated by reference. If not otherwise indicated, the publications, patents and patent applications mentioned in this specification are incorporated herein by reference in their entirety. Summary of the invention

[0009] As described below, the present invention is characterized in that a composition and method for accurately correcting pathogenic amino acids using a programmable nucleobase editor. In particular, the compositions and methods of the present invention can be used to treat Rett syndrome (RTT or RETT). Therefore, the present invention provides a composition and method for treating Rett syndrome, which uses an adenosine (A) base editor (ABE) (e.g., ABE8) to accurately correct single nucleotide polymorphisms in the endogenous Mecp2 gene to correct harmful mutations (e.g., R106W, R133C, T158M, R255*, R270*, R306C).

[0010] In one aspect, a method for editing a subject's methyl CpG binding protein 2 (MECP2) gene or a regulatory element thereof is provided, wherein the method comprises administering to a subject in need thereof: (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid replacement located at amino acid position 82 or 166 or a position corresponding thereto relative to a TadA reference sequence, and wherein the guide polynucleotide directs the adenosine base editor to produce an A to G nucleobase change within a MECP2 gene or a regulatory element thereof comprising a SNP associated with Rett syndrome (RETT); wherein the A to G nucleobase change is located at a SNP associated with RETT, and the SNP results in an R133C or R306C amino acid mutation in a MECP2 polypeptide or a variant thereof encoded by the MECP2 gene. In one embodiment, the TadA reference sequence does not comprise the amino acid sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD. In one embodiment, the A to G nucleobase change changes the SNP associated with RETT to a wild-type nucleobase. In one embodiment, the A to G nucleobase change changes the SNP associated with RETT to a non-wild-type nucleobase, which reduces one or more symptoms of RETT. In one embodiment, the A to G nucleobase change at the SNP associated with RETT changes a cysteine ​​in a methyl CpG binding protein 2 (MECP2) polypeptide to an arginine or changes a stop codon to an arginine. In one embodiment, the SNP associated with RETT results in the expression of a MECP2 polypeptide comprising an arginine at amino acid position 133 and / or 306. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence complementary to a MECP2 gene comprising a SNP associated with RETT, or a regulatory element thereof. In one embodiment, the adenosine base editor is complexed with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to a MECP2 gene comprising a SNP associated with RETT, or a regulatory element thereof.In one embodiment, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of: 5'-AGAGCAAAAGGCUUUUCCCU-3', 5'-UAGAGCAAAAGGCUUUUCCC-3', 5'-UAGAGCAAAAGGCUUUUCCCU-3', 5'-UUUAGAGCAAAAGGCUUUUCCCU-3', 5'-UCUUGCACUUCUUGAUGGGG-3', 5'-CUUGCACUUCUUGAUGGGGAG-3', or 5'-GUCUUGCACUUCUUGAUGGGGAG-3'.

[0011] In one aspect, a base editor system is provided, wherein the base editor system comprises: (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid replacement at amino acid position 82 or 166 or a corresponding position thereof relative to the TadA reference sequence, and wherein the guide polynucleotide guides the adenosine base editor to produce an A to G nucleobase change within a methyl CpG binding protein 2 (MECP2) gene or a regulatory element thereof containing a SNP associated with Rett syndrome (RETT); wherein the A to G nucleobase change is located at a SNP associated with RETT, and the SNP causes an R133C or R306C amino acid mutation in a MECP2 polypeptide encoded by the MECP2 gene or a variant thereof. In one embodiment, the A to G nucleobase change causes the SNP associated with RETT to become a wild-type nucleobase. In one embodiment, the A to G nucleobase change changes the SNP associated with RETT to a non-wild-type nucleobase, which reduces one or more symptoms of RETT. In one embodiment, the SNP associated with RETT results in the expression of a MECP2 polypeptide comprising an arginine at amino acid position 133 and / or 306. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence complementary to a MECP2 gene or a regulatory element thereof comprising a SNP associated with RETT. In one embodiment, the adenosine base editor is complexed with a single guide RNA (sgRNA), wherein the sgRNA comprises a nucleic acid sequence complementary to a MECP2 gene or a regulatory element thereof comprising a SNP associated with RETT. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of: 5'-AGAGCAAAAGGCUUUUCCCU-3', 5'-UAGAGCAAAAGGCUUUUCCC-3', 5'-UAGAGCAAAAGGCUUUUCCCU-3', 5'-UUUAGAGCAAAAGGCUUUUCCCU-3', 5'-UCUUGCACUUCUUGAUGGGG-3', 5'-CUUGCACUUCUUGAUGGGGAG-3', or 5'-GUCUUGCACUUCUUGAUGGGGAG-3'.

[0012] On the other hand, a method for editing a MECP2 polynucleotide comprising a single nucleotide polymorphism (SNP) associated with Rett syndrome (RETT) is provided, wherein the method comprises: contacting the MECP2 polynucleotide with a complex of an adenosine deaminase base editor 8 (ABE8) and one or more guide polynucleotides, wherein ABE8 comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein the one or more guide polynucleotides target the base editor to cause an A·T to G·C change in the SNP in the MECP2 polynucleotide associated with RETT, wherein the change is one or both of R133C or R306C. In one embodiment, the contact is in a cell, a eukaryotic cell, a mammalian cell, or a human cell. In one embodiment, the cell is in vivo or in vitro. In one embodiment, the A·T to G·C change at the SNP associated with RETT changes the cysteine ​​in the methyl CpG binding protein 2 (Mecp2) polypeptide to arginine or changes the stop codon to arginine. In one embodiment, the SNP associated with RETT results in the expression of a MECP2 polypeptide comprising an arginine at amino acid position 133 and / or 306. In one embodiment, the polynucleotide programmable DNA binding domain is a Cas9 selected from the following: Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), Streptococcus canis Cas9 (ScCas9), or variants thereof. In one embodiment, the polynucleotide programmable DNA binding domain comprises a modified SpCas9 bound to an altered protospacer adjacent motif (PAM). In one embodiment, the modified SpCas9 binds to a PAM comprising a nucleic acid sequence selected from 5'-NGT-3' or 5'-NGG-3'. In one embodiment, the modified SpCas9 binds to an NGT PAM variant. In one embodiment, the NGT PAM variant comprises an amino acid substitution at one or more amino acid residues 1335, 1337, 1135, 1136, 1218 and / or 1219 of the modified SpCas9, or an amino acid substitution corresponding thereto.In one embodiment, the modified SpCas9 comprises one or more of the amino acid substitutions L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R and L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T1337, T1337L, T1337Q, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337H, T1337Q, and T1337M, or their corresponding amino acid substitutions. In one embodiment, the modified SpCas9 comprises one or more of the amino acid substitutions D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, and T1337, and L1111R, G1218R, E1219F, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M, or their corresponding amino acid substitutions. In some embodiments, the polynucleotide programmable DNA binding domain is a nuclease-inactive or nickase variant. In one embodiment, the nickase variant comprises an amino acid replacement D10A or its corresponding amino acid replacement. In one embodiment, the adenosine deaminase domain is capable of deaminating adenosine in deoxyribonucleic acid (DNA). In one embodiment, the adenosine deaminase domain comprises an amino acid position 82 and / or 166 change located at MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD. In one embodiment, the adenosine deaminase domain comprises an change located at M amino acid positions 82 and 166. In one embodiment, the adenosine deaminase domain comprises a change selected from the following: a V82S change, a T166R change, or both a V82S and a T166R change. In some embodiments, the adenosine deaminase domain further comprises one or more of the following mutations: Y147T, Y147R, Q154S, Y123H, and Q154R.In some embodiments, the adenosine deaminase domain comprises a combination of alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123 In one embodiment, ABE8 comprises an adenosine deaminase variant monomer, wherein the adenosine deaminase monomer comprises V82S and T166R changes. In one embodiment, ABE8 comprises an adenosine deaminase heterodimer, wherein the heterodimer comprises a wild-type adenosine deaminase domain and an adenosine deaminase variant. In one embodiment, the adenosine deaminase variant monomer further comprises one or more changes selected from the group consisting of: Y147T, Y147R, Q154S, Y123H, V82S, T166R and Q154R. In one embodiment, ABE8 comprises an adenosine deaminase heterodimer comprising a TadA*8 domain and a wild-type TadA domain. In one embodiment, the adenosine deaminase variant further comprises a change selected from the group consisting of: Y147T, Y147R, Q154S, Y123H, V82S, T166R and Q154R. In one embodiment, the ABE8 base editor comprises a heterodimer comprising a wild-type TadA domain and an adenosine deaminase variant comprising a combination of changes 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.In one embodiment, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of AGAGCAAAAGGCUUUUCCCU-3', 5'-UAGAGCAAAAGGCUUUUCCC-3', 5'-UAGAGCAAAAGGCUUUUCCCU-3', 5'-UUUAGAGCAAAAGGCUUUUCCCU-3', 5'-UCUUGCACUUCUUGAUGGGG-3', 5'-CUUGCACUUCUUGAUGGGGAG-3', or 5'-GUCUUGCACUUCUUGAUGGGGAG-3'. In one embodiment, the adenosine deaminase is a TadA deaminase. In one embodiment, the TadA deaminase is a TadA*8 variant. In one embodiment, the TadA*8 variant is selected from the group consisting of: TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8. 11. TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, Tad A*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23 and TadA*8.24.In one embodiment, the ABE8 base editor is selected from the group consisting of ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, ABE8.5-m, ABE8.6-m, ABE8.7-m, ABE8.8-m, ABE8.9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.13-m, ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8. 23-m, 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, ABE8.1 2-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 and ABE8.24. In one embodiment, the one or more guide RNAs comprise a CRISPR RNA (crRNA) and a trans-encoding small RNA (tracrRNA), wherein the crRNA comprises a nucleic acid sequence complementary to a MECP2 nucleic acid sequence comprising a SNP associated with RETT. In one embodiment, the ABE8 base editor is complexed with a single guide RNA (sgRNA), wherein the sgRNA comprises a nucleic acid sequence complementary to a MECP2 nucleic acid sequence comprising a SNP associated with RETT. In some embodiments, the ABE8 base editor comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:.

[0013] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0014] On the other hand, a cell is provided, which is produced by introducing the following items into the cell or its ancestor: (i) an ABE8 base editor or a polynucleotide encoding the base editor, wherein the ABE8 base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and (ii) one or more guide polynucleotides that target the base editor to cause a SNP in a MECP2 polynucleotide associated with RETT syndrome (RETT) to undergo an A·T to G·C change, wherein the change is one or both of R133C or R306C. In one embodiment, the cell is a neuron. In one embodiment, the neuron expresses a MECP2 polypeptide. In one embodiment, the cell is from a subject with RETT. In one embodiment, the cell is a mammalian cell or a human cell. In one embodiment, the A·T to G·C change at a SNP associated with RETT changes a cysteine ​​in a methyl CpG binding protein 2 (MECP2) polypeptide to an arginine and a stop codon to an arginine. In one embodiment, the SNP associated with RETT results in the expression of a MECP2 polypeptide comprising an arginine at amino acid position 133 and / or 306. In one embodiment, the polynucleotide programmable DNA binding domain is a Cas9 selected from the following: Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), Streptococcus canis Cas9 (ScCas9), or variants thereof. In one embodiment, the polynucleotide programmable DNA binding domain comprises a modified SpCas9 bound to an altered protospacer adjacent motif (PAM). In one embodiment, the modified SpCas9 binds to a PAM comprising a nucleic acid sequence selected from 5'-NGT-3' or 5'-NGG-3'. In one embodiment, the modified SpCas9 binds to an NGT PAM variant. In one embodiment, the NGT PAM variant comprises an amino acid substitution at one or more amino acid residues 1335, 1337, 1135, 1136, 1218 and / or 1219 of the modified SpCas9, or an amino acid substitution corresponding thereto.In one embodiment, the modified SpCas9 comprises one or more of the amino acid substitutions L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R and L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T1337, T1337L, T1337Q, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337H, T1337Q, and T1337M, or their corresponding amino acid substitutions. In one embodiment, the modified SpCas9 comprises one or more of the amino acid substitutions D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, and T1337, and L1111R, G1218R, E1219F, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M, or their corresponding amino acid substitutions. In some embodiments, the polynucleotide programmable DNA binding domain is a nuclease-inactive or nickase variant. In one embodiment, the nickase variant comprises an amino acid replacement D10A or its corresponding amino acid replacement. In one embodiment, the adenosine deaminase domain is capable of deaminating adenosine in deoxyribonucleic acid (DNA). In one embodiment, the adenosine deaminase domain comprises an amino acid position 82 and / or 166 change located at MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD. In some embodiments, the adenosine deaminase further comprises one or more of the following mutations: Y147T, Y147R, Q154S, Y123H, and Q154R.In some embodiments, the adenosine deaminase domain comprises a combination of combinations of alterations selected from the group consisting of: Y147T + Q154R; Y147T + Q154S; Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y147R + Q154R + Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76Y; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R. In one embodiment, ABE8 comprises an adenosine deaminase variant monomer, wherein the adenosine deaminase monomer comprises V82S and T166R alterations. In one embodiment, ABE8 comprises an adenosine deaminase heterodimer comprising a wild-type adenosine deaminase domain and an adenosine deaminase variant. In one embodiment, the adenosine deaminase variant monomer further comprises one or more alterations selected from the group consisting of: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R. In one embodiment, ABE8 comprises an adenosine deaminase heterodimer comprising a TadA*7.10 domain and a TadA*8 domain. In one embodiment, the adenosine deaminase variant monomer further comprises one or more alterations selected from the group consisting of: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R. In one embodiment, the ABE8 base editor comprises a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant, the adenosine deaminase variant comprising alterations selected from the group consisting of: Y147T + Q154R; Y147T + Q154S; Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y147R + Q154R + Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76Y; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R.In one embodiment, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of 5'-AGAGCAAAAGGCUUUUCCCU-3', 5'-UAGAGCAAAAGGCUUUUCCC-3', 5'-UAGAGCAAAAGGCUUUUCCCU-3', 5'-UUUAGAGCAAAAGGCUUUUCCCU-3', 5'-UCUUGCACUUCUUGAUGGGG-3', 5'-CUUGCACUUCUUGAUGGGGAG-3', or 5'-GUCUUGCACUUCUUGAUGGGGAG-3'. In one embodiment, the adenosine deaminase is a TadA deaminase. In one embodiment, the TadA deaminase is a TadA*8 variant. In one embodiment, the TadA*8 variant is selected from the group consisting of: TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8. 11. TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, Tad A*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23 and TadA*8.24.In one embodiment, the ABE8 base editor is selected from the group consisting of ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, ABE8.5-m, ABE8.6-m, ABE8.7-m, ABE8.8-m, ABE8.9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.13-m, ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8. 23-m, 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, ABE8.1 2-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 and ABE8.24. In one embodiment, the one or more guide RNAs comprise a CRISPR RNA (crRNA) and a trans-encoding small RNA (tracrRNA), wherein the crRNA comprises a nucleic acid sequence complementary to a MECP2 nucleic acid sequence comprising a SNP associated with RETT. In one embodiment, the base editor is complexed with a single guide RNA (sgRNA), wherein the sgRNA comprises a nucleic acid sequence complementary to a MECP2 nucleic acid sequence comprising a SNP associated with RETT. In some embodiments, the ABE8 base editor comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:.

[0015] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD. In one embodiment, the IgRNA comprises a scaffold having the following sequence: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT.

[0016] On the other hand, a method for treating RETT syndrome (RETT) in a subject is provided, wherein the method comprises administering to the subject: (i) an ABE8 base editor or a polynucleotide encoding the base editor, wherein the ABE8 base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and (ii) one or more guide polynucleotides that target the base editor to cause a SNP in a MECP2 polynucleotide associated with RETT syndrome (RETT) to undergo an A·T to G·C change, wherein the change is one or both of R133C or R306C. In one embodiment, the subject is a mammal or a human. In one embodiment, the method comprises delivering an ABE8 base editor or a polynucleotide encoding the ABE8 base editor, and the one or more guide polynucleotides to a cell of the subject, optionally, wherein the cell is a neuron. In one embodiment of the method, the A·T to G·C change at a SNP associated with RETT changes a cysteine ​​in a methyl CpG binding protein 2 (MECP2) polypeptide to an arginine or changes a stop codon to an arginine. In one embodiment, the SNP associated with RETT results in the expression of a MECP2 polypeptide comprising an arginine at amino acid position 133 and / or 306. In one embodiment, the polynucleotide programmable DNA binding domain is a Cas9 selected from the following: Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), Streptococcus canis Cas9 (ScCas9), or variants thereof. In one embodiment, the polynucleotide programmable DNA binding domain comprises a modified SpCas9 bound to an altered protospacer adjacent motif (PAM). In one embodiment, the modified SpCas9 binds to a PAM comprising a nucleic acid sequence selected from 5'-NGT-3' or 5'-NGG-3'. In one embodiment, the modified SpCas9 binds to an NGT PAM variant. In one embodiment, the NGT PAM variant comprises an amino acid substitution at one or more amino acid residues 1335, 1337, 1135, 1136, 1218 and / or 1219 of the modified SpCas9, or an amino acid substitution corresponding thereto.In one embodiment, the modified SpCas9 comprises one or more of the amino acid substitutions L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R and L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, R1335Q, T1337, T1337L, T1337Q, T1337I, T1337V, T1337F and T1337M, or their corresponding amino acid substitutions. In one embodiment, the modified SpCas9 comprises amino acid substitutions D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, and T1337, and L1111R, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332 In some embodiments, the polynucleotide programmable DNA binding domain is a nucleic acid-free or nickase variant. In one embodiment, the nickase variant comprises an amino acid replacement D10A or its corresponding amino acid replacement. In one embodiment, the adenosine deaminase domain is capable of deaminating adenosine in deoxyribonucleic acid (DNA). In one embodiment, the adenosine deaminase domain comprises an alteration at amino acid position 82 and / or 166 of MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD. In one embodiment, the adenosine deaminase domain comprises an alteration at amino acid position 82 and 166 of M. In one embodiment, the adenosine deaminase domain comprises an alteration selected from the group consisting of a V82S alteration, a T166R alteration, or both a V82S and a T166R alteration. In some embodiments, the adenosine deaminase domain further comprises one or more of the following mutations: Y147T, Y147R, Q154S, Y123H, and Q154R.In some embodiments, the adenosine deaminase domain comprises a combination of alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123 In one embodiment, ABE8 comprises an adenosine deaminase variant monomer, wherein the adenosine deaminase monomer comprises V82S and T166R changes. In one embodiment, ABE8 comprises an adenosine deaminase heterodimer, wherein the heterodimer comprises a wild-type adenosine deaminase domain and an adenosine deaminase variant. In one embodiment, the adenosine deaminase variant monomer further comprises one or more changes selected from the group consisting of: Y147T, Y147R, Q154S, Y123H, V82S, T166R and Q154R. In one embodiment, ABE8 comprises an adenosine deaminase heterodimer comprising a TadA*7.10 domain and a TadA*8 domain. In one embodiment, the adenosine deaminase variant monomer further comprises one or more changes selected from the group consisting of: Y147T, Y147R, Q154S, Y123H, V82S, T166R and Q154R. In one embodiment, the ABE8 base editor comprises a heterodimer comprising a TadA7.10 domain and an adenosine deaminase variant comprising an alteration selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y 123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.In one embodiment, the guide polynucleotide has a nucleic acid sequence selected from the group consisting of: 5'-AGAGCAAAAGGCUUUUCCCU-3', 5'-UAGAGCAAAAGGCUUUUCCC-3', 5'-UAGAGCAAAAGGCUUUUCCCU-3', 5'-UUUAGAGCAAAAGGCUUUUCCCU-3', 5'-UCUUGCACUUCUUGAUGGGG-3', 5'-CUUGCACUUCUUGAUGGGGAG-3', or 5'-GUCUUGCACUUCUUGAUGGGGAG-3'. In one embodiment, the adenosine deaminase is a TadA deaminase. In one embodiment, the TadA deaminase is a TadA*8 variant. In one embodiment, the TadA*8 variant is selected from the group consisting of: TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8. 11. TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, Tad A*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23 and TadA*8.24.In one embodiment, the ABE8 base editor is selected from the group consisting of ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, ABE8.5-m, ABE8.6-m, ABE8.7-m, ABE8.8-m, ABE8.9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.13-m, ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8. 23-m, 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, ABE8.1 2-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 and ABE8.24. In one embodiment, the one or more guide RNAs comprise a CRISPR RNA (crRNA) and a trans-encoding small RNA (tracrRNA), wherein the crRNA comprises a nucleic acid sequence complementary to a MECP2 nucleic acid sequence comprising a SNP associated with RETT. In one embodiment, the base editor is complexed with a single guide RNA (sgRNA), wherein the sgRNA comprises a nucleic acid sequence complementary to a MECP2 nucleic acid sequence comprising a SNP associated with RETT.

[0017] On the other hand, a method for treating Rett syndrome (RETT) in a subject is provided, wherein the method comprises administering to a subject in need thereof: (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid replacement at amino acid position 82 or 166 or a corresponding position thereof relative to a TadA reference sequence, wherein the guide polynucleotide directs the adenosine base editor to produce an A to G nucleobase change within a methyl CpG binding protein 2 (MECP2) gene or a regulatory element thereof comprising a SNP associated with Rett syndrome (RETT) in the subject, thereby treating RETT in the subject; and wherein the SNP associated with RETT causes an R133C or R306C amino acid mutation in a MECP2 polypeptide encoded by the MECP2 gene or a variant thereof. In one embodiment of the method, administration alleviates at least one symptom associated with RETT. In one embodiment of the method, administration results in a reduction in at least one symptom associated with RETT compared to treatment with a base editor without an amino acid substitution in adenosine deaminase. In one embodiment of the method, the A to G nucleobase change causes the SNP associated with RETT to become a wild-type nucleobase. In one embodiment, the A to G nucleobase change changes the SNP associated with Rett syndrome to a non-wild-type nucleobase, which reduces the symptoms of RETT syndrome. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence complementary to a MECP2 gene or a regulatory element thereof comprising a SNP associated with RETT. In one embodiment, an adenosine base editor is complexed with a single guide RNA (sgRNA), the sgRNA comprising a nucleic acid sequence complementary to a MECP2 gene or a regulatory element thereof comprising a SNP associated with RETT. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of: 5'-AGAGCAAAAGGCUUUUCCCU-3', 5'-UAGAGCAAAAGGCUUUUCCC-3', 5'-UAGAGCAAAAGGCUUUUCCCU-3', 5'-UUUAGAGCAAAAGGCUUUUCCCU-3', 5'-UCUUGCACUUCUUGAUGGGG-3', 5'-CUUGCACUUCUUGAUGGGGAG-3', or 5'-GUCUUGCACUUCUUGAUGGGGAG-3'.

[0018] In the above-mentioned editing method embodiments and the above-mentioned aspects of the base editor system and embodiments thereof, the guide polynucleotide comprises a nucleic acid sequence comprising at least 10 consecutive nucleotides complementary to the MECP2 gene or its regulatory element. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence comprising 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 or 40 consecutive nucleotides complementary to the MECP2 gene or its regulatory element.

[0019] In the above embodiments of the editing or treatment methods and the above aspects of the cells and embodiments thereof, the guide polynucleotide comprises a nucleic acid sequence comprising at least 10 consecutive nucleotides that are complementary to a MECP2 polynucleotide. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence comprising 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 or 40 consecutive nucleotides that are complementary to a MECP2 polynucleotide.

[0020] In embodiments of the above-mentioned methods for treating RETT and embodiments thereof, the SNP associated with RETT causes R133C and / or R306C amino acid mutations in a MECP2 polypeptide encoded by a MECP2 gene or a variant thereof. In one embodiment, the SNP associated with RETT causes an R133C amino acid mutation in a MECP2 polypeptide encoded by a MECP2 gene or a variant thereof. In one embodiment, the SNP associated with RETT causes an R306C amino acid mutation in a MECP2 polypeptide encoded by a MECP2 gene or a variant thereof.

[0021] In an embodiment of the above-mentioned base editor system and embodiments thereof, the A to G nucleobase change at the SNP associated with RETT results in an R133C amino acid mutation in a MECP2 polypeptide encoded by a MECP2 gene or a variant thereof. In one embodiment of the base editor system, the A to G nucleobase change at the SNP associated with RETT results in an R306C amino acid mutation in a MECP2 polypeptide encoded by a MECP2 gene or a variant thereof.

[0022] In the above-mentioned method of editing MECP2 polynucleotides or the method of treating RETT syndrome (RETT) and embodiments thereof, the change of the SNP associated with RETT comprises both R133C and R306C. In one embodiment, the change of the SNP associated with RETT is R133C. In one embodiment, the change of the SNP associated with RETT is R306C.

[0023] In an embodiment of the above cells and embodiments thereof, the change in the SNP associated with RETT syndrome (RETT) is R133C. In one embodiment, the change in the SNP associated with RETT syndrome (RETT) is R306C.

[0024] In another aspect, a guide polynucleotide or guide RNA is provided, wherein the guide polynucleotide or guide RNA (gRNA) comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 consecutive nucleotides that are perfectly complementary to a MECP2 gene encoding a MECP2 protein. In one embodiment, the guide polynucleotide or guide RNA comprises a nucleic acid sequence selected from the group consisting of 5'-AGAGCAAAAGGCUUUUCCCU-3', 5'-UAGAGCAAAAGGCUUUUCCC-3', 5'-UAGAGCAAAAGGCUUUUCCCU-3', 5'-UUUAGAGCAAAAGGCUUUUCCCU-3', 5'-UCUUGCACUUCUUGAUGGGG-3', 5'-CUUGCACUUCUUGAUGGGGAG-3' or 5'-GUCUUGCACUUCUUGAUGGGGAG-3'. In one embodiment, the guide polynucleotide or guide RNA further comprises a scaffold sequence, wherein the scaffold sequence is optionally as follows: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT.

[0025] On the other hand, a composition comprising an adenosine deaminase base editor 8 (ABE8) and a guide RNA is provided, wherein in the composition, ABE8 comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein the guide RNA targets the base editor to cause a SNP in a MECP2 polynucleotide associated with RETT syndrome to undergo an A·T to G·C change, and wherein the change is one or both of R133C or R306C. In one embodiment of the composition, the A·T to G·C change at the SNP associated with RETT changes a cysteine ​​in a methyl CpG binding protein 2 (MECP2) polypeptide to an arginine or changes a stop codon to an arginine. In one embodiment, the SNP associated with RETT results in the expression of a MECP2 polypeptide comprising an arginine at amino acid positions 133 and / or 306. In one embodiment, the polynucleotide programmable DNA binding domain is a Cas9 selected from the following: Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), Streptococcus canis Cas9 (ScCas9), or variants thereof. In one embodiment, the polynucleotide programmable DNA binding domain comprises a modified SpCas9 bound to an altered protoplast adjacent motif (PAM). In some embodiments, the polynucleotide programmable DNA binding domain is a nuclease-inactive or nickase variant. In one embodiment, the nickase variant comprises an amino acid substitution D10A or its corresponding amino acid substitution. In one embodiment, the adenosine deaminase domain is capable of deaminating adenosine in deoxyribonucleic acid (DNA). In one embodiment of the composition, the adenosine deaminase domain comprises an alteration at amino acid position 82 and / or 166 of MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD. In one embodiment, the adenosine deaminase domain comprises an alteration selected from the group consisting of a V82S alteration, a T166R alteration, or both a V82S and a T166R alteration. In some embodiments, the adenosine deaminase domain further comprises one or more of the following mutations: Y147T, Y147R, Q154S, Y123H, and Q154R.In some embodiments, the adenosine deaminase domain comprises a combination of alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123 In one embodiment, ABE8 comprises an adenosine deaminase variant monomer, wherein the adenosine deaminase monomer comprises V82S and T166R changes. In one embodiment, ABE8 comprises an adenosine deaminase heterodimer, wherein the heterodimer comprises a wild-type adenosine deaminase domain and an adenosine deaminase variant. In one embodiment, the adenosine deaminase variant monomer further comprises one or more changes selected from the group consisting of: Y147T, Y147R, Q154S, Y123H, V82S, T166R and Q154R. In one embodiment, ABE8 comprises an adenosine deaminase heterodimer comprising a TadA*8 domain and a wild-type TadA domain. In one embodiment, the adenosine deaminase variant monomer further comprises one or more changes selected from the group consisting of: Y147T, Y147R, Q154S, Y123H, V82S, T166R and Q154R. In one embodiment, the ABE8 base editor comprises a heterodimer comprising a wild-type TadA domain and an adenosine deaminase variant comprising a combination of changes 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.In one embodiment, the guide RNA comprises a nucleic acid sequence selected from the group consisting of AGAGCAAAAGGCUUUUCCCU-3', 5'-UAGAGCAAAAGGCUUUUCCC-3', 5'-UAGAGCAAAAGGCUUUUCCCU-3', 5'-UUUAGAGCAAAAGGCUUUUCCCU-3', 5'-UCUUGCACUUCUUGAUGGGG-3', 5'-CUUGCACUUCUUGAUGGGGAG-3', or 5'-GUCUUGCACUUCUUGAUGGGGAG-3'. In one embodiment, the adenosine deaminase is a TadA deaminase. In one embodiment, the TadA deaminase is a TadA*8 variant. In one embodiment, the TadA*8 variant is selected from the group consisting of: TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8. 11. TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, Tad A*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23 and TadA*8.24.In one embodiment, the ABE8 base editor is selected from the group consisting of ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, ABE8.5-m, ABE8.6-m, ABE8.7-m, ABE8.8-m, ABE8.9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.13-m, ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8. 23-m, 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, ABE8.1 2-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 and ABE8.24. In one embodiment of the composition, the guide RNA comprises a CRISPR RNA (crRNA) and a trans-encoding small RNA (tracrRNA), wherein the crRNA comprises a nucleic acid sequence complementary to a MECP2 nucleic acid sequence comprising a SNP associated with RETT. In one embodiment, the ABE8 base editor is complexed with a single guide RNA (sgRNA), and the sgRNA comprises a nucleic acid sequence complementary to a MECP2 nucleic acid sequence comprising a SNP associated with RETT. In some embodiments, the ABE8 base editor comprises or is essentially composed of the following sequence or a fragment thereof having adenosine deaminase activity: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD. In one embodiment, the composition further comprises a lipid, optionally wherein the lipid is a cationic lipid.

[0026] In embodiments of the above aspects of the composition and its embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient or diluent. In one embodiment, the pharmaceutical composition is used to treat RETT syndrome. In one embodiment of the pharmaceutical composition, gRNA and ABE8 base editors are formulated together or separately. In one embodiment of the pharmaceutical composition, a vector suitable for expression in a mammalian cell is further included, wherein the vector comprises a polynucleotide encoding an ABE8 base editor. In one embodiment of the pharmaceutical composition, the vector is a viral vector. In one embodiment of the pharmaceutical composition, the viral vector is a retroviral vector, an adenoviral vector, a lentiviral vector, a herpes virus vector, or an adeno-associated virus vector (AAV). In one embodiment, the pharmaceutical composition further comprises ribonucleic acid particles suitable for expression in mammalian cells.

[0027] In another aspect, a method of treating RETT syndrome is provided, wherein the method comprises administering to a subject in need thereof a pharmaceutical composition as described in any of the above aspects and embodiments. In one embodiment of the method, the subject is a mammal or a human.

[0028] In another aspect, there is provided a use of the pharmaceutical composition described in any of the above aspects and embodiments for treating RETT syndrome in a subject. In one embodiment of the use, the subject is a mammal or a human.

[0029] In one aspect, a composition comprising the cells described in any of the above aspects and embodiments is provided. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier or diluent.

[0030] On the other hand, a pharmaceutical composition is provided, comprising: (i) a nucleic acid encoding an ABE8 base editor; and (ii) a guide polynucleotide or guide RNA according to the above aspects and embodiments. In one embodiment, the pharmaceutical composition further comprises a lipid. In one embodiment, the lipid is a cationic lipid. In one embodiment of the pharmaceutical composition, the nucleic acid encoding the base editor is mRNA.

[0031] In another aspect, an ABE8 base editor is provided, wherein the ABE8 base editor comprises: (i) a modified SpCas9 comprising amino acid substitutions L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R and L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D133 one or more of 32T, D1332V, D1332L, D1332K, D1332R, R1335Q, T1337, T1337L, T1337Q, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337H, T1337Q and T1337M, or their corresponding amino acid substitutions, and (ii) TadA*8 adenosine deaminase.

[0032] In another aspect, an ABE8 base editor is provided, wherein the ABE8 base editor comprises: (i) a modified SpCas9 comprising amino acid substitutions D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q and T1337, and L1111R, G1218R, E1219F, D1332A, D1332S, D1333 one or more of 32T, D1332V, D1332L, D1332K, D1332R, T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q and T1337M, or their corresponding amino acid substitutions, and (ii) TadA*8 adenosine deaminase. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure may be obtained with reference to the following detailed description of exemplary embodiments in which the principles of the present disclosure are utilized, and with reference to the accompanying drawings, in which:

[0034] Figure 1 is a graph depicting the evaluation of motor behavior based on RTT mutations.

[0035] Figure 2 The functions of each domain of MECP2 protein and the location of common RTT mutations are illustrated.

[0036] Figure 3Graph depicting the percentage of accurate correction of the R106W RTT mutation by making ABE8 base editor variants using gRNA 1, gRNA 2, and gRNA 5. Results for each base editor paired with each gRNA are shown, from left to right ABE8.14 (far left) to Neg.Ct (far right). DETAILED DESCRIPTION

[0037] The invention features compositions and methods for using programmable nucleobase editors (e.g., ABE8) to precisely correct pathogenic amino acids associated with RTT.

[0038] The present invention is based at least in part on the following discovery: a base editor characterized by an adenosine deaminase variant (referred to herein as adenosine base editor 8 or "ABE8") accurately corrects single nucleotide polymorphisms (SNPs) in the endogenous Mecp2 gene (e.g., R106W, R133C, T158M, R255*, R270*, R306C). In one embodiment, the SNP in the Mecp2 gene is R133C. In one embodiment, the SNP in the Mecp2 gene is R306C.

[0039] Described herein are compositions and methods that provide base editors and base editing systems to precisely correct one or more mutations in the methyl-CpG-binding protein 2 (Mecp2) gene that are causally associated with the progressive neurodevelopmental disorder Rett syndrome (RTT or RETT) and its symptoms. RTT is an X-linked dominant disorder that primarily affects females, is associated with 96% of affected individuals with a mutation in the Mecp2 gene, and is characterized by apparently normal early development followed by regression with loss of fine motor function and the ability to communicate effectively, rigidity of movement, and apraxia or complete lack of gait (see Figure 1 ). Other clinical features of affected individuals include abnormal deceleration of head growth velocity after birth, periodic breathing, gastrointestinal dysfunction, seizures, and scoliosis.

[0040] The most common mutation causing RTT is a cytosine to thymine (C→T) transition mutation, which results in a C·G to T·A base pair substitution. This substitution can be reversed back to the wild-type, non-pathogenic genomic sequence using an adenosine base editor (ABE) that catalyzes an A·T to G·C substitution. By extension, common RTT-causing mutations are potential targets for reversal to wild-type sequences using ABEs, without the risk of causing overexpression of the Mecp2 gene as might occur using gene therapy. Therefore, A●T to G●C DNA base editing has the potential to precisely correct one or more of the common RTT-causing mutations in the Mecp2 gene.

[0041] The following description and examples illustrate the embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and is therefore variable. It should be understood by those skilled in the art that there are a large number of changes and modifications about the present disclosure, and these changes and modifications are also within the scope of the present disclosure.

[0042] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0043] Although multiple features of the present disclosure may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described in the context of separate embodiments herein for clarity, the present disclosure may also be implemented in a single embodiment.

[0044] definition

[0045] The following definitions supplement those in the art and are for the present application, and are not attributed to any relevant or irrelevant circumstances, e.g., any commonly owned patents or applications. Although methods and materials similar or equivalent to those disclosed herein can be used to test the practice of the present disclosure, preferred methods and materials are described herein. Accordingly, the technology used herein is only used for the purpose of describing specific embodiments, rather than attempting to limit.

[0046] Unless otherwise defined, all scientific and technological terms used in the present disclosure and its embodiments have the meanings generally understood by those skilled in the art. The following references provide general definitions of multiple terms used in the present disclosure to technicians: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); Cambridge Dictionary of Science and Technology (Walker ed., 1988); Rieger et al., The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0047] In this application, unless specifically specified otherwise, the use of the singular includes the plural. It must be noted that as used in this specification, the singular forms "a", "an", and "the" include plural referents unless the context clearly excludes them. In this application, the use of "or" means "and / or" and is understood to be inclusive unless otherwise specified. In addition, the term "including" and other forms (such as active and passive forms) are not limiting.

[0048] As used in this specification and claims, the words "comprising" (and other forms of comprising, such as plural and singular), "having" (and other forms of having, such as plural and singular), "including" (and other forms of including, such as plural and singular), or "containing" (and other forms of containing, such as singular and plural) are inclusive or open-ended, and do not exclude additional, unreferenced elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0049] The term "about" or "approximately" means within an acceptable error range for a particular value, as determined by one skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within one (1) or more than one (1) standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, preferably within 5-fold, more preferably within 2-fold. If a specific value is described in the application and claims, it should be assumed that the term "about" means within an acceptable error range for the specific value unless otherwise specified.

[0050] 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0051] Reference in the specification to "some embodiments," "embodiments," "one embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments of the present disclosure, but not necessarily all embodiments.

[0052] "Adenosine deaminase" means a polypeptide or fragment thereof that can catalyze the hydrolytic deamination reaction of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination reaction of adenosine to inosine or the deoxygenation reaction of adenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination reaction of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) can be from any organism such as bacteria.

[0053] In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain 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 to a naturally occurring deaminase.

[0054] 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. The wild-type TadA (wt) adenosine deaminase has the following sequence (also referred to as the TadA reference sequence):

[0055] MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD.

[0056] In some embodiments, the adenosine deaminase comprises an alteration in the following sequence:

[0057] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0058] (also known as TadA*7.10).

[0059] In some embodiments, TadA*7.10 comprises at least one change. In some embodiments, TadA*7.10 comprises changes at amino acids 82 and / or 166. In specific embodiments, variants of the sequences referenced above comprise one or more of the following changes: Y147T, Y147R, Q154S, Y123H, V82S, T166R and / or Q154R. In other embodiments, variants of the TadA*7.10 sequence comprise a combination of changes selected from the following: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H, I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123 H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166 R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; or I76Y+V82S+Y123H+Y147R+Q154R.

[0060] In other embodiments, the adenosine deaminase variant is provided as comprising a deletion comprising a C-terminal deletion starting at residues 149, 150, 151, 152, 153, 154, 155, 156 or 157 relative to TadA*7.10 (TadA reference sequence), or a corresponding mutation in another TadA. In other embodiments, the adenosine deaminase variant is a TadA (e.g., TadA*8) monomer comprising one or more of the following changes: Y147T, Y147R, Q154S, Y123H, V82S, T166R and / or Q154R relative to TadA*7.10 (TadA reference sequence), or a corresponding mutation in another TadA. In other embodiments, the adenosine deaminase variant is a TadA (e.g., TadA*8) monomer comprising a combination of changes selected from the following: relative to TadA*7.10 (TadA reference sequence), Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H, I76Y+V82S; V82S+Y123H+Y147 T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; or I76Y+V82S+Y123H+Y147R+Q154R, or a corresponding mutation in another TadA.

[0061] In yet other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each adenosine deaminase domain having one or more of the following mutations: Y147T, Y147R, Q154S, Y123H, V82S, T166R and / or Q154R, or corresponding mutations in another TadA, relative to TadA*7.10 (TadA reference sequence). In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each of which has a combination of changes selected from the following: relative to TadA*7.10 (TadA reference sequence), Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H, I76Y+V82S; V82S+ Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; or I76Y+V82S+Y123H+Y147R+Q154R, or a corresponding mutation in another TadA.

[0062] 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 mutations: Y147T, Y147R, Q154S, Y123H, V82S, T166R and / or Q154R, or corresponding mutations in another TadA, relative to TadA*7.10 (TadA reference sequence). 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 a combination of changes 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, or the corresponding mutations in another TadA.

[0063] 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 mutations: Y147T, Y147R, Q154S, Y123H, V82S, T166R and / or Q154R, or corresponding mutations in another TadA, relative to TadA*7.10 (TadA reference sequence). 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 a combination of the following changes relative to TadA*7.10 (TadA reference sequence): 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, or the corresponding mutations in another TadA.

[0064] In one embodiment, the adenosine deaminase is TadA*8, which comprises or consists essentially of the following sequence, or a fragment thereof having adenosine deaminase activity:

[0065] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0066] In some embodiments, TadA*8 is truncated. In some embodiments, the truncated TadA*8 loses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19 or 20 N-terminal amino acid residues relative to the full-length TadA*8. In some embodiments, the truncated TadA*8 loses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19 or 20 C-terminal amino acid residues relative to the full-length TadA*8. In some embodiments, the adenosine deaminase variant is full-length TadA*8.

[0067] In certain embodiments, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from:

[0068] Staphylococcus aureus (S. aureus) TadA:

[0069] MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAH AEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN

[0070] Bacillus subtilis (B. subtilis) TadA:

[0071] MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEML VIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE

[0072] Salmonella typhimurium (S. typhimurium) TadA:

[0073] MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV

[0074] Shewanella putrefaciens (S. putrefaciens) TadA:

[0075] MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE

[0076] Haemophilus influenzae (H. influenzae) F3031 TadA:

[0077] MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTΑΗAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK

[0078] Caulobacter crescentus (C. crescentus) TadA:

[0079] MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI

[0080] Geobacter sulfurreducens (G.sulfurreducens) TadA:

[0081] MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP

[0082] TadA*7.10

[0083] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0084] "Adenosine deaminase base editor 8 (ABE8) polypeptide" or "ABE8" means a base editor as defined herein comprising an adenosine deaminase variant comprising an alteration at amino acid position 82 and / or 166 of the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0085] In some embodiments, ABE8 comprises further alterations, as described herein, relative to the reference sequence.

[0086] "Adenosine deaminase base editor 8 (ABE8) polynucleotide" means a polynucleotide encoding ABE8.

[0087] Herein, "administration" refers to providing one or more compositions described herein to a patient or subject. For example and without limitation, composition administration (e.g., injection) can be performed by intravenous (iv) injection, subcutaneous (sc) injection, intradermal (id) injection, intraperitoneal (ip) injection, or intramuscular (im) injection. One or more such routes can be used. For example, parenteral administration can be performed by a single rapid injection or by progressive perfusion over time. In some embodiments, parenteral administration includes intravascular, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, and intrasternal infusion or injection. Alternatively, or simultaneously, administration can be by oral route.

[0088] "Agent" means any small molecule chemical compound, antibody, nucleic acid molecule, or peptide, or fragment thereof.

[0089] "Alter" means a change (increase or decrease) in the expression level or activity of a gene or polypeptide, as detected by methods known in the standard art, such as those described herein. As used herein, alterations include expression level changes of at least 10%, preferably 25%, more preferably 40%, and most preferably expression level changes of 50% or more. "Relieve" means to reduce, suppress, attenuate, reduce, retard or stabilize the development or progression of a disease.

[0090] "Analog" means a molecule that is not identical but has similar functional or structural features. For example, a polypeptide analog retains the biological activity of the corresponding native polypeptide, but has certain biochemical modifications that enhance the function of the analog relative to the native polypeptide. Such biochemical modifications will increase the protease resistance, membrane permeability, and / or half-life of the analog without changing, for example, ligand binding. Analogs can include non-natural amino acids.

[0091] "Methyl CpG binding protein 2 (Mecp2) protein" means a polypeptide or fragment thereof having at least about 95% amino acid sequence identity to NCBI Accession No. NP_004983 and having demethylase activity. In certain embodiments, the Mecp2 protein comprises one or more changes relative to the following reference sequence. In certain embodiments, the Mecp2 protein associated with RTT comprises one or more mutations selected from R106W, R168*, R133C, T158M, R255*, R270*, and R306C. Exemplary Mecp amino acid sequences are provided below.

[0092]

[0093]

[0094] "Mecp2 polynucleotide" means a nucleic acid molecule encoding a Mecp2 protein or a fragment thereof. The sequence of an exemplary Mecp2 polynucleotide (which can be obtained as NCBI reference sequence: NM_004992) is provided below. In a specific embodiment, the Mecp2 polynucleotide comprises one or more changes relative to the following reference sequence. In a specific embodiment, the Mecp2 polynucleotide associated with RTT comprises one or more mutations selected from 316C>T, 397C>T, 473C>T, 763C>T, 808C>T and 916C>T.

[0095]

[0096]

[0097]

[0098]

[0099]

[0100] "Base editor (BE)" or "nucleobase editor (NBE)" means an agent that binds to a polynucleotide and has nucleobase modification activity. In various embodiments, a base editor comprises a nucleobase modification polypeptide (e.g., a deaminase) and a nucleic acid-programmable nucleotide binding domain, which cooperates with a guide polynucleotide (e.g., a guide RNA). In various embodiments, the agent is a biomolecular complex comprising a protein domain with base editing activity, i.e., a domain capable of modifying a base (e.g., A, T, C, G, or U) in a nucleic acid molecule (e.g., DNA). In some embodiments, the polynucleotide-programmable DNA binding domain is fused or linked to a deaminase domain. In one embodiment, the reagent is a fusion protein comprising a domain with base editing activity. In another embodiment, the protein domain with base editing activity is connected to a guide RNA (e.g., via an RNA binding motif on the guide RNA and fused to an RNA binding domain of a deaminase). In some embodiments, the domain with base editing activity can deaminize bases in nucleic acid molecules. In some embodiments, a base editor can deaminize one or more bases in a DNA molecule. In some embodiments, a base editor can deaminize adenosine (A) in DNA. In some embodiments, a base editor is an adenosine base editor (ABE).

[0101] In some embodiments, a base editor (e.g., ABE8) is produced by cloning an adenosine deaminase variant (e.g., TadA*8) into a scaffold including a circular full mutant Cas9 (e.g., spCAS9 or saCAS9) and a bipartite nuclear localization sequence. Circular full mutant Cas9 is known in the art and is described in, for example, Oakes et al., Cell 176, 254–267, 2019. Exemplary circular full mutants are as follows, where bold sequences indicate sequences derived from Cas9, italic sequences represent linker sequences, and underlined sequences represent bipartite nuclear localization sequences.

[0102] CP5 (with MSP "NGC = Pam variant with regular Cas9 mutations such as NGG", PID = protein interaction domain and "D10A" nickase):

[0103]

[0104] In some embodiments, the base editor is adenosine base editor 8 (ABE8). In some embodiments, BE8 is selected from base editors from Table 9 above. In some embodiments, ABE8 contains an adenosine deaminase variant evolved from TadA. In some embodiments, the adenosine deaminase variant of ABE8 is a TadA*8 variant as described in Table 9 above. In some embodiments, the adenosine deaminase variant is a TadA*7.10 variant (e.g., TadA*8) comprising one or more changes selected from the group consisting of: Y147T, Y147R, Q154S, Y123H, V82S, T166R and / or Q154R. In various embodiments, ABE8 comprises a TadA*7.10 variant (e.g., TadA*8) having a combination of alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H, I76Y+V82S; V82S+Y123H+Y147T ; V82S+Y123H+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 monomer construct. In some embodiments, ABE8 is a heterodimer construct. In some embodiments, the ABE8 base editor comprises the sequence:

[0105] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0106] In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-related (e.g., Cas or Cpf1) enzyme. In some embodiments, the base editor is a catalytically dead Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. In some embodiments, the base editor is fused to an inhibitor of base excision repair (BER). In some embodiments, the inhibitor of base excision repair is a uracil DNA glycosidase inhibitor (UGI). In some embodiments, the inhibitor of base excision repair is an inosine base excision repair inhibitor. The details of the base editor are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 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 excision repair inhibition and bacteriophage Mu Gamprotein yields C:G-to-T: A base editors with higher efficiency and productpurity" 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.

[0107] For example, the adenosine base editor ABE used in the base editing compositions, systems and methods described herein has a nucleic acid sequence (8877 base pairs) as provided below (Addgene, Watertown, MA.; Gaudelli NM, et al., Nature. 2017 Nov 23; 551(7681): 464-471. doi: 10.1038 / nature24644; Koblan LW, et al., Nat Biotechnol. 2018 Oct; 36(9): 843-846. doi: 10.1038 / nbt.4172.). Polynucleotide sequences having at least 95% or higher identity to the ABE nucleic acid sequence are also included.

[0108]

[0109] "Base editing activity" means acting to chemically change a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is adenosine or adenine deaminase activity, for example, converting A·T to G·C.

[0110] In some embodiments, base editing activity is assessed by the efficiency of 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 by the total percentage of sequencing reads with nuclear base conversions caused by base editors, for example, the total percentage of sequencing reads with target A·T base pairs converted to G·C base pairs. In some embodiments, when base editing is performed in a cell population, the base editing efficiency is measured by the total percentage of cells with nuclear base conversions caused by base editors.

[0111] The term "base editor system" refers to a system for editing the nucleobase of a target nucleotide sequence. In various embodiments, the base editor (BE) 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 an adenosine base editor (ABE8). In some embodiments, ABE8 is a monomer construct. In some embodiments, ABE8 is 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. In some embodiments, ABE8 is a heteromeric construct. In some embodiments, ABE8 is 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.

[0112] In some embodiments, the base editor system comprises more than one base editing component. For example, the base editor system can include one or more adenosine deaminases. In some embodiments, a single guide polynucleotide can be used to target different deaminases to a target nucleic acid sequence. In some embodiments, a pair of guide polynucleotides can be used to target different deaminases to a target nucleic acid sequence.

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

[0114] The base editor system may further include a guide polynucleotide component. It should be understood that the components of the base editor system may be associated with each other via covalent bonds, non-covalent interactions, or any combination of their association and interaction. In some embodiments, the deaminase domain can be targeted to the target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the deaminase domain may include an additional heterologous portion or domain (e.g., a polynucleotide binding domain such as RNA or DNA binding protein), which is capable of interacting, associating or forming a complex with a portion or segment (e.g., a polynucleotide motif) of the guide polynucleotide. In some embodiments, an additional heterologous portion or domain (e.g., a polynucleotide binding domain such as RNA or DNA binding protein) may be fused or connected to the deaminase domain. In some embodiments, an additional heterologous portion may be able to bind to, interact with, associate with, or form a complex with a polypeptide. In some embodiments, an additional heterologous portion may be able to bind to, interact with, associate with, or form a complex with a polynucleotide. In some embodiments, an additional heterologous portion may be able to bind to a guide polynucleotide. In some embodiments, the additional heterologous moiety may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous moiety may be capable of binding to a polynucleotide linker. The additional heterologous moiety may be a protein domain. In some embodiments, the additional heterologous moiety may be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0115] In some embodiments, the base editor system may further include an inhibitor of a base excision repair (BER) component. It should be understood that the components of the base editor system may be associated with each other via covalent bonds, non-covalent interactions, or any combination of their association and interaction. The inhibitor of the BER component may include a base excision repair (BER) inhibitor. In some embodiments, the inhibitor of base excision repair (BER) may be a uracil DNA glycosidase inhibitor (UGI). In some embodiments, the inhibitor of base excision repair may be an inosine base excision repair (BER) inhibitor. In some embodiments, the inhibitor of base excision repair (BER) may be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain may be fused or connected to an inhibitor of base excision repair (BER). In some embodiments, the polynucleotide programmable nucleotide binding domain may be fused or connected to a deaminase domain and an inhibitor of base excision repair (BER). In some embodiments, a polynucleotide programmable nucleotide binding domain can target an inhibitor of base excision repair (BER) to a nucleotide sequence by non-covalently interacting or associating with an inhibitor of base excision repair. For example, in some embodiments, an inhibitor of a base excision repair (BER) component can include an additional heterologous portion or domain that is capable of interacting, associating, or forming a complex with an additional heterologous portion or domain that is part of a polynucleotide programmable nucleotide binding domain. In some embodiments, an inhibitor of base excision repair (BER) can be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, an inhibitor of base excision repair (BER) can include an additional heterologous portion or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) that is capable of interacting, associating, or forming a complex with a portion or segment (e.g., a polynucleotide motif) of a guide polynucleotide. In some embodiments, an additional heterologous portion or domain of a guide polynucleotide (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or connected to an inhibitor of base excision repair (BER). In some embodiments, the additional heterologous moiety may be capable of binding, interacting, associating, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous moiety may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous moiety may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous moiety may be capable of binding to a polynucleotide linker. The additional heterologous moiety may be a protein domain.In some embodiments, the additional heterologous portion can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0116] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease or a fragment thereof (e.g., a protein comprising an active, inactivated or partially active DNA cleavage domain of Cas9 and / or a gRMA binding domain of Cas9) comprising a Cas9 protein. Cas9 nucleases are sometimes also referred to as Casn1 nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)-related nucleases. CRISPR is an adaptive immune system that provides protection against mobile genetic factors (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacer sequences, sequences complementary to ancestral mobile factors, and nucleic acids that invade targets. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 proteins are required for correct processing of pre-crRNA. TracrRNA acts as a guide in ribonuclease 3-assisted pre-crRNA processing. Subsequently, Cas9 / crRNA / tracrRNA endonucleases and dissolves to cleave linear or circular dsDNA targets complementary to the spacer sequence. The target chain that is not complementary to crRNA is first cut by endonucleases and then trimmed by 3'-5' exonucleases. In essence, DNA binding and cleavage generally require protein and two RNAs. However, single guide RNA ("sgRNA", or simply "gRNA") can be engineered to merge various aspects of both crRNA and tracrRNA into a single RNA species. See, for example, Jinek M., et al., Science 337: 816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes short motifs (PAM or pre-spacer adjacent motifs) in CRISPR repeats to help distinguish self from non-self.Cas9 nuclease sequence and structure are known to those skilled in the art (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., Proc. 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 "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., et al., Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference). Cas9 direct homologs have been described in a variety of species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus. Other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0117] An exemplary Cas9 is Streptococcus pyogenes Cas9 (spCas9), the amino acid sequence of which is provided below:

[0118] (Single underline: HNH domain; double underline: RuvC domain)

[0119] The Cas9 protein inactivated by nucleases can be interchangeably referred to as "dCas9" protein (Cas9 "dead" for nucleases) or catalytically inactive Cas9. Methods for producing Cas9 proteins (or fragments thereof) with inactive 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 each 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 in these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337: 816-821 (2012); Qi et al., Cell. 28; 152 (5): 1173-83 (2013)). In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, in other words, Cas9 is a nickase, referred to as a "nCas9" protein (for "nickase" Cas9). In some embodiments, a protein comprising a fragment of Cas9 is provided. For example, in some embodiments, the protein comprises one or two Cas9 domains: (1) a gRNA binding domain of Cas9; or (2) a DNA cleavage domain of Cas9. In some embodiments, a protein comprising Cas9 or a fragment thereof is referred to as a "Cas9 variant". Cas9 variants share homology with Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9.In some embodiments, the Cas9 variant can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical to the corresponding fragment of wild-type Cas9, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of the corresponding wild-type Cas9.

[0120] 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.

[0121] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, nucleotide and amino acid sequences are as follows).

[0122]

[0123]

[0124] (Single underline: HNH domain; double underline: RuvC domain)

[0125] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence:

[0126]

[0127]

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

[0129] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2 (nucleotide sequence below); and Uniprot Reference Sequence: Q99ZW2 (amino acid sequence below)).

[0130]

[0131] (Single underline: HNH domain; double underline: RuvC domain)

[0132] In some embodiments, Cas9 refers to Cas9 from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense, China (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021847.1); Ref: NC_021314.1; Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: Yp_002344900.1); or Neisseria meningitidis (NCBI Ref: YP_002342100.1) or refers to Cas9 from any other organism.

[0133] In some embodiments, Cas9 is from Neisseria meningitidis (Nme). In some embodiments, Cas9 is Nme1, Nme2, or Nme3. In some embodiments, the PAM interaction domain for Nme1, Nme2, or Nme3 is Nme1, Nme2, or Nme3, respectively. 4 GAT, N4 CC and N 4 CAAA (see, e.g., Edraki, A., et al., A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing, Molecular Cell (2018)). An exemplary Neisseria meningitidis Cas9 protein, Nme1Cas9 (NCBI reference: WP_002235162.1; type II CRISPR RNA-guided endonuclease Cas9) has the following amino acid sequence:

[0134]

[0135] Another exemplary Neisseria meningitidis Cas9 protein, Nme2Cas9 (NCBI reference: WP_002230835; type II CRISPR RNA-guided endonuclease Cas9) has the following amino acid sequence:

[0136]

[0137] In some embodiments, dCas9 partially or completely corresponds to or comprises a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises D10A and H840A mutations or corresponding mutations in another Cas9. In some embodiments, dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A):

[0138]

[0139] (Single underline: HNH domain; double underline: RuvC domain).

[0140] In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains as a histidine in the amino acid sequences provided above, or at the corresponding position in any of the amino acid sequences provided herein.

[0141] In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which mutations, for example, result in nuclease-inactivated Cas9 (dCas9). For example, such mutations include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs of dCas9 are provided, which are at least about 70% identical, at least about 80% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants of dCas9 are provided, wherein the amino acid sequence of the variant is shorter or longer, shorter or longer 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.

[0142] In some embodiments, the Cas9 fusion protein as provided herein comprises the full-length amino acid sequence of the Cas9 protein, for example, one of the Cas9 sequences provided herein. However, in other embodiments, the fusion protein as provided herein does not comprise the full-length Cas9 sequence, but only comprises one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and other suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0143] It should be understood that other Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including variants and homologs thereof, are within the scope of the present disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is a nuclease-dead Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease-active Cas9.

[0144] Exemplary catalytically inactive Cas9 (dCas9):

[0145]

[0146] Exemplary Catalytic Cas9 Nickase (nCas9):

[0147]

[0148] Exemplary catalytically active Cas9:

[0149]

[0150] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., Nanoarchaea), which constructs domains and kingdoms of unicellular prokaryotic microorganisms. In some embodiments, Cas9 refers to CasX or CasY, which have been described in, for example, Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genome-analyzed metagenomics, a large number of CRISPR-Cas systems have been identified, including Cas9 reported for the first time in the archaeal life domain. This divergent Cas9 protein was discovered in Nanoarchaea, which has rarely been studied, as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, were found, which are one of the most compact systems discovered so far. In some embodiments, Cas9 refers to a variant of CasX or CasX. In some embodiments, Cas9 refers to a variant of CasY or CasY. It will be appreciated that other RNA-guided DNA binding proteins may be used as nucleic acid programmable DNA binding proteins (napDNAbps) and are within the scope of the present disclosure.

[0151] In certain embodiments, napDNAbp useful in the disclosed methods include circular full mutants, which are known in the art and described, for example, by Oakes et al., Cell 176, 254–267, 2019. Exemplary circular full mutants are as follows, where bold sequences indicate sequences derived from Cas9, italic sequences represent linker sequences, and underlined sequences represent bipartite nuclear localization sequences.

[0152] CP5 (with MSP "NGC = Pam variant with regular Cas9 mutations such as NGG", PID = protein interaction domain and "D10A" nickase):

[0153]

[0154] Non-limiting examples of polynucleotide programmable nucleotide-binding domains that can be incorporated into base editors include domains derived from CRISPR proteins, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs).

[0155] 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, and the amino acid sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to a naturally occurring CasX or CasY protein. In some embodiments, napDNAbp is a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any CasX or CasY protein described herein. It should be appreciated that Cas12b / C2c1, CasX, and CasY from other bacterial species may also be used according to the present disclosure.

[0156] Cas12b / C2c1(uniprot.org / uniprot / T0D7A2#2)

[0157] sp|T0D7A2|C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acido-terrestris (strain ATCC49025 / DSM 3922 / CIP 106132 / NCIMB13137 / GD3B) GN=c2c1 PE=1 SV=1

[0158]

[0159] CasX(uniprot.org / uniprot / F0NN87;uniprot.org / uniprot / F0NH53)

[0160] >tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx proteins OS=Sulfolobus islandicus (strain HVE10 / 4) GN=SiH_0402 PE=4 SV=1

[0161] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0162] >tr|F0NH53|F0NH53_SULIR CRISPR-associated protein, Casx OS=Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4SV=1

[0163] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTSNIILPLSGNDKNPWTETLKCYNFPTTTVALSEVFKNFSQVKECEEVSAPSFVKPFEYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0164] δ spp.CasX

[0165] MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQK WYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0166] CasY(ncbi.nlm.nih.gov / protein / APG80656.1)

[0167] >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacteria]

[0168]

[0169] The term "Cas12" or "Cas12 domain" refers to an RNA-guided nuclease or a fragment thereof comprising a Cas12 protein (e.g., a protein comprising an active, inactivated, or partially active DNA cleavage domain of Cas12 and / or a gRMA binding domain of Cas12). Cas12 belongs to a Class 2 V-type CRISPR / Cas system. Cas12 nucleases are sometimes also referred to as CRISPR (clustered regularly interspaced short palindromic repeats)-associated nucleases. The sequence of an exemplary Bacillus hisashii Cas 12b (BhCas12b) Cas 12 domain is provided below:

[0170]

[0171] Amino acid sequences having at least 85% or higher identity to the BhCas12b amino acid sequence can also be used in the methods of the present disclosure.

[0172] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid by another amino acid with common properties. A functional approach to define common properties between individual amino acids is to analyze the standardized 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)). According to such analysis, multiple groups of amino acids can be defined, in which the amino acids within one group are preferentially exchanged with each other, and therefore are most similar to each other in the impact on the overall protein structure (Schulz, GE and Schirmer, RH, as above). Non-limiting examples of conservative mutations include amino acid substitutions of the following amino acids: for example, amino acid substitutions of arginine and vice versa, so that the positive charge can be maintained; glutamic acid substitutions of aspartic acid and vice versa, so that the negative charge can be maintained; serine substitutions of threonine, so that free -OH can be maintained; and glutamine substitutions of asparagine, so that free -NH 2 Can be maintained.

[0173] The terms "coding sequence" or "protein coding sequence" as used interchangeably herein 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 referred to as an open reading frame.

[0174] As used herein, the term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination reaction of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination reaction of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the deoxygenation reaction of adenosine or deoxyinosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination reaction of adenosine in deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) can be from any organism such as bacteria. In some embodiments, the adenosine deaminase is from a bacterium, such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Caulobacter crescentus.

[0175] In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is a TadA*7.10 variant. In some embodiments, the TadA*7.10 variant is TadA*8. In some embodiments, TadA*8 is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8 .12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23 or TadA*8.24. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain 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 to a naturally occurring deaminase. For example, deaminase domains are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is incorporated herein by reference in its entirety.See also Komor, AC, et al., "Programmable base editing of A·T to G·Cin genomic DNA without DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, NM, et al., "Programmable base editing of A·T to G·Cin 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 base editors with higher efficiency and product purity" Science Advances 3:eaao4774 (2017)) and Rees, HA, et al., "Base editing: precisionchemistry 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.

[0176] "Detectable label" means a composition that, when attached to a molecule of interest, renders the latter detectable via spectroscopic, photochemical, biochemical, immunochemical or chemical means. For example, useful labels include radioisotopes, magnetic microbeads, metal microbeads, colloidal ions, fluorescent dyes, high electron density reagents, enzymes (e.g., commonly used in enzyme-linked immunosorbent assays (ELISA)), biotin, digoxigenin or haptens.

[0177] "Disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. An example of a disease includes Rett syndrome.

[0178] As used herein, the term "effective amount" refers to an amount of a bioactive agent sufficient to cause a biological response. In some embodiments, an effective amount means the amount required to alleviate the symptoms of a disease relative to an untreated patient. The effective amount of an active compound for therapeutic treatment of a disease used to practice the methods and uses described herein is variable, depending on the mode of administration and the age, weight and general health of the subject. Basically, the attending physician or veterinarian will determine the appropriate amount and medication regimen. This amount is referred to as an "effective" amount. In one embodiment, an effective amount is a base editor described herein (e.g., a fusion protein comprising a programmable DNA binding protein, a nuclear base editor, and a gRNA) sufficient to introduce changes to a gene of interest (e.g., Mecp2) in a cell (e.g., a cell in vitro or in vivo). In one embodiment, an effective amount is the amount of a base editor required to achieve a therapeutic effect (e.g., weakening or controlling Rett syndrome or its symptoms or symptoms). Such therapeutic effects need not be sufficient to alter Mecp2 in all cells of a subject, tissue or organ, but only in about 1%, 5%, 10%, 25%, 50%, 75% or more of the cells present in a subject, tissue or organ. In some embodiments, the effective amount is sufficient to alleviate one or more symptoms of Rett syndrome.

[0179] In some embodiments, an effective amount of a fusion protein (e.g., a nucleobase editor comprising an nCas9 domain and a deaminase domain (e.g., adenosine deaminase)) provided herein refers to an amount of the fusion protein sufficient to induce editing of a target site specifically bound and edited by a nucleobase editor as described herein. As will be appreciated by those skilled in the art, the effective amount of an agent, such as a fusion protein, a nucleobase, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide can vary based on a variety of factors, for example, based on the desired biological response, for example, based on the specific allele, genome, or target to be edited, based on the targeted cell or assembly, and / or based on the agent used.

[0180] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion preferably contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of a reference nucleic acid molecule or polypeptide. A fragment may 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.

[0181] "Guide RNA" or "gRNA" means a polynucleotide that can be specific for a target sequence and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cas12). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs, or as a single RNA molecule. A gRNA that exists as a single RNA molecule can be referred to as a single guide RNA (sgRNA), but "gRNA" is used to interchangeably refer to a guide RNA that exists as a single molecule or as a complex of two or more molecules. Typically, a gRNA that exists as a single RNA substance comprises two domains: (1) a domain that shares homology with a target nucleic acid (e.g., and guides the Cas9 or Cas12 complex to bind to a target); and (2) a domain that binds to a Cas9 or Cas12 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and includes a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA provided in Jinek et al., Science 337: 816-821 (2012), the entire contents of which are incorporated herein by reference. gRNAs (e.g., those including domain (2)) can be found in U.S. Provisional Patent Application Serial No. 61 / 874,682 entitled "Switchable Cas9 Nucleases and Uses Thereof" and U.S. Provisional Patent Application Serial No. 61 / 874,746 entitled "Delivery System For Functional Nucleases", filed on September 6, 2013, the entire contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the gRNA comprises two or more domains (1) and (2), and can be referred to as "extended gRNA". The extended gRNA will bind to two or more Cas9 or Cas12 proteins and bind to the target nucleic acid in two or more different regions, as described herein. The gRNA comprises a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site and provides sequence specificity for the nuclease:RNA complex.

[0182] "Heterodimer" means a fusion protein comprising two domains, such as a wild-type TadA domain and a variant TadA domain (e.g., TadA*8), or two variant TadA domains (e.g., TadA*7.10 and TadA*8, or two TadA*8 domains).

[0183] "Hybridization" means hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen and reversed Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds.

[0184] By "increase" is meant a positive change of at least 10%, 25%, 50%, 75% or 100%.

[0185] The term "inhibitor of base repair" or "IBR" refers to a protein that can inhibit the activity of a nucleic acid repair enzyme (e.g., a base excision repair enzyme). In some embodiments, IBR is an inhibitor of inosine base excision repair (BER). Exemplary base repair inhibitors include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGG1, hNEIL1, T7 Endol, T4PDG, UDG, hSMUG1, and hAAG. In some embodiments, IBR is an inhibitor of Endo V or hAAG. In some embodiments, IBR is a catalytically inactive EndoV or a catalytically inactive hAAG. In some embodiments, a base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, a base repair inhibitor is a catalytically inactive EndoV or a catalytically inactive hAAG.

[0186] In some embodiments, the base repair inhibitor is a uracil glycosidase inhibitor (UGI). UGI refers to a protein that can inhibit uracil-DNA glycosidase base excision repair enzymes. In some embodiments, the UGI domain comprises a fragment of wild-type UGI or a fragment of wild-type UGI. In some embodiments, the UGI protein provided herein includes a fragment of UGI and a protein homologous to UGI or a fragment of UGI. In some embodiments, the base repair inhibitor is an inhibitor of inosine base excision repair. In some embodiments, the base repair inhibitor is a "catalytically inactive inosine-specific nuclease" or a "dead inosine-specific nuclease". Without being bound by any particular theory, a catalytically inactive inosine glycosidase (e.g., alkyladenine glycosidase (AAG)) can bind to inosine, but cannot create a baseless site or remove the inosine, thereby spatially blocking the effect of DNA damage / repair mechanisms on the newly formed inosine portion. In some embodiments, a catalytically inactive inosine-specific nuclease can be able to bind to inosine in a nucleic acid but does not cleave the nucleic acid. Non-limiting exemplary catalytically inactive inosine-specific nucleases include, for example, catalytically inactive alkyladenosine glycosidase (AAG nuclease) from humans, and, for example, catalytically inactive endonuclease V (EndoV nuclease) from Escherichia coli. In some embodiments, the catalytically inactive AAG nuclease comprises an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0187] "Intein" is a fragment of a protein that can excise itself and join the remaining fragment (extein) by a peptide bond in a process called protein splicing. Intein is also called "protein intron". The process in which intein excises itself and joins the remainder of a protein is referred to herein as "protein splicing" or "intein-mediated protein splicing". In some embodiments, the intein of a precursor protein (a protein containing intein before intein-mediated protein splicing) comes from two genes. Herein, such intein is referred to as split intein (e.g., split intein-N and split intein-C). For example, in cyanobacteria, DnaE (catalytic subunit of DNA polymerase III) is encoded by two independent genes (dnaE-n and dnaE-c). The intein encoded by the dnaE-n gene may be referred to herein as "intein-N". The intein encoded by the dnaE-c gene may be referred to herein as "intein-C".

[0188] Other intein systems may also be used. For example, synthetic inteins based on the dnaE intein, Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C) intein pairs, have been described (e.g., in Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7): 2162-5, which is incorporated herein by reference). Non-limiting examples of intein pairs that may be used according to the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, and Cne Prp8 intein (e.g., as described in U.S. Pat. No. 8,394,604, which is incorporated herein by reference).

[0189] Exemplary nucleotide and amino acid sequences of inteins are provided.

[0190] DnaE intein-N DNA: TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTCTGCCAATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCGATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGGG GAGAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAGGGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTTCCTAAT

[0191] DnaE intein-N protein: CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN

[0192] DnaE intein-C DNA: ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGATATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAGCTTCTAAT

[0193] Intein-C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN

[0194] Cfa-N DNA: TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCCTATTGGAAAGATTGTCGAAGAGAGAATTGAATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGGCACAATCGCGGCGAACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACGAGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAATAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTGCCA

[0195] Cfa-N protein:

[0196] CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP

[0197] Cfa-C DNA: ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAGGAAAGTAAAGATAATATCTCGAAAAAGTCTTGGTACCCAAAATGTCTATGATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTAGCCAGCAAC

[0198] Cfa-C protein: MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN

[0199] Intein-N and intein-C can be fused to the N-terminal portion of split Cas9 and the C-terminal portion of split Cas9, respectively, to join the N-terminal portion of split Cas9 and the C-terminal portion of split Cas9. For example, in some embodiments, intein-N is fused to the C-terminal of the N-terminal portion of split Cas9, that is, to form a structure of N-[N-terminal portion of split Cas9]-[intein-N]--C. In some embodiments, intein-C is fused to the N-terminal of the C-terminal portion of split Cas9, that is, to form a structure of N-[intein-C]--[C-terminal portion of split Cas9]-C. The mechanism of intein-mediated protein splicing for joining the protein (e.g., split Cas9) to which the intein is fused is known in the art, for example, as described in Shah et al., Chem Sci. 2014; 5(1): 446-461, which is incorporated herein by reference. Methods for designing and using inteins are known in the art and are described in, for example, WO2014004336, WO2017132580, US20150344549, and US20180127780, each of which is incorporated herein by reference in its entirety.

[0200] The terms "isolated", "purified" or "biologically pure" refer to substances that are free of varying degrees of normal accompanying components found in their natural state. "Isolated" means the degree of separation from the original source or surrounding material. "Purification" means a degree of separation higher than "isolated". "Purified" or "biologically pure" proteins are free of other materials so that any impurities do not materially affect the biological properties of the protein or cause other negative consequences. In other words, if the nucleic acid or peptide described herein is substantially free of cellular material, viral material or culture medium when produced by recombinant DNA technology, or free of chemical precursors or other chemicals when chemically synthesized, the nucleic acid or peptide is purified. Analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography are typically used to determine purity and homogeneity. The term "purified" can mean that the nucleic acid or protein gives essentially one band in the electrophoresis gel. For proteins that can be modified such as phosphorylation or glycosylation, different modifications can give different isolated proteins, which can be purified independently.

[0201] "Isolated polynucleotide" means a nucleic acid (e.g., DNA) that is free of the genes that flank the gene in the naturally occurring genome of the organism from which the nucleic acid molecule described herein is derived. The term thus includes, for example, recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryotic or eukaryotic organism, or exists as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments prepared by PCR or restriction endonuclease digestion). In addition, the term includes RNA molecules transcribed from DNA molecules, as well as recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0202] "Isolated polypeptide" means a polypeptide described herein that has been separated from its naturally associated components. Typically, a polypeptide is isolated when at least 60% by weight of the polypeptide is free of its naturally associated proteins and naturally occurring organic molecules. Preferably, the preparation contains at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the polypeptide described herein. An isolated polypeptide of the present disclosure can be obtained, for example, by extraction from a natural source, expression of a recombinant nucleic acid encoding the polypeptide, or chemical synthesis of the protein. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0203] As used herein, the term "linker" may refer to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group or a molecule that connects two molecules or parts, for example, two components of a protein complex or a ribonucleic acid complex, or two domains of a fusion protein, for example, a polynucleotide programmable DNA binding domain (e.g., dCas9) and a deaminase domain (e.g., adenosine deaminase). The linker can engage different components of a base editor system or different parts of multiple components. For example, in some embodiments, the linker can engage the guide polynucleotide binding domain of a polynucleotide programmable nucleotide binding domain with the catalytic domain of a deaminase. In some embodiments, the linker can engage a CRISPR polypeptide with a deaminase. In some embodiments, the linker can engage Cas9 with a deaminase. In some embodiments, the linker can engage dCas9 with a deaminase. In some embodiments, the linker can engage nCas9 with a deaminase. In some embodiments, the linker can engage a guide polynucleotide with a deaminase. In some embodiments, the linker can engage the deamination component of the base editor system with the polynucleotide programmable nucleotide binding component. In some embodiments, the linker can bind to the RNA binding portion of the deamination component of the base editor and the polynucleotide programmable nucleotide binding component. In some embodiments, the linker can bind to the RNA binding portion of the deamination component of the base editor and the RNA binding portion of the polynucleotide programmable nucleotide binding component. The linker can be positioned between two groups, molecules or other parts or have two groups, molecules or other parts on the flank, and is linked to each group, molecule or other part via a covalent bond or non-covalent interaction, thereby linking the two. In some embodiments, the linker can be an organic molecule, group, polymer or chemical part. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, the linker can include an aptamer capable of binding to a ligand. In some embodiments, the ligand can be a carbohydrate, a peptide, a protein or a nucleic acid. In some embodiments, the linker can include an aptamer that can be derived from a riboswitch. The riboswitch from which the aptamer is derived 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-deazaguanosine 1 (pre-queosine 1 (PreQ1)) riboswitch. In some embodiments, the linker can comprise an aptamer that is bonded to a polypeptide or protein domain such as a polypeptide ligand.In some embodiments, the polypeptide ligand can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and a Ku protein, a telomerase Sm7 binding motif and a Sm7 protein, or an RNA recognition motif. In some embodiments, the polypeptide ligand can be part of a base editor system component. For example, a nucleobase editing component can include a deaminase domain and an RNA recognition motif.

[0204] 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 a length of about 5-100 amino acids, 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 a length of 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 can also be envisioned.

[0205] In some embodiments, the linker engages the gRNA engagement domain of the RNA programmable nuclease (including the Cas9 nuclease domain) and the catalytic domain of the nucleic acid editing protein (e.g., adenosine deaminase). In some embodiments, the linker engages dCas9 and the nucleic acid editing protein. For example, the linker can be positioned between two groups, molecules or other parts or have two groups, molecules or other parts on the flanks, and is linked to each group, molecule or other part via a covalent bond, thereby linking the two. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer or chemical part. In some embodiments, the linker is 5-200 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90, 95, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 175, 180, 190, or 200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES, which may also be referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker comprises (SGGS) n 、(GGGS) n 、(GGGGS) n 、(G) n 、(EAAAK) n 、(GGS) n ,SGSETPGTSESATPES or (XP) n motif, or a combination of any of these motifs, 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. In some embodiments, the linker comprises a plurality of proline residues and is 5-21, 5-14, 5-9, 5-7 amino acids in length, e.g., PAPAP, PAPAPA, PAPAPAP, PAPAPAPA, P(AP) 4 、P(AP) 7 、P(AP) 10 Such proline-rich linkers are also called "rigid" linkers.

[0206] In some embodiments, multiple domains of a base editor are fused via a linker comprising the amino acid sequence:

[0207] SGGSSGSETPGTSESATPESSGGS,

[0208] SGGSSGGSSGSETPGTSESATPESSGGSSGGS or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS.

[0209] In some embodiments, multiple domains of the base editor are fused via a linker, which comprises the amino acid sequence SGSETPGTSESATPES, which may also be referred to as an XTEN linker. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS.

[0210] In some embodiments, the linker is 92 amino acids in length.In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0211] As used herein, the term "mutation" refers to a residue in a sequence (e.g., a nucleic acid or amino acid sequence) being replaced with another residue, or a deletion or insertion of one or more residues in a sequence. Typically, mutations are described herein by identifying the original residue, then identifying the position of the residue in the sequence and the identity of the newly replaced residue. The method for performing amino acid substitution (mutation) provided herein is well known in the art and is provided in, for example, "Molecular Cloning: Laboratory Manual (Fourth Edition)" (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 disclosed herein can effectively produce "expected mutations" such as point mutations in nucleic acids (e.g., nucleic acids in the subject's genome) without producing a significant number of unexpected mutations such as unexpected point mutations. In some embodiments, the expected mutation is a mutation generated by a specific base editor (e.g., adenosine base editor) bound to a guide polynucleotide (e.g., gRNA), and the specific base editor is specifically involved to produce an expected mutation. Typically, a mutation made or identified in a sequence (e.g., an amino acid sequence as described herein) is numbered relative to a reference (or wild-type) sequence, i.e., a sequence that does not contain the mutation. Those skilled in the art will readily understand how to determine the position of a mutation in an amino acid sequence and a nucleic acid sequence relative to a reference sequence.

[0212] The term "non-conservative mutation" includes amino acid substitutions between different groups, for example, lysine substitutions for tryptophan, or phenylalanine substitutions for serine, etc. In this case, it is preferred that the non-conservative amino acid substitutions do not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, so that the biological activity of the functional variant is increased compared to the wild-type protein.

[0213] The term "nuclear localization sequence", "nuclear localization signal" or "NLS" refers to an amino acid sequence that causes a protein to enter the nucleus of a cell. Nuclear localization sequences are known in the art and are described, for example, in International PCT Application PCT / EP2000 / 011690 filed by Plank et al. on November 23, 2000 and published as WO / 2001 / 038547 on May 31, 2001, which is incorporated herein by reference for its disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS, for example, as described in Koblan et al., Nature Biotech.2018doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence

[0214] KRTADGSEFESPKKKRKV,

[0215] KRPAATKKAGQAKKKK,

[0216] KKTELQTTNAENKTKKL,

[0217] KRGINDRNFWRGENGRKTR,

[0218] RKSGKIAAIVVKRPRK,

[0219] PKKKRKV or

[0220] MDSLLMNRKFLYQFKNVRWAKGRRETYLC.

[0221] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a compound comprising a core base and an acidic portion, for example, a polymer of a nucleoside, a nucleotide or a nucleotide. Typically, a polymeric nucleic acid (for example, a nucleic acid molecule) comprising three or more nucleotides is a linear molecule in which adjacent nucleotides are linked to each other via a phosphodiester link. In some embodiments, "nucleic acid" refers to an individual nucleic acid residue (for example, a nucleotide and / or a nucleoside). 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" can be used interchangeably and refer to a polymer of nucleotides (for example, a string of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA and single-stranded and / or double-stranded DNA. Nucleic acid can occur naturally in the context of, for example, genomes, transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plasmids, cosmids, chromosomes, chromatids or other naturally occurring nucleic acid molecules. On the other hand, nucleic acid molecules can be molecules that occur in non-natural, for example, recombinant DNA or RNA, artificial chromosomes, genomes or fragments thereof through engineering approaches, or synthetic DNA, RNA, DNA / RNA hybrids, or nucleotides or nucleosides that comprise non-natural occurrences. In addition, the terms "nucleic acid", "DNA", "RNA" and / or similar terms include nucleic acid analogs, for example, analogs with the backbone except for phosphodiester. Nucleic acid can be purified from natural sources, produced using a recombinant expression system and optionally purified, chemically synthesized, etc. If appropriate, for example, in the case of chemically synthesized molecules, nucleic acid can include nucleoside analogs such as analogs with chemically modified bases or sugars and backbone modifications. Unless otherwise noted, nucleotide sequences are presented in 5' to 3' directions. In some embodiments, the nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyuridine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (2' modifications, e.g., fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0222] Herein, the terms "nucleobase", "nitrogenous base" or "base" are used interchangeably and refer to biological compounds containing nitrogen that form nucleosides, which in turn become components of nucleotides. The ability of nucleobases to form base pairs and stack one after another directly leads to long-chain helical structures, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Five nucleobases, i.e., adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), are referred to as basic bases or standard bases. Adenine and guanine are derived from purines, while cytosine, uracil and thymine are derived from pyrimidines. DNA and RNA may also contain other (non-basic) bases that are modified. Non-limiting exemplary modified nucleobases may include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C) and 5-hydroxymethylcytosine. Hypoxanthine and xanthine can be produced by the presence of mutagen, and both are produced by deamination reaction (replacing amino group with carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be obtained from the deamination reaction of cytosine. "Nucleoside" consists of a nucleobase and a pentose (or ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. Examples of nucleosides with modified nucleobases include inosine (I), xanthine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C) and pseudouridine (Ψ). "Nucleotide" consists of a nucleobase, a pentose (or ribose or deoxyribose) and at least one phosphate group.

[0223] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" can be used interchangeably with "polynucleotide programmable nucleotide binding domain" and refers to a protein (e.g., DNA or RNA) associated with a nucleic acid, such as a guide nucleic acid (e.g., gRNA) that guides napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. The Cas9 protein can be associated with a guide RNA that guides the Cas9 protein to a specific DNA sequence, and the DNA sequence is complementary to the guide RNA. In some embodiments, napDNAbp is a Cas9 domain, for example, a nuclease-active Cas9, a Cas9 nickase (nCas9), or a Cas9 without nuclease activity (dCas9). Non-limiting examples of nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i.Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2 , Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a type II Cas effector protein, a type V Cas effector protein, a type VI Cas effector protein, CARF, DinG, a homolog thereof, or a modified or engineered version thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, but they may not be specifically listed in the present disclosure. See, for example, Makarova et al. "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR J. 2018 Oct; 1: 325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., "Functionally diverse type V CRISPR-Cas systems" Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, the entire contents of each of which are incorporated herein by reference.

[0224] As used herein, the term "nucleobase editing domain" or "nucleobase editing protein" refers to a protein or enzyme that can catalyze nucleobase modifications in RNA or DNA, such as cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine) and adenine (or adenosine) to hypoxanthine (or inosine), as well as non-templated nucleotide additions and insertions. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., adenine deaminase or adenosine deaminase). In some embodiments, the nucleobase editing domain may be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase editing domain may be a nucleobase editing domain engineered or evolved from a naturally occurring nucleobase editing domain. The nucleobase editing domain may be from any organism, such as bacteria, humans, chimpanzees, gorillas, monkeys, cows, dogs, rats, or mice.

[0225] As used herein, "obtaining" as in "obtaining a dose" includes synthesizing, purchasing or otherwise obtaining the dose.

[0226] As used herein, "patient" or "subject" refers to a mammalian subject or individual who has been diagnosed with, is at risk of, or is susceptible to or develops a disease or disorder. In some embodiments, the term "patient" refers to a mammalian subject who has a higher than average likelihood of developing a disease or disorder. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cows, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), and other mammals that may benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female.

[0227] As used herein, a "patient in need thereof" or a "subject in need thereof" refers to a patient who has been diagnosed with, is at risk of, is previously identified as having, or is susceptible to a disease or disorder, such as, but not limited to, Rett syndrome (RTT).

[0228] The terms "pathogenic mutation", "pathogenic variant", "disease-causing mutation", "disease-causing variant", "deleterious mutation" or "predisposing mutation" refer to a genetic mutation or change that increases an individual's susceptibility or predisposition to a disease or condition. In some embodiments, the pathogenic mutation comprises at least one wild-type amino acid in a protein encoded by a gene being replaced by at least one pathogenic amino acid.

[0229] The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate, zinc stearate or stearic acid) or solvent encapsulation material, which is involved in carrying or transporting the compound from one site in the body (e.g., delivery site) to another site in the body (e.g., an organ, tissue or part of a peptide). A pharmaceutically acceptable carrier is "acceptable" in terms of compatibility with the other ingredients of the formulation and is not harmful to the tissues of the subject (e.g., physiologically compatible, sterile, physiological pH, etc.). Terms such as "excipient", "carrier", "pharmaceutically acceptable carrier", "vehicle", etc. are used interchangeably herein.

[0230] The term "pharmaceutical composition" means a composition formulated for pharmaceutical use.

[0231] As used herein, the terms "protein", "peptide", "polypeptide" and their grammatical equivalents are used interchangeably and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. These terms refer to proteins, peptides or polypeptides of any size, structure or function. Typically, the length of a protein, peptide or polypeptide will be at least three amino acids. A protein, peptide or polypeptide may refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide or polypeptide may be modified, for example, by the addition of chemical entities such as carbohydrate groups, hydroxyls, phosphates, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization or other modifications, or other modifications. A protein, peptide or polypeptide may also be a single molecule or may be a multimolecular complex. A protein, peptide or polypeptide may be only a fragment of a naturally occurring protein or peptide. A protein, peptide or polypeptide may be naturally occurring, recombinant or synthetic, or any combination thereof. As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. A protein can be positioned at the amino terminal (N-terminal) part of the fusion protein or positioned at the carboxyl terminal (C-terminal) protein, thereby forming an amino terminal fusion protein or a carboxyl terminal fusion protein, respectively. The protein can include different domains, for example, a nucleic acid binding domain (for example, a gRNA binding domain of Cas9 that guides the protein to bind to the target site) and a nucleic acid cleavage domain or catalytic domain of a nucleic acid editing protein. In some embodiments, the protein includes a protein portion (for example, an amino acid sequence constituting a nucleic acid binding domain) and an organic compound (for example, a compound that can serve as a nucleic acid cleavage agent). In some embodiments, the protein is compounded or associated with nucleic acids such as RNA or DNA. Any protein provided herein can be produced by any method known in the art. For example, the protein provided herein can be produced via recombinant protein expression and purification, and the method is particularly suitable for fusion proteins comprising peptide connectors. Methods for recombinant protein expression and purification are well known and include those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), the entire contents of which are incorporated herein by reference.

[0232] The polypeptides and proteins disclosed herein (including functional parts and functional variants thereof) may comprise synthetic amino acids to replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2 ... -carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminoallonic acid, aminoallonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine. Polypeptides and proteins can be associated with post-transcriptional modifications of one or more amino acids of the polypeptide construct. Non-limiting examples of post-transcriptional modifications include phosphorylation, acylation (including acetylation and formylation), glycosylation (including N-linked glycosylation and O-linked glycosylation), amidation, hydroxylation, alkylation (including methylation and ethylation), ubiquitination, addition of pyrrolidonecarboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, prenylation, farnesylation, geranylation, glycosylphosphatidylinositolization, lipidation, and iodination.

[0233] As used herein, the term "recombinant" in the context of a protein or nucleic acid refers to a protein or nucleic acid that does not occur in nature but is an artificially engineered product. For example, in some embodiments, a recombinant protein or nucleic acid comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any naturally occurring sequence.

[0234] By "reduced" is meant a negative change of at least 10%, 25%, 50%, 75% or 100%.

[0235] "Reference" means a standard or control condition. In one embodiment, the reference is a wild-type or healthy cell. In other embodiments and without limitation, the reference is an untreated cell that has not been subjected to the test condition, or has been subjected to a placebo or saline, culture medium, buffer, and / or control vector that does not carry the polynucleotide of interest.

[0236] A "reference sequence" is a defined sequence used as a basis for sequence alignment. A reference sequence can be a portion or the entirety of a particular sequence; for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer close to or therebetween. In some embodiments, the reference sequence is the wild-type sequence of a protein of interest. In other embodiments, the reference sequence is a polynucleotide sequence encoding a wild-type protein.

[0237] The terms "RNA programmable nuclease" and "RNA guided nuclease" are used in conjunction with (e.g., bound to or associated with) one or more RNAs that are not cleavage targets. In some embodiments, when complexed with RNA, the RNA programmable nuclease can be referred to as a nuclease: RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs, or as a single RNA molecule. A gRNA that exists as a single RNA molecule can be referred to as a single guide RNA (sgRNA), but "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or as a complex of two or more molecules. Typically, a gRNA that exists as a single RNA species contains two domains: (1) a domain that shares homology with a target nucleic acid (e.g., and guides the Cas9 complex to bind 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 includes a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. gRNAs (e.g., those including domain (2)) can be found in U.S. Provisional Patent Application Serial No. 61 / 874,682 entitled "Switchable Cas9 Nucleases and Uses Thereof" and U.S. Provisional Patent Application Serial No. 61 / 874,746 entitled "Delivery System For Functional Nucleases" filed on September 6, 2013, the entire contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the gRNA comprises two or more domains (1) and (2), and can be referred to as "extended gRNA". For example, the extended gRNA will, for example, bind to two or more Cas9 proteins and bind to target nucleic acids in two or more different regions, as described herein. The gRNA comprises a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site and provides sequence specificity for the nuclease:RNA complex.

[0238] In some embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 from Streptococcus pyogenes (Casn1) (see, e.g., Complete genome sequence of an Ml strain of Streptococcus pyogenes. Ferretti JJ, et al., Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); CRISPR RNA maturation by trans-encoded small RNA and hostfactor RNase III. Deltcheva E., et al., Nature 471:602-607 (2011)).

[0239] Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins can in principle be targeted to any sequence that is specific to the guide RNA. Methods for site-specific cleavage (e.g., to modify the genome) using RNA programmable nucleases such as Cas9 are known in the art (see, e.g., Cong, L. et al., Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, WY et al., Efficient genome editing inzebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference).

[0240] The term "single nucleotide polymorphism (SNP)" is a single nucleotide variation that occurs at a specific position of the genome, wherein each variation exists in a population to a certain appreciable degree (e.g., >1%). For example, at a specific base position in the human genome, a C nucleotide may appear in most individuals, but in a few individuals, the position is occupied by A. This means that there is a SNP at this specific position, and two possible nucleotide variations, i.e., C or A, are called alleles at this position. SNPs form the basis for differences in susceptibility to disease. The severity of the disease and the way our bodies respond to treatment are also manifestations of genetic variation. SNPs can fall into the coding region of a gene, the non-coding region of a gene, or into an intergenic region (the region between genes). In some embodiments, due to the degeneracy of the genetic code, SNPs in the coding sequence do not necessarily change the amino acid sequence of the produced protein. There are two types of SNPs in the coding region: synonymous SNPs and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, while non-synonymous SNPs change the amino acid sequence of the protein. There are two types of non-synonymous SNPs: missense and nonsense. SNPs that are not in protein coding regions may still affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNA. The gene expression affected by this type of SNP is called eSNP (expression SNP) and can be located upstream or downstream of the gene. Single nucleotide variants (SNVs) are variations in single nucleotides, without any frequency restrictions, and can occur in somatic cells. Somatic single nucleotide variations (e.g., related to cancer) can also be referred to as single nucleotide changes.

[0241] "Specifically binds" means that nucleic acid molecules, polypeptides, or complexes thereof (e.g., nucleic acid programmable DNA binding domains and guide nucleic acids), compounds, or molecules recognize and bind to the polypeptides and / or nucleic acid molecules of the present disclosure, but they do not substantially recognize and bind to other molecules in a sample, such as a biological sample.

[0242] Nucleic acid molecules that can be used in the methods described herein include any nucleic acid molecules encoding polypeptides or fragments thereof disclosed herein. Such nucleic acid molecules need not be 100% identical to endogenous nucleic acid sequences, but typically will exhibit basic consistency. Polynucleotides having "basic consistency" with endogenous sequences are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules that can be used in the methods described herein include any nucleic acid molecules encoding polypeptides or fragments thereof disclosed herein. Such nucleic acid molecules need not be 100% identical to endogenous nucleic acid sequences, but typically will exhibit basic consistency. Polynucleotides having "basic consistency" with endogenous sequences are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridization" means pairing of complementary polynucleotide sequences (e.g., genes described herein) or portions thereof under a variety of stringent conditions to form double-stranded molecules. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152: 399; Kimmel, AR (1987) Methods Enzymol. 152: 507).

[0243] For example, stringent salt concentrations are generally less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be 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 and more preferably at least about 50% formamide. Stringent temperature conditions will generally include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Variable additional factors, such as hybridization time, concentration of detergents such as sodium dodecyl sulfate (SDS), and inclusion or non-saturation of carrier DNA, are well known to those skilled in the art. Various levels of stringency are implemented by combining these various conditions as needed. In certain embodiments, hybridization will occur in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30°C. In a more preferred embodiment, hybridization will occur in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C. In a most preferred embodiment, hybridization will occur in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations on these conditions will be apparent to those skilled in the art.

[0244] For most applications, the stringency of the washing step after hybridization will also vary. Washing stringency conditions can be defined by salt concentration and temperature. As above, washing stringency can be increased by reducing salt concentration or increasing temperature. For example, the stringent salt concentration of the washing step is preferably less than about 30mM NaCl and 3mM trisodium citrate, and most preferably less than about 15mM NaCl and 1.5mM trisodium citrate. The stringent temperature conditions for the washing step will generally include a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, the washing step will occur in 30mM NaCl, 3mM trisodium citrate and 0.1% SDS at 25°C. In a more preferred embodiment, the washing step will occur in 15mM NaCl, 1.5mM trisodium citrate and 0.1% SDS at 42°C. In a more preferred embodiment, the washing step will occur in 15mM NaCl, 1.5mM trisodium citrate and 0.1% SDS at 68°C. Additional variations on these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are disclosed in, for example, 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.

[0245] "Split" means to divide into two or more fragments.

[0246] "Split Cas9 protein" or "split Cas9" refers to a Cas9 protein that is provided as an N-terminal fragment and a C-terminal fragment encoded by two independent nucleotide sequences. The polypeptides corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein can be split to form a "reconstructed" Cas9 protein. In a specific embodiment, the Cas9 protein is divided into two fragments within the disordered region of the protein, for example, as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014 or as described in Jiang et al. (2016) Science 351: 867-871. PDB file: 5F9R, each of which is incorporated herein by reference. In some embodiments, the protein is divided into two fragments at any C, T, A or S in the region between about amino acids A292-G364, F445-K483 or E565-T637 of SpCas9, or at the corresponding position of any other Cas9, Cas9 variant (e.g., nCas9, dCas9) or other napDNAbp. In some embodiments, the protein is divided into two fragments at SpCas9 T310, T313, A456, S469 or C574. In some embodiments, the process of dividing a protein into two fragments is called "splicing" the protein.

[0247] In other embodiments, the N-terminal portion of the Cas9 protein comprises amino acids 1-573 or 1-637 of Streptococcus pyogenes Cas9 wild type (SpCas9) (NCBI reference sequence: NC_002737.2, Uniprot reference sequence: Q99ZW2), and the C-terminal portion of the Cas9 protein comprises amino acids 574-1368 or 638-1368 of the SpCas9 wild type, or corresponding positions thereof.

[0248] The C-terminal portion of the split Cas9 can be joined to the N-terminal portion of the split Cas9 to form a complete Cas9 protein. In some embodiments, the C-terminal portion of the Cas9 protein begins where the N-terminal portion of the Cas9 protein ends. Therefore, in some embodiments, the C-terminal portion of the split Cas9 comprises the amino acid (551-651)-1368 portion of spCas9. "(551-651)-1368" means starting at the amino acid between amino acids 551-651 (inclusive) and ending at amino acid 1368.For example, the C-terminal portion of the split-Cas9 can include amino acids 551-1368, 552-1368, 553-1368, 554-1368, 555-1368, 556-1368, 557-1368, 558-1368, 559-1368, 560-1368, 561-1368, 562-1368, 563-1368, 564-1368, 565-1368, 566-1368, 567-1368, 568-1368, 569-1368, 570-1368, 571-1368, 572-1368, 573-1368, 574-1368 74-1368、575-1368、576-1368、577-1368、578-1368、579-1368、580-1368、581-1368、582-1368、583-1368、584-1368、585-1368、586-1368、587-1368、588-1368、589-1368、590-1368、591-1368、592-1368、593-1368、594-1368、595-1368、596-1368、597-1368、598-1368、599-1368、600 -1368, 601-1368, 602-1368, 603-1368, 604-1368, 605-1368, 606-1368, 607-1368, 608-1368, 609-1368, 610-1368, 611-1368, 612-1368, 613-1368, 614-1368, 615-1368, 616-1368, 617-1368, 618-1368, 619-1368, 620-1368, 621-1368, 622-1368, 623-1368, 624-1368, 625-1368, 626-1368 or a portion of any of: 627-1368, 628-1368, 629-1368, 630-1368, 631-1368, 632-1368, 633-1368, 634-1368, 635-1368, 636-1368, 637-1368, 638-1368, 639-1368, 640-1368, 641-1368, 642-1368, 643-1368, 644-1368, 645-1368, 646-1368, 647-1368, 648-1368, 649-1368, 650-1368, or 651-1368. In some embodiments, the C-terminal portion of the split-Cas9 protein comprises amino acids 574-1368 or 638-1368 of spCas9.

[0249] "Subject" means mammals, including but not limited to humans and non-human mammals, such as cattle, horses, dogs, sheep or cats. Subjects include livestock, domesticated animals used to produce labor and provide commodities such as food, including but not limited to cattle, goats, chickens, horses, pigs, rabbits and sheep.

[0250] "Substantially identical" means that a polypeptide or nucleic acid molecule exhibits at least 50% identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, this sequence is at least 60% identical to the sequence used for comparison at the amino acid level or nucleic acid level, more preferably 80% or 85%, and more preferably 90%, 95% or even 99%.

[0251] Sequence identity is usually measured using sequence analysis software (e.g., BLAST, BESTFIT, COBALT, EMBOSS Needle, GAP, or PILEUP / PRETTYBOX programs from Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). The software matches identical or similar sequences by setting the degree of homology of various substitutions, deletions, and / or other modifications. Conservative substitutions typically include intra-group substitutions of the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program can be used, wherein between e -3 With e -100 Likelihood scores between indicate closely related sequences. COBALT, for example, with the following parameters was used:

[0252] a) Alignment parameters: gap penalty -11, -1, and end gap penalty -5, -1,

[0253] b) CDD parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved columns and Recompute on, and

[0254] c) Query clustering parameters: Use query clustering enabled; word length 4; maximum inter-cluster distance 0.8; self-style is Regular.

[0255] For example, the EMBOSS Needle with the following parameters was used:

[0256] a) Matrix: BLOSUM62;

[0257] b) GAP OPEN: 10;

[0258] c) GAP EXTEND: 0.5;

[0259] d) Output format: Yes;

[0260] e) End gap penalty: False;

[0261] f) beginning of terminal gap: 10; and

[0262] g) Terminal gap extension: 0.5.

[0263] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a base editor. In one embodiment, the target site is deaminated by a deaminase or a fusion protein comprising a deaminase (e.g., adenine deaminase).

[0264] As used herein, "treat" (verb, gerund, name) and the like refer to alleviating or relieving the pathology and / or one or more symptoms associated therewith or obtaining the desired pharmacological and / or physiological effect. It should be understood that, although not excluded, treating a disorder or condition does not require the complete elimination of the disorder, condition or symptom associated with the disorder or condition. In some embodiments, the effect is therapeutic, that is, without limitation, partially or completely weakening, reducing, abolishing, weakening, alleviating, reducing the intensity of the disease and / or the adverse symptoms attributable to the disease or curing the disease and / or the adverse symptoms attributable to the disease. In some embodiments, the effect is preventive, that is, the effect of protecting or preventing the onset or recurrence of a disease or condition. In this regard, the method disclosed in the present invention comprises administering a therapeutically effective amount of a composition as described herein.

[0265] "Uracil glycosidase inhibitor" or "UGI" means an agent that inhibits the uracil excision repair system. In one embodiment, the agent is a protein or a fragment thereof that binds to a host uracil-DNA glycosidase and prevents uracil residues from being removed from DNA. In one embodiment, UGI refers to a protein, a fragment thereof, or a domain that is capable of inhibiting a uracil-DNA glycosidase base excision repair enzyme. In some embodiments, the UGI domain comprises a wild-type UGI or a modified version thereof. In some embodiments, the UGI domain comprises a fragment of the exemplary amino acid sequence described in detail below. In some embodiments, the UGI fragment comprises an amino acid sequence comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the exemplary UGI sequence provided below. In some embodiments, UGI comprises an amino acid sequence homologous to an exemplary GI amino acid sequence or a fragment thereof, as described in detail below. In some embodiments, the UGI or a portion thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a wild-type UGI or a UGI sequence or a portion thereof, as described in detail below. An exemplary UGI comprises the following amino acid sequence:

[0266] >splP14739IUNGI_BPPB2 Uracil-DNA glycosidase inhibitor

[0267] MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML.

[0268] The term "vector" refers to the introduction of a nucleic acid sequence into a cell to obtain a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, and episomes. An "expression vector" is a nucleic acid sequence that contains a nucleotide sequence to be expressed in a recipient cell. An expression vector may include additional nucleic acid sequences to promote and / or facilitate expression of the introduced sequence, such as initiators, terminators, promoters, and secretory sequences.

[0269] Herein, the description of a series of chemical groups in any variable definition includes the definition of the variable as any single group or as a combination of the listed groups. The description of an embodiment of a variable or aspect herein includes the embodiment as any single delivery scheme or as a combination with any other embodiment or part thereof.

[0270] Any composition or method provided and described herein may be combined with one or more of any other compositions and methods provided and described herein.

[0271] DNA editing has emerged as a viable means to correct disease states by correcting pathogenic mutations at the genetic level. Until recently, all DNA editing platforms functioned by introducing DNA double-strand breaks (DSBs) into specific genomic loci and relying on endogenous DNA repair pathways to determine product outcomes in a semi-random manner, resulting in a complex population of gene products. Although precise, user-defined repair outcomes can be achieved through the homology-directed repair (HDR) pathway, numerous challenges prevent the use of HDR to achieve efficient repair in therapeutically relevant cell types. In practice, this pathway is inefficient relative to the competing, 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 in post-mitotic cells. As a result, it has proven difficult or impossible to efficiently change genomic sequences in these populations in a user-defined, programmable manner.

[0272] Nucleobase editors

[0273] Disclosed herein is a base editor or a nuclear base editor for editing, modifying or changing a target nucleotide sequence of a polynucleotide. A nuclear base editor or a base editor is described herein, comprising a polynucleotide programmable nucleotide binding domain and a nuclear base editing domain (e.g., adenosine deaminase). When combined with a guide polynucleotide (e.g., gRNA), the polynucleotide programmable nucleotide binding domain can specifically bind to a target polynucleotide sequence (i.e., via complementary base pairing between the base of the combined guide nucleic acid and the base of the target polynucleotide sequence), and thus the base editor is positioned at the target nucleic acid sequence where it is desired to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid.

[0274] Polynucleotide programmable nucleotide binding domain

[0275] It should be understood that the polynucleotide programmable nucleotide binding domain may also include a nucleic acid programmable protein that binds to RNA. For example, the polynucleotide programmable nucleotide binding domain may be associated with a nucleic acid that guides the polynucleotide programmable nucleotide binding domain to RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, but they are not specifically listed in the present disclosure.

[0276] The polynucleotide programmable nucleotide binding domain of the base editor itself may include one or more domains. For example, the polynucleotide programmable nucleotide binding domain may include one or more nuclease domains. In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain may include an endonuclease or an exonuclease. Herein, the term "exonuclease" refers to a protein or polypeptide capable of digesting a nucleic acid (e.g., RNA or DNA) from a free end, and the term "endonuclease" refers to a protein or polypeptide capable of catalyzing (e.g., cleaving) an internal region in a nucleic acid (e.g., DNA or RNA). In some embodiments, an endonuclease may cleave one strand of a double-stranded nucleic acid. In some embodiments, an endonuclease may cleave two 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.

[0277] In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain can cut zero, one or two chains of the target polynucleotide. In some embodiments, the polynucleotide programmable nucleotide binding domain can include a nickase domain. Herein, the term "nickase" refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain, which can only cleave one of the two chains in a duplex nucleic acid molecule (e.g., DNA). In some embodiments, a nickase can be derived from a polynucleotide programmable nucleotide binding domain in a fully catalytically active (e.g., natural) form by introducing one or more mutations into an active polynucleotide programmable nucleotide binding domain. For example, if a polynucleotide programmable nucleotide binding domain includes a nickase domain derived from Cas9, the nickase domain derived from Cas9 can include a D10A mutation and a histidine at position 840. In such embodiments, residue H840 maintains catalytic activity and can therefore cleave a chain of a nucleic acid duplex. In another embodiment, a nickase domain derived from Cas9 can include an H840A mutation, while the amino acid at position 10 remains D. In some embodiments, the nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide programmable nucleotide binding domain by removing all or part of the nuclease domain not required for nickase activity. For example, if the polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the nickase domain derived from Cas9 can comprise all or part of a RuvC domain or a HNH domain.

[0278] The amino acid sequence of an exemplary catalytically active Cas9 is as follows:

[0279]

[0280] A base editor comprising a polynucleotide programmable nucleotide binding domain comprising a nickase domain is capable of producing single-stranded DNA breaks (nicks) at a specific (e.g., determined by the complementary sequence of the bound guide nucleic acid) polynucleotide target sequence. In some embodiments, the chain of the nucleic acid duplex target polynucleotide sequence cleaved by a base editor comprising a nickase domain (e.g., a nickase domain derived from Cas9) is a chain not edited by the base editor (i.e., the chain cleaved by the base editor is opposite to the chain comprising the methyl to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., a nickase domain derived from Cas9) can cleave the chain of a DNA molecule targeted for editing. In such embodiments, the non-targeted chain is not cleaved.

[0281] Also provided herein is a base editor comprising a polynucleotide programmable nucleotide binding domain that is catalytically dead (ie, incapable of cleaving a target polynucleotide sequence). Herein, the terms "catalytically dead" and "nuclease dead" are used interchangeably and refer to a polynucleotide programmable nucleotide binding domain having one or more mutations and / or deletions, resulting in the loss of the ability to cleave nucleic acid chains. In some embodiments, as a result of specific point mutations in one or more nuclease domains, a catalytically dead polynucleotide programmable nucleotide binding domain base editor may lack nuclease activity. For example, in the case where a base editor comprises a Cas9 domain, the Cas9 may comprise both a D10A mutation and an H840A mutation. Such mutations inactivate two nuclease domains, resulting in the 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 (eg, a RuvC1 domain and / or an HNH domain). In further embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (eg, D10A or H840A) and a deletion of all or part of the nuclease domain.

[0282] Mutations of polynucleotide programmable nucleotide binding domains that can produce catalytically dead polynucleotide programmable nucleotide binding domains from previously functional versions of the polynucleotide programmable nucleotide binding domains are also contemplated herein. For example, in the case of catalytically dead Cas9 ("dCas9"), variants with mutations other than D10A and H840A are provided, which result in Cas9 without nuclease activity. For example, such mutations include other amino acid replacements at D10 and H840, or other replacements within the nuclease domain of Cas9 (e.g., replacements in HNH nuclease subdomains and / or RuvC1 subdomains). Based on the disclosure and knowledge in the art, other suitable dCas9 domains without nuclease activity are apparent to those skilled in the art and are within the scope of the disclosure. Such other exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9):833-838, the entire contents of which are incorporated herein by reference).

[0283] Non-limiting examples of polynucleotide programmable nucleotide binding domains that can be incorporated into base editors include domains derived from CRISPR proteins, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some embodiments, the base editor comprises a polynucleotide programmable nucleotide binding domain having a natural or modified protein or part thereof, which can bind to a nucleic acid sequence via a bound guide nucleic acid during nucleic acid modification mediated by CRISPR (i.e., clustered regularly interspaced short palindromic repeats). Herein, such proteins are referred to as "CRISPR proteins". Accordingly, disclosed herein are base editors comprising polynucleotide programmable nucleotide binding domains having all or part of a CRISPR protein (i.e., a base editor comprising all or part of a CRISPR protein as a domain, the domain also being referred to as a "domain derived from a CRISPR protein" of a base editor). The domain derived from a CRISPR protein incorporated into a base editor can be modified compared to a wild-type or natural version of a CRISPR protein. For example, as described below, a domain derived from a CRISPR protein can comprise one or more mutations, insertions, deletions, rearrangements and / or recombinations relative to a wild-type or naturally occurring version of a CRISPR protein.

[0284] CRISPR is an adaptive immune system that provides protection against mobile genetic factors (viruses, transposable elements, and conjugative plasmids). The CRISPR cluster contains a spacer sequence, a sequence complementary to the ancestral mobile factor, and a nucleic acid that invades the target. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In the type II CRISPR system, the correct processing of pre-crRNA requires trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. TracrRNA acts as a guide in the ribonuclease 3-assisted pre-crRNA processing. Subsequently, Cas9 / crRNA / tracrRNA endonucleases dissolve and cleave linear or circular dsDNA targets complementary to the spacer sequence. The target chain that is not complementary to crRNA is first cut by endonucleases dissolving, and then trimmed by 3'-5' exonucleases dissolving. In essence, DNA binding and cleavage generally require protein and two RNAs. However, a single guide RNA ("sgRNA", or simply "gRNA") can be engineered to incorporate aspects of both crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif (PAM or pre-spacer adjacent motif) in the CRISPR repeat sequence to help distinguish self from non-self.

[0285] In some embodiments, the methods described herein can utilize engineered Cas proteins. Guide RNA (gRNA) is a short synthetic RNA consisting of a scaffold sequence necessary for Cas binding and a user-defined spacer sequence of about 20 nucleotides that defines the genomic target to be modified. Thus, a technician can change the genomic target of the Cas protein, with specificity determined in part by how specific the gRNA targeting sequence is for the genomic target versus the rest of the genome.

[0286] In some embodiments, the gRNA scaffold sequence is as follows: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAUAAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU.

[0287] In some embodiments, the domain derived from the CRISPR protein incorporated into the base editor is an endonuclease (e.g., a deoxyribonuclease or a ribonuclease), which can bind to the target polynucleotide when acting in concert with the bound guide nucleic acid. In some embodiments, the domain derived from the CRISPR protein incorporated into the base editor is a nickase, which can bind to the target polynucleotide when acting in concert with the bound guide nucleic acid. In some embodiments, the domain derived from the CRISPR protein incorporated into the base editor is a catalytically dead domain, which can bind to the target polynucleotide when acting in concert with the bound guide nucleic acid. In some embodiments, the target polynucleotide bound by the CRISPR protein derived from the base editor is DNA. In some embodiments, the target polynucleotide bound by the CRISPR protein derived from the base editor is RNA.

[0288] Cas proteins that can be used herein include class 1 and class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, C Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h and Cas12i, CARF, DinG, homologs thereof, or modified versions thereof. Unmodified CRISPR enzymes may have DNA cleavage activity, such as Cas9, which has two functional endonuclease domains: RuvC and HNH. The CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within the target sequence and / or within the complement of the target sequence. For example, the CRISPR enzyme can direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence.

[0289] A vector can be used that encodes a CRISPR enzyme that is mutated with respect to a 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 can refer to a polypeptide having at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from Streptococcus pyogenes). Cas9 can refer to a polypeptide having at most or at most about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and / or sequence homology with a wild-type exemplary Cas9 polypeptide (e.g., from Streptococcus pyogenes). Cas9 can refer to a wild-type or modified form of a Cas9 protein, which can include amino acid changes such as deletions, insertions, substitutions, variations, mutations, fusions, chimeras, or any combination thereof.

[0290] In some embodiments, the domain derived from a CRISPR protein of a base editor may include all or part of Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense, China (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021847.1); Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningitidis (NCBI Ref: YP_002342100.1); Streptococcus pyogenes or Staphylococcus aureus.

[0291] Cas9 domains of nucleobase editors

[0292] Cas9 nuclease sequence and structure are known to those skilled in the art (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., Proc. 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 "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., et al., Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference). Cas9 direct homologs have been described in a variety of species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus. Other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0293] In some aspects, nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting exemplary Cas9 domains are provided herein. The Cas9 domain can be a nuclease activity Cas9 domain, a Cas9 domain without nuclease activity (dCas9) or a Cas9 nickase (nCas9). In some embodiments, the Cas9 domain is a nuclease activity domain. For example, the Cas9 domain can be a Cas9 domain that cuts two chains of a duplex nucleic acid (e.g., two chains of a duplex DNA molecule). In some embodiments, the Cas9 domain includes any one of the amino acid sequences described in detail herein. In some embodiments, the Cas9 domain includes an amino acid sequence, and the amino acid sequence is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 99.5% identical to any one of the amino acid sequences described in detail herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of the amino acid sequences detailed herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any one of the amino acid sequences detailed herein.

[0294] In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, the protein comprises one or two Cas9 domains: (1) a gRNA binding domain of Cas9; or (2) a DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or a fragment thereof are referred to as "Cas9 variants". Cas9 variants share homology with Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70% identical to wild-type Cas9, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the Cas9 variant can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9.

[0295] In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical to the corresponding fragment of wild-type Cas9, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% of the amino acid length of the corresponding wild-type Cas9. In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250 or at least 1300 amino acids in length.

[0296] In some embodiments, the Cas9 fusion protein as provided herein comprises the full-length amino acid sequence of the Cas9 protein, for example, one of the Cas9 sequences provided herein. However, in other embodiments, the fusion protein as provided herein does not comprise the full-length Cas9 sequence, but only comprises one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and other suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0297] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1). Exemplary nucleotide and amino acid sequences are as follows:

[0298]

[0299] (Single underline: HNH domain; double underline: RuvC domain)

[0300] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence:

[0301]

[0302] (Single underline: HNH domain; double underline: RuvC domain).

[0303] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2 (nucleotide sequence below); and Uniprot Reference Sequence: Q99ZW2 (amino acid sequence below)):

[0304]

[0305] (Single underline: HNH domain; double underline: RuvC domain).

[0306] In some embodiments, Cas9 refers to Cas9 from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense, China (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021847.1); Ref: NC_021314.1; Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: Yp_002344900.1); or Neisseria meningitidis (NCBI Ref: YP_002342100.1) or refers to Cas9 from any other organism.

[0307] It should be understood that other Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including variants and homologs thereof, are within the scope of the present disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is a nuclease-active Cas9. In some embodiments, the Cas9 protein is a nuclease-dead Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9).

[0308] In some embodiments, the Cas9 domain is a Cas9 domain without nuclease activity (dCas9). For example, the dCas9 domain can be bound to a duplex nucleic acid molecule (e.g., via a gRNA molecule) without cleaving any chain of the duplex nucleic acid molecule. The Cas9 protein inactivated by nuclease can be interchangeably referred to as "dCas9" protein (Cas9 "dead" for nuclease) or Cas9 without catalytic activity. Methods for producing Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains are known (see, e.g., Jinek et al., Science.337:816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell.28; 152 (5): 1173-83, the entire contents of each 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 in these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337: 816-821 (2012); Qi et al., Cell. 28; 152 (5): 1173-83 (2013)).

[0309] In some embodiments, dCas9 partially or completely corresponds to or comprises a Cas9 amino acid sequence with one or more mutations, and the mutation inactivates the Cas9 nuclease activity. In some embodiments, the dCas9 domain without nuclease activity comprises a D10X mutation and a H840X mutation of the amino acid sequence described in detail herein, or a corresponding mutation of any amino acid sequence provided herein, wherein X is any amino acid change. In some embodiments, the dCas9 domain without nuclease activity comprises a D10A mutation and a H840A mutation of the amino acid sequence described in detail herein, or a corresponding mutation of any amino acid sequence provided herein. In some embodiments, the Cas9 domain without nuclease activity comprises an amino acid sequence described in detail in the cloning vector pPlatTET-gRNA2 (accession number BAV54124). In some embodiments, dCas9 comprises the amino acid sequence (D10A and H840A) of dCas9:

[0310]

[0311] (Single underline: HNH domain; double underline: RuvC domain).

[0312] In some embodiments, the amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows:

[0313]

[0314] (See, e.g., Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.” Cell. 2013; 152(5): 1173-83, the entire contents of which are incorporated herein by reference).

[0315] In some embodiments, the amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows:

[0316]

[0317] In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains as the histidine in the amino acid sequence provided above, or is located at the corresponding position in any amino acid sequence provided herein. In other embodiments, a dCas9 variant having a mutation other than D10A and H840A is provided, the mutation, for example, resulting in a nuclease-inactivated Cas9 (dCas9). For example, such mutations include other amino acid replacements at D10 and H840, or other replacements in the nuclease domain of Cas9 (e.g., replacements in HNH nuclease subdomains and / or RuvC1 subdomains). In some embodiments, a variant or homologue of dCas9 is provided, the variant or homologue being at least about 70% identical, at least about 80% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants of dCas9 are provided, wherein the amino acid sequence of the variant is shorter or longer, shorter or longer 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.

[0318] Based on the present disclosure and the knowledge in the art, other suitable nuclease-free dCas9 domains will be apparent to those skilled in the art and are within the scope of the present disclosure. Such other exemplary suitable nuclease-free Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A and D10A / D839A / H840A / N863A mutant domains (see, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference).

[0319] In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, in other words, Cas9 is a nickase, referred to as a "nCas9" protein (for "nickase" Cas9). In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase can be a Cas9 protein that can cleave only one strand of a duplex nucleic acid molecule (e.g., a duplex DNA molecule). In some embodiments, the Cas9 nickase cleaves the target strand of a duplex nucleic acid molecule, meaning that the Cas9 nickase cleaves a strand that is base-paired (complementary) with a gRNA (e.g., sgRNA) bound to Cas9. In some embodiments, the Cas9 nickase comprises a D10A mutation and has a histidine at position 840. In some embodiments, the Cas9 nickase cleaves a non-target, non-base-edited strand of a duplex nucleic acid molecule, meaning that the Cas9 nickase cleaves a strand that is not base-paired (complementary) with a gRNA (e.g., sgRNA) bound to Cas9. In some embodiments, the Cas9 nickase comprises an H840A mutation and has an aspartic acid residue at position 10, or a corresponding mutation. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 nickases provided herein. Based on the present disclosure and the knowledge in the art, other suitable Cas9 nickases will be apparent to those skilled in the art and are within the scope of the present disclosure.

[0320] The amino acid sequence of an exemplary catalytic Cas9 nickase (nCas9) is as follows:

[0321]

[0322] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., Nanoarchaea), which constructs domains and kingdoms of unicellular prokaryotic microorganisms. In some embodiments, the nucleic acid programmable DNA binding protein can be a CasX or CasY protein, which has been described in, for example, Burstein et al., "New CRISPR-Cas systems from uncultivatedmicrobes." Cell Res. 2017Feb 21. doi: 10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genome-resolved metagenomics, a large number of CRISPR-Cas systems have been identified, including Cas9, which was first reported in the archaeal life domain. This divergent Cas9 protein was discovered in Nanoarchaea, which has been little studied, as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, were found, which are among the most compact systems discovered so far. In some embodiments, in the base editor system described herein, Cas9 is replaced with a variant of CasX or CasX. In some embodiments, in the base editor system described herein, Cas9 is replaced with CasY or a variant of CasY. It should be understood that other RNA-guided DNA binding proteins can be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0323] 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, and the amino acid sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to a naturally occurring CasX or CasY protein. In some embodiments, napDNAbp is a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any CasX or CasY protein described herein. It should be appreciated that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.

[0324] An exemplary CasX ((uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53)tr|F0NN87|F0NN87_SULIHCRISPR-associated Casx protein OS=Sulfolobus icelandica (strain HVE10 / 4) GN=SiH_0402PE=4SV=1) amino acid sequence is as follows:

[0325] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0326] An exemplary CasX (>tr|FONH53|FONH53_SULIR CRISPR-associated protein, Casx OS=Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4SV=1) amino acid sequence is as follows:

[0327] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAK VSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0328] Deltaproteobacterial CasX

[0329] MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQ KWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0330] An exemplary CasY ((ncbi.nlm.nih.gov / protein / APG80656.1)>APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacteria]) amino acid sequence is as follows:

[0331]

[0332] The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Cas9 undergoes a conformational change when targeting those positions of the nuclease domain to cleave the opposite strand of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (approximately 3-4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired by one of two conventional repair pathways: (1) the efficient but error-prone non-homologous end joining (NHEJ) pathway; or (2) the inefficient but high-fidelity homology-directed repair (HDR) pathway.

[0333] The "efficiency" of non-homologous end joining (NHEJ) and / or homology-guided repair (HDR) can be calculated by any conventional method. For example, efficiency can be expressed as a percentage of successful HDR. For example, an inspector nuclease assay can be used to generate a cleavage result, and the ratio of product to substrate can be used to calculate the percentage. For example, an inspector nuclease can be used that directly cleaves DNA containing newly incorporated restriction sequences as a result of successful HDR. More cleaved substrates indicate a higher percentage of HDR (higher HDR efficiency). As an illustrative example, the score (percentage) of HDR can be calculated using the following equation: [(cleavage product) / (substrate plus cleavage product)] (e.g., (b+c) / (a+b+c), where "a" is the band intensity of the DNA substrate, and "b" and "c" are cleavage products).

[0334] In some embodiments, efficiency can be expressed as a percentage of successful NHEJ. For example, a T7 endonuclease I assay can be used to generate cleavage results, and the ratio of product to substrate can be used to calculate the percentage NHEJ. T7 endonuclease I cleaves mismatched heteroduplex DNA from hybridization of wild-type and mutant DNA chains (NHEJ produces small random insertions or deletions (indels) at the site of the original paragraph). More cleavage indicates a higher percentage of NHEJ (higher NHEJ efficiency). As an illustrative example, the score (percentage) of NHEJ can be calculated using the following equation: (1-(1-(b+c) / (a+b+c)) 1 / 2 )×100, where “a” is the band intensity of the DNA substrate, and “b” and “c” are the cleavage products (Ranet. al., Cell. 2013 Sep. 12; 154(6): 1380-9; and Ran et al., Nat Protoc. 2013 Nov.; 8(11): 2281–2308).

[0335] The NHEJ repair pathway is the most active repair mechanism, and it often causes small nucleotide insertions or deletions (indels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical significance because cell populations or guide polynucleotides expressing Cas9 and gRNA can lead to diverse mutations. In some embodiments, NHEJ gives small indels in the target DNA, which lead to amino acid deletions, insertions, or frameshift mutations that result in the extraction of stop codons within the open reading frame (ORF) of the targeted gene. The ideal end result is a disabling mutation within the targeted gene.

[0336] Although NHEJ-mediated DSB repair often disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to generate specific nucleotide changes ranging from single nucleotide changes to large insertions such as the addition of fluorophores or tags.

[0337] In order to utilize HDR for gene editing, a DNA repair template containing the desired sequence can be delivered to the cell type of interest using gRNA and Cas9 or Cas9 nickase. The repair template may contain the desired editing and additional homologous sequences (referred to as left homology arms and right homology arms) located immediately upstream or downstream of the target. The length of each homology arm may depend on the introduced variable size, and the larger the insertion, the longer the homology arm. The repair template may be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. Even in cells expressing Cas9, gRNA, and exogenous information service templates, the efficiency of HDR is generally low (<10% of modified alleles). The efficiency of HDR can be improved by synchronizing the cells because HDR occurs in the S and G2 phases of the cell cycle. The chemical or genetic inhibition of genes involved in NHEJ may also increase the frequency of HDR.

[0338] In some embodiments, Cas9 is a modified Cas9. Throughout the genome where there is partial homology, a given gRNA targeting sequence may have additional sites. These sites are called off-target, and these sites need to be considered when designing gRNA. In addition to optimizing gRNA design, CRISPR specificity can also be increased by modifying Cas9. Cas9 produces double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase, the D10A mutant of SpCas9, retains a nuclease domain and produces DNA nicks instead of DSBs. The nickase system can also be combined with HDR-mediated gene editing for specific gene editing.

[0339] In some embodiments, Cas9 is a variant Cas9 protein. The variant Cas9 polypeptide has an amino acid sequence that differs by one amino acid (e.g., with a deletion, insertion, substitution, fusion) when compared to the amino acid sequence of the wild-type Cas9 protein. In some examples, the variant Cas9 polypeptide has an amino acid change (e.g., a deletion, insertion, or substitution) that reduces the nuclease activity of the Cas9 polypeptide. For example, in some examples, the variant Cas9 polypeptide has a nuclease activity of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the corresponding wild-type Cas9 protein. In some embodiments, the variant Cas9 protein has no substantial nuclease activity. When the tested Cas9 protein is a variant Cas9 protein without substantial nuclease activity, it may be referred to as "dCas9".

[0340] 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 (e.g., wild-type Cas9 protein).

[0341] In some embodiments, the variant Cas9 protein can cleave the complementary strand of the guide target sequence, but the ability to cleave the non-complementary strand of the double-stranded guide target sequence is reduced. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, the variant Cas9 protein has D10A (aspartic acid at amino acid position 10 becomes alanine), and therefore can cleave the complementary strand of the double-stranded guide target sequence but the ability to cleave the non-complementary strand of the double-stranded guide target sequence is reduced (therefore, when the variant Cas9 protein cleaves the double-stranded target nucleic acid, it causes a single-strand break (SSB) rather than a double-strand break (DSB)) (see, for example, Jinek et al., Science. 2012 Aug. 17; 337 (6096): 816-21).

[0342] In some embodiments, the variant Cas9 protein can cleave the non-complementary strand of the double-stranded guide target sequence, but the ability to cleave the complementary strand of the guide target sequence is reduced. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif). As a non-limiting example, in some embodiments, the variant Cas9 protein has an H840A (histidine at amino acid position 840 becomes alanine) mutation, and therefore can cleave the non-complementary strand of the guide target sequence but the ability to cleave the complementary strand of the guide target sequence is reduced (therefore, when the variant Cas9 protein cleaves the target sequence, it causes SSB rather than DSB). Such Cas9 proteins have a reduced ability to cleave guide target sequences (e.g., single-stranded guide target sequences), but retain the ability to bind to guide target sequences (e.g., single-stranded guide target sequences).

[0343] In some embodiments, the variant Cas9 protein has a reduced ability to cleave both the complementary and non-complementary strands of a double-stranded target DNA. As a non-limiting example, in some embodiments, the variant Cas9 protein has two mutations, D10A and H840A, so that the polypeptide has a reduced ability to cleave the complementary and non-complementary strands of a double-stranded target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0344] As another non-limiting example, in some embodiments, the variant Cas9 protein carries W476A and W1126A mutations, so that the ability of the polypeptide to cleave target DNA is reduced. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0345] As another non-limiting example, in some embodiments, the variant Cas9 protein carries P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, so that the ability of the polypeptide to cleave target DNA is reduced. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0346] As another non-limiting example, in some embodiments, the variant Cas9 protein has H840A, W476A and W1126A mutations, so that the ability of the polypeptide to cleave target DNA is reduced. The ability of such Cas9 protein to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind target DNA (e.g., single-stranded target DNA) is retained. As another non-limiting example, in some embodiments, the variant Cas9 protein has H840A, D10A, W476A and W1126A mutations, so that the ability of the polypeptide to cleave target DNA is reduced. The ability of such Cas9 protein to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind target DNA (e.g., single-stranded target DNA) is retained. In some embodiments, the variant Cas9 has a repaired His residue (A840H) at position 840 in the Cas9 HNH domain.

[0347] As another non-limiting example, in some embodiments, the variant Cas9 protein has H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, so that the ability of the polypeptide to cleave the target DNA is reduced. The ability of such Cas9 protein to cleave the target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to the target DNA (e.g., single-stranded target DNA) is retained. As another non-limiting example, in some embodiments, the variant Cas9 protein has D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, so that the ability of the polypeptide to cleave the target DNA is reduced. The ability of such Cas9 protein to cleave the target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to the target DNA (e.g., single-stranded target DNA) is retained. In some embodiments, when the variant Cas9 protein carries W476A and W1126A mutations or when the variant Cas9 protein carries P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, the variant Cas9 protein does not effectively bind to the PAM sequence. Therefore, in some such embodiments, when such variant Cas9 proteins are used in the binding method, the method does not require a PAM sequence. In other words, in some embodiments, when such variant Cas9 proteins are used in the grafting method, the method can include a guide RNA, but the method can be performed in the absence of a PAM sequence (thus, the specificity of binding is provided by the targeting segment of the guide RNA). Other residues can be mutated to achieve the above effects (i.e., partially inactivating one or another strand of the nuclease). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986 and / or A987 may be altered (ie, substituted). Furthermore, mutations other than alanine substitutions are suitable.

[0348] In some embodiments, a variant Cas9 protein with reduced catalytic activity (e.g., when the Cas9 protein has D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986 and / or A987 mutations such as D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A and / or D986A), the variant Cas9 protein can still bind to the target DNA in a site-specific manner (because it is still guided to the target DNA sequence by the guide RNA) as long as it retains the ability to interact with the guide RNA.

[0349] In some embodiments, the variant Cas protein can be spCas9, spCas9-VRQR, spCas9-VRER, xCas9(sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.

[0350] In some embodiments, a modified SpCas9 comprising amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and having specificity for an altered PAM 5'-NGC-3' is used.

[0351] As an alternative to Streptococcus pyogenes Cas9, an RNA-guided endonuclease from the Cpf1 family can be included, which exhibits cleavage activity in mammalian cells. CRISPR (CRISPR / Cpf1) from Prevotella and Francisella 1 is a DNA editing technology homolog of the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of the Class II CRISPR / Cas system. This acquired immunity mechanism is found in Prevotella and Francisella bacteria. The Cpf1 gene is associated with the CRISPR locus, encoding an endonuclease that uses guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some limitations of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, the DNA cleavage result mediated by Cpf1 is a double-strand break with short 3' nucleotides. The staggered cleavage pattern of Cpf1 can open the possibility of targeted gene transfer, similar to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Similar to the above-mentioned Cas9 variants and orthologs, Cpf1 can also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack NGGPAM sites favored by SpCas9. The Cpf1 locus contains a mixed α / β domain, RuvC-I, RuvC-II, and a zinc finger-like domain followed by a helical region. The Cpf1 protein has a RuvC-like nuclease domain similar to the RuvC domain of Cas9.

[0352] Furthermore, unlike Cas9, Cpf1 does not have an HNH endonuclease domain, and the N-terminus of Cpf1 does not have the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionally unique and is classified as a Class 2 Type V CRISPR system. The Cas1, Cas2, and Cas4 proteins encoded by the Cpf1 locus are more similar to Type I and Type III systems than to Type II systems. Functional Cpf1 does not require a reverse activating CRISPR RNA (tracrRNA), and therefore, only CRISPR (crRNA) is required. This is beneficial for genome editing because Cpf1 is not only smaller than Cas9, but it also has a smaller sgRNA molecule (about half the number of nucleotides of Cas9). Unlike the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex cleaves the target DNA or RNA by identifying the protospacer adjacent motif 5'-YTN-3' or 5'-TTN-3'. After identifying the PAM, Cpf1 introduces sticky-end-like DNA double-strand breaks with 4 or 5 nucleotide overhangs.

[0353] In some embodiments, Cas9 is a Cas9 variant having specificity for a changed PAM sequence. In some embodiments, additional Cas9 variants and PAM sequences are described in Miller, SM, et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs, Nat. Biotechnol. (2020), the entire contents of which are incorporated herein by reference. In some embodiments, Cas9 variants do not have specific PAM requirements. In some embodiments, Cas9 variants, for example, SpCas9 variants have specificity for NRNH PAM, wherein R is A or G, and H is A, C or T. In some embodiments, SpCas9 variants have specificity for PAM sequences AAA, TAA, CAA, GAA, TAT, GAT or CAC. In some embodiments, the SpCas9 variant comprises an amino acid replacement at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1218, 1219, 1221, 1249, 1256, 1264, 1290, 1318, 1317, 1320, 1321, 1323, 1332, 1333, 1335, 1337, or 1339, as numbered in SEQ ID NO: 1, or a corresponding position thereof. In some embodiments, the SpCas9 variant comprises an amino acid replacement at position 1114, 1135, 1218, 1219, 1221, 1249, 1320, 1321, 1323, 1332, 1333, 1335, or 1337, or a corresponding position thereof, as numbered in SEQ ID NO: 1. In some embodiments, the SpCas9 variant comprises an amino acid replacement at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1219, 1221, 1256, 1264, 1290, 1318, 1317, 1320, 1323, 1333, or a corresponding position thereof, as numbered in SEQ ID NO: 1. In some embodiments, the SpCas9 variant comprises an amino acid replacement at position 1114, 1131, 1135, 1150, 1156, 1180, 1191, 1218, 1219, 1221, 1227, 1249, 1253, 1286, 1293, 1320, 1321, 1332, 1335, 1339, or a corresponding position thereof as numbered in SEQ ID NO: 1.In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1127, 1135, 1180, 1207, 1219, 1234, 1286, 1301, 1332, 1335, 1337, 1338, 1349, or a corresponding position thereof as numbered in SEQ ID NO: 1. Exemplary amino acid substitutions and PAM specificities of SpCas9 variants are shown in Tables 1A to 1D.

[0354] Table 1A

[0355]

[0356] Table 1B

[0357]

[0358] Table 1C

[0359]

[0360] Table 1D

[0361]

[0362] In some embodiments, Cas9 is Neisseria meningitidis Cas9 (NmeCas9) or a variant thereof. In some embodiments, NmeCas9 has specificity for NNNNGAYW PAM, wherein Y is C or T, and W is A or T. In some embodiments, NmeCas9 has specificity for NNNNGYTT PAM, wherein Y is C or T. In some embodiments, the NmeCas9 domain has specificity for NNNNGTCT PAM. In some embodiments, NmeCas9 is Nme1Cas9. In some embodiments, NmeCas9 has specificity for NNNNGATT PAM, NNNNCCTA PAM, NNNNCCTC PAM, NNNNCCTT PAM, NNNNCCTG PAM, NNNNCCGT PAM, NNNCCGGPAM, NNNNCCCA PAM, NNNNCCCT PAM, NNNNCCCC PAM, NNNNCCAT PAM, NNNNCCAG PAM, NNNNCCAT PAM or NNNGATT PAM. In some embodiments, Nme1Cas9 has specificity for NNNGATT PAM, NNNNCTA PAM, NNNCCTC PAM, NNNNCCTTPAM or NNNCCTG PAM. In some embodiments, NmeCas9 has specificity for CAA PAM, CAAA PAM or CCA PAM. In some embodiments, NmeCas9 is Nme2 Cas9. In some embodiments, NmeCas9 has specificity for NNNNCC (N4CC) PAM, wherein N is any one of A, G, C or T. In some embodiments, NmeCas9 has specificity for NNNNCCGT PAM, NNNNCCGGPAM, NNNNCCCA PAM, NNNNCCCT PAM, NNNNCCCCPAM, NNNNCCAT PAM, NNNNCCAG PAM, NNNNCCAT PAM or NNNGATT PAM. In some embodiments, NmeCas9 is Nme3Cas9. In some embodiments, NmeCas9 has specificity for NNNNCAAA PAM, NNNNCCPAM or NNNNCNNN PAM. Additional NmeCas9 features and PAM sequences as described in Edraki et al. Mol. Cell. (2019) 73(4): 714-726 are incorporated herein by reference in their entirety.

[0363] An exemplary Nme1Cas9 amino acid sequence is provided below:

[0364] Type II CRISPR RNA-guided endonuclease Cas9 [Neisseria meningitidis] WP_002235162.1

[0365]

[0366] An exemplary Nme2Cas9 amino acid sequence is provided below:

[0367] Type II CRISPR RNA-guided endonuclease Cas9 [Neisseria meningitidis] WP_002230835.1

[0368]

[0369]

[0370] Cas12 domains of nucleobase editors

[0371] Typically, microbial CRISPR-Cas systems are divided into Class 1 systems 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, but belong to 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, e.g., 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 a RuvC (or RuvC-like) nuclease domain. Although the production of mature CRISPR RNA (crRNA) is generally independent of tracrRNA, for example, Cas12b / C2c1 requires tracrRNA to produce crRNA. Cas12b / C2c1 relies on both crRNA and tracrRNA for DNA cleavage.

[0372] The nucleic acid programmable DNA binding protein contemplated by the present disclosure includes Cas proteins (Cas12 proteins) classified as 2 types of V. Non-limiting examples of 2 types of V-type Cas proteins include Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h and Cas12i, their homologues 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 the present disclosure comprises an amino acid sequence interrupted by an internally fused protein domain such as a deaminase domain.

[0373] In some embodiments, the Cas12 domain is a Cas12 domain or a Cas12 nickase without nuclease activity. In some embodiments, the Cas12 domain is a nuclease activity domain. For example, the Cas12 domain can be a Cas12 domain that cuts a strand of a duplex nucleic acid (e.g., a duplex DNA molecule). In some embodiments, the Cas12 domain includes any one of the amino acid sequences described in detail herein. In some embodiments, the Cas12 domain includes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 99.5% identical to any one of the amino acid sequences described in detail herein. In some embodiments, the Cas12 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of the amino acid sequences detailed herein. In some embodiments, the Cas12 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any one of the amino acid sequences detailed herein.

[0374] In some embodiments, a protein comprising a fragment of Cas12 is provided. For example, in some embodiments, the protein comprises one or two Cas12 domains: (1) a gRNA binding domain of Cas12; or (2) a DNA cleavage domain of Cas12. In some embodiments, a protein comprising Cas12 or a fragment thereof is referred to as a "Cas12 variant". Cas12 variants share homology with Cas12 or a fragment thereof. For example, a Cas12 variant is at least about 70% identical to wild-type Cas12, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the Cas12 variant can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas12. In some embodiments, the Cas12 variant comprises a fragment of Cas12 (e.g., a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical to the corresponding fragment of wild-type Cas12, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% of the amino acid length of the corresponding 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.

[0375] In some embodiments, Cas12 partially or completely corresponds to or comprises a Cas12 amino acid sequence having one or more mutations, and the mutations change the Cas12 nuclease activity. For example, such mutations include amino acid replacements within the RuvC nuclease domain of Cas2. In some embodiments, variants or homologs of Cas12 are provided, which are at least about 70% identical to wild-type Cas12, at least about 80% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants of Cas12 are provided, wherein the amino acid sequence of the variant is shorter or longer, shorter or longer 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.

[0376] In some embodiments, the Cas12 fusion protein as provided herein comprises the full-length amino acid sequence of the Cas12 protein, for example, one of the Cas12 sequences provided herein. However, in other embodiments, the fusion protein as provided herein does not comprise the full-length Cas12 sequence, but only comprises one or more fragments thereof. Exemplary amino acid sequences of suitable Cas12 domains are provided herein, and other suitable sequences of Cas12 domains and fragments will be apparent to those skilled in the art.

[0377] Typically, Class 2 V-type Cas proteins have a single functional RuvC endonuclease domain (see, e.g., Chen et al., “CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity,” Science 360:436-439 (2018)). In some cases, the Cas12 protein is a variant Cas12 protein. (See, Strecker et al., Nature Communications, 2019, 10 (1): Art. No.: 212). In one embodiment, the variant Cas12 polypeptide has an amino acid sequence that differs by 1, 2, 3, 4, 5 or more amino acids (e.g., having a deletion, insertion, substitution, fusion) when compared to the amino acid sequence of the wild-type Cas12 protein. In some examples, the variant Cas12 polypeptide has an amino acid change (e.g., a deletion, insertion or substitution) that reduces the activity of the Cas12 polypeptide. For example, in some instances, the variant Cas12 polypeptide is a Cas2b polypeptide having less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 protein. In some cases, the variant Cas12b protein has no substantial nickase activity.

[0378] In some cases, the variant Cas12b protein has reduced nickase activity. For example, the variant Cas12 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% nickase activity of the wild-type Cas12b protein.

[0379] In some embodiments, the Cas12 protein includes an RNA-guided endonuclease from the Cas12a / Cpf1 family, which exhibits activity in mammalian cells. CRISPR (CRISPR / Cpf1) from Prevotella and Francisella 1 is a DNA editing technology homolog of the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of the Class II CRISPR / Cas system. This acquired immunity mechanism is found in Prevotella and Francisella bacteria. The Cpf1 gene is associated with the CRISPR locus, encoding an endonuclease that uses guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some limitations of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, the DNA cleavage result mediated by Cpf1 is a double-stranded break with short 3' nucleotides. The staggered cleavage pattern of Cpf1 can open up the possibility of targeted gene transfer, similar to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Similar to the above-mentioned Cas9 variants and orthologs, Cpf1 can also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack NGGPAM sites favored by SpCas9. The Cpf1 locus contains a mixed α / β domain, RuvC-I, RuvC-II, and zinc finger-like domains followed by a helical region. The Cpf1 protein has a RuvC-like nuclease domain similar to the RuvC domain of Cas9. In addition, unlike Cas9, Cpf1 does not have a HNH endonuclease domain, and the N-terminus of Cpf1 does not have the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionally unique and is classified as a Class 2 type V CRISPR system. The Cas1, Cas2, and Cas4 proteins encoded by the Cpf1 locus are more similar to type I and type III systems than type II systems. Functional Cpf1 does not require reverse activation of CRISPR RNA (tracrRNA), therefore, only CRISPR (crRNA) is required. This is beneficial for genome editing because Cpf1 is not only smaller than Cas9, but it also has a smaller sgRNA molecule (about half the number of nucleotides of Cas9). Unlike the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex cleaves the target DNA or RNA by identifying the protospacer adjacent motif 5'-YTN-3' or 5'-TTTN-3'. After identifying the PAM, Cpf1 introduces a sticky end-like DNA double-strand break with a 4 or 5 nucleotide overhang.

[0380] In some aspects of the present disclosure, a vector can be used that encodes a CRISPR enzyme that is mutated with respect to a 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. Cas12 can refer to a polypeptide having at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas12 polypeptide (e.g., Cas12 from Bacillus subtilis). Cas12 can refer to a polypeptide having at most or at most about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and / or sequence homology with a wild-type exemplary Cas12 polypeptide (e.g., from Bacillus spp. (BhCas12b), from Bacillus V3-13 (BvCas12b), and from Alicyclobacillus acidiphilus (AaCas12b). Cas12 can refer to a wild-type or modified form of a Cas12 protein, which can include amino acid changes such as deletions, insertions, substitutions, variations, mutations, fusions, chimeras, or any combination thereof.

[0381] Nucleic acid programmable DNA binding protein Some aspects of the present disclosure provide fusion proteins comprising a domain acting as a nucleic acid programmable DAN binding protein, which can be used to guide proteins such as base editors to specific nucleic acid (e.g., DNA or RNA) sequences. In certain embodiments, the fusion protein comprises a nucleic acid programmable DNA binding protein domain or a deaminase domain. Non-limiting examples of nucleic acid programmable DNA binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2 , Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a type II Cas effector protein, a type V Cas effector protein, a type VI Cas effector protein, CARF, DinG, a homolog thereof, or a modified or engineered version thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, but they may not be specifically listed in the present disclosure.See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct; 1: 325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, the entire contents of each of which are incorporated herein by reference.

[0382] An example of a nucleic acid programmable DNA binding protein with a different PAM specificity than Cas9 is the clustered regularly interspaced short palindromic repeats from Prevotella and Francisella 1 (Cpf1). Similar to Cas9, Cpf1 is also a Class 2 CRISPR effector. It has been shown that Cpf1 mediates robust DNA interference, with characteristics that are distinct from Cas9. Cpf1 is a single RNA-guided endonuclease-deficient tracrRNA, and it utilizes a T-rich protospacer adjacent motif (TTN, TTTN, or YTN). In addition, Cpf1 cleaves DNA via staggered DNA double-strand breaks. Of the 16 Cpf1 family proteins, two enzymes from Acidaminococcus and Lachnospiraceae were shown to have efficient genome editing activity in human cells. Cpf1 protein is known in the art and has been previously described, for example, in Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962, the entire contents of which are incorporated herein by reference.

[0383] Can be used in the compositions and methods of the present invention is a Cpf1 (dCpf1) variant without nuclease activity, these variants can be used as guide nucleotide sequence programmable DNA binding protein domain.Cpf1 protein has a RuvC-like nuclease domain similar to the RuvC domain of Cas9 but does not have a HNH nuclease domain, and the N-terminus of Cpf1 does not have the α-helical recognition leaf of Cas9.Zetsche et al., Cell, 163, 759-771, 2015 (which is incorporated herein by reference) show that the main responsibility of the RuvC-like domain of Cpf1 is to cleave two DNA chains, and the inactivation of the RuvC-like domain inactivates the Cpf1 nuclease activity.For example, the mutation corresponding to D917A, E1006A or D1255A in Francisella novicida Cpf1 inactivates the Cpf1 nuclease activity. In some embodiments, dCpf1 of the present disclosure comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It should be understood that any mutation that inactivates the RuvC domain of Cpf1, e.g., a substitution mutation, deletion, or insertion, can be used according to the present disclosure.

[0384] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In some embodiments, the Cpf1 protein is a Cpf1 without nuclease activity (dCpf1). In some embodiments, Cpf1, nCpf1 or dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to the Cpf1 sequence disclosed herein. In some embodiments, dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a Cpf1 sequence disclosed herein and comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It will be appreciated that Cpf1 from other bacterial species may also be used in accordance with the present disclosure.

[0385] Wild-type Francisella novo Cpf1 (D917, E1006 and D1255 are bold and underlined)

[0386]

[0387] Francisella novo Cpf1 D917A (A917, E1006 and D1255 are bold and underlined)

[0388]

[0389] Francisella novo Cpf1 E1006A (D917, A1006 and D1255 are bold and underlined)

[0390]

[0391] Francisella novo Cpf1 D1255A (D917, E1006 and A1255 are bold and underlined)

[0392]

[0393] Francisella novo Cpf1 D917A / E1006A (A917, A1006 and D1255 are bold and underlined)

[0394]

[0395] Francisella novo Cpf1 D917A / D1255A (A917, E1006 and A1255 are bold and underlined)

[0396]

[0397] Francisella novo Cpf1 E1006A / D1255A (D917, A1006 and A1255 are bold and underlined)

[0398]

[0399] Francisella novo Cpf1 D917A / E1006A / D1255A (A917, A1006 and A1255 are bold and underlined)

[0400]

[0401] In some embodiments, one of the Cas9 domains present in the fusion protein can be replaced with a guide nucleotide sequence programmable DNA binding protein domain that does not require a PAM sequence.

[0402] In some embodiments, nucleic acid programmable DNA binding protein (napDNAbp) is a single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, but are not limited to, Cas9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 systems 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 distinct Class 2 CRISPR-Cas systems (Cas12b / C2c1 and Cas12c / C2c3) have been described by Shmakov et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems", Mol. Cell, 2015 Nov. 5; 60 (3): 385-397, the entire contents of which are incorporated herein by reference. The effectors of two systems, Cas12b / C2c1 and Cas12c / C2c3, contain a Ruv-like nuclease domain associated with Cpf1. The third system contains an effector with two domains called HEPN RNase. Unlike the production of CRISPR RNA by Cas12b / C2c1, the production of mature CRISPR RNA is independent of tracrRNA. Cas12b / C2c1 relies on both CRISPR RNA and tracrRNA for DNA cleavage.

[0403] The crystal structure of A. acidophilic thermotolerant Alicyclobaccillus (Alicyclobaccillus acidoterrastris) Cas12b / C2c1 (AacC2c1) has been reported as 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 been reported as a ternary complex of A. acidophilic thermotolerant Alicyclobaccillus (Alicyclobaccillus acidoterrastris) C2c1 bound to a target DNA. See, e.g., Yang et al., "PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease", Cell, 2016 Dec. 15; 167(7): 1814-1828, the entire contents of which are incorporated herein by reference. A catalytically active conformation of AacC2c1 with target and non-target DNA strands has been captured and independently localized in a single RuvC catalytic zone, and Cas12b / C2c1-mediated cleavage results in staggered heptanucleotide breaks of the target DNA. Structural comparisons between the Cas12b / C2c1 ternary complex and previously identified Cas9 and Cpf1 partners demonstrate the diversity of mechanisms used by the CRISPR-Cas9 system.

[0404] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can be Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, napDNAbp is Cas12b / C2c1 protein. In some embodiments, napDNAbp is Cas12c / C2c3 protein. In some embodiments, napDNAbp comprises an amino acid sequence, and the amino acid sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to the naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, napDNAbp is a naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the napDNAbp sequences provided herein. It should be appreciated that Cas12b / C2c1 or Cas12c / C2c3 from other bacterial species can also be used according to the present disclosure.

[0405] Cas12b / C2c1 ((uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2|C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acido-terrestris (strain ATCC49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) GN=c2c1 PE=1SV=1) amino acid sequence is as follows:

[0406]

[0407] BhCas12b (Bacillus hisashii) NCBI reference sequence: WP_095142515

[0408]

[0409] In some embodiments, Cas12b is BvCas12b V4, which is a variant of BhCas12b and comprises the following changes relative to BhCas12B: S893R, K846R, and E837G. BhCas12b (V4) is expressed as follows: 5'mRNA Cap --- 5'UTR --- bhCas12b --- STOP sequence --- 3'UTR --- 120polyA tail

[0410] 5'UTR:

[0411] GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC

[0412] 3'UTR (TriLink standard UTR)

[0413] GCTGGAGCCTCGGTGGCCATGCTTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGA

[0414] Nucleic acid sequence of bhCas12b(V4)

[0415]

[0416] In some embodiments, Cas12b is BvCas12B. In some embodiments, Cas12b comprises amino acid substitutions S893R, K846R, and E837G, as numbered in the BvCas12b exemplary sequence provided below.

[0417] BvCas12b (Bacillus sp. V3-13) NCBI reference sequence: WP_101661451.1

[0418]

[0419] In some embodiments, Cas12b is BTCas12b. BTCas12b (Bacillus thermoamylovorans) NCBI reference sequence: WP_041902512

[0420]

[0421] In some embodiments, napDNAbp refers to Cas12c. In some embodiments, Cas12c protein is a variant of Cas12c1 or Cas12c1. In some embodiments, Cas12 protein is a variant of Cas12c2 or Cas12c2. In some embodiments, Cas12 protein is a Cas12c protein (i.e., OspCas12c) or a variant of OspCas12c from Oleiphilus sp. HI0009. These Cas12c molecules have been described in Yan et al., "Functionally DiverseType V CRISPR-Cas Systems," Science, 2019 Jan. 4; 363: 88-91, the entire contents of which are incorporated herein by reference. In some embodiments, napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to naturally occurring Cas12c1, Cas12c2, or OspCas12c proteins. In some embodiments, napDNAbp is a naturally occurring Cas12c1, Cas12c2, or OspCas12c protein. In some embodiments, napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any Cas12c1, Cas12c2, or OspCas12c protein described herein. It will be appreciated that Cas12c1, Cas12c2 or OspCas12c from other bacterial species may also be used according to the present disclosure.

[0422] Cas12c1

[0423]

[0424] Cas12c2

[0425]

[0426] OspCas 12c

[0427]

[0428] In some embodiments, napDNAbp refers to Cas12g, Cas12h or Cas12i, which have been described in, for example, Yan et al., "Functionally Diverse Type V CRISPR-Cas Systems," Science, 2019 Jan. 4; 363: 88-91, and the entire contents of each are incorporated herein by reference. By assembling more than 10TB of sequence data, a new classification of V-type Cas proteins was identified, which showed weak similarities to previously characterized V-type proteins (including Cas12g, Cas12h and Cas12i). In some embodiments, Cas12 protein is a variant of Cas12g or Cas12g. In some embodiments, Cas12 protein is a variant of Cas12h or Cas12h. In some embodiments, Cas12 protein is a variant of Cas12i or Cas12i. It should be understood that other RNA-guided DNA binding proteins can be used as napDNAbp and are within the scope of the present disclosure. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring Cas12g, Cas12h, or Cas12i protein. In some embodiments, the napDNAbp is a naturally occurring Cas12g, Cas12h, or Cas12i protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any Cas12g, Cas12h, or Cas12i protein described herein. It should be understood that Cas12g, Cas12h or Cas12i from other bacterial species can also be used according to the present disclosure. In some embodiments, the Cas12i protein is Cas12i1 or Cas12i2.

[0429] Cas12g1

[0430] MAQASSTPAVSPRPRPRYREERTLVRKLLPRPGQSKQEFRENVKKLRKAFLQFNADVSGVCQWAIQFRPRYGKPAEPTETFWKFFLEPETSLPPNDSRSPEFRRLQAFEAAAGINGAAALDDPAFTNELRDSILAVASRPKTKEAQRLFSRLKDYQPAHRMILAKVAAEWIESRYRRAHQNWERNYEEWKKEKQEWEQNHPELTPEIREAFNQIFQQLEVKEKRVRICPAARLLQNKDNCQYAGKNKHSVLCNQFNEFKKNHLQGKAIKFFYKDAEKYLRCGLQSLKPNVQGPFREDWNKYLRYMNLKEETLRGKNGGRLPHCKNLGQECEFNPHTALCKQYQQQLSSRPDLVQHDELYRKWRREYWREPRKPVFRYPSVKRHSIAKIFGENYFQADFKNSVVGLRLDSMPAGQYLEFAFAPWPRNYRPQPGETEISSVHLHFVGTRPRIGFRFRVPHKRSRFDCTQEELDELRSRTFPRKAQDQKFLEAARKRLLETFPGNAEQELRLLAVDLGTDSARAAFFIGKTFQQAFPLKIVKIEKLYEQWPNQKQAGDRRDASSKQPRPGLSRDHVGRHLQKMRAQASEIAQKRQELTGTPAPETTTDQAAKKATLQPFDLRGLTVHTARMIRDWARLNARQIIQLAEENQVDLIVLESLRGFRPPGYENLDQEKKRRVAFFAHGRIRRKVTEKAVERGMRVVTVPYLASSKVCAECRKKQKDNKQWEKNKKRGLFKCEGCGSQAQVDENAARVLGRVFWGEIELPTAIP

[0431] Cas12h1

[0432] MKVHEIPRSQLLKIKQYEGSFVEWYRDLQEDRKKKFASLLFRWAAFGYAAREDDGATYSPSQALLERRLLLGDAEDVAIKFLDVLFKGGAPSSSCYSLFYEDFALRDKAKYSGAKREFIEGLATMPLDKIIERIRQDEQLSKIPAEEWLILGAEYSPEEIWEQVAPRIVNVDRSLGKQLRERLGIKCRRPHDAGYCKILMEVVARQLRSHNETYHEY LNQTHEMKTKVANNLTNEFDLVCEFAEVLEEKNYGLGWYVLWQGVKQALKEQKKPTKIQIAVDQLRQPKFAGLLTAKWRALKGAYDTWKLKKRLEKRKAFPYMPNWDNDYQIPVGLTGGLGVFTLEVKRTEVVVDLKEHGKLFCSHSHYFGDLTAEKHPSRYHLKFRHKLKLRKRDSRVEPTIGPWIEAALREITIQKKPNWGLFYLGLPYALSHGIDNF QIAKRFFSAAKPDKEVINGLPSEMVVGAADLNLSNIVAPVKARIGKGLEGPLHALDYGYGELIDGPKILTPDGPRCGELISLKRDIVEIKSAIKEFKACQREGLTMSEETTTWLSEVESPDSPRCMIQSRIADTSRRLNSFKYQMNKEGYQDLAEALRLLDAMDSYNSLLESYQRMHLSPGEQSPKEAKFDTKRASFRDLRRRRVAHTIVEYFDDCDIVFFEDLDGPSDSDSRNNALVKLLSPRTLLLYIRQALEKRGIGMVEVAKDGTSQNNPISGHVGWRNKQNKSEIYFYEDKELLVMDADEVGAMNILCRGLNHSVCPYSFVTKAPEKKNDEKKEGDYGKRVKRFLKDRYGSSNVRFLVASMGFVTVTTKRPKDALVGKRLYYHGGELVTHDLHNRMKDEIKYLVEKEVLARRVSLSDSTIKSYKSFAHV

[0433] Cas12i 1

[0434]

[0435] Cas12i2

[0436]

[0437] Representative nucleic acid and protein sequences of base editors are as follows:

[0438] BhCas12b GGSGGS-ABE8-Xten20 at P153

[0439]

[0440]

[0441]

[0442]

[0443] BhCas12b GGSGGS-ABE8-Xten20 at K255

[0444]

[0445]

[0446]

[0447]

[0448] BhCas12b GGSGGS-ABE8-Xten20 at D306

[0449]

[0450]

[0451]

[0452]

[0453] BhCas12b GGSGGS-ABE8-Xten20 at D980

[0454]

[0455]

[0456]

[0457]

[0458] BhCas12b GGSGGS-ABE8-Xten20 at K1019

[0459]

[0460]

[0461]

[0462]

[0463] For the above sequences, the Kozak sequence is bolded and underlined; The N-terminal nuclear localization signal (NLS) is marked; lowercase letters represent the GGGSGGS linker; the sequence encoding ABE8 is marked, and the unmodified sequence encodes BhCas12b; double underlines represent the Xten20 linker; single underlines represent the C-terminal NLS; represents a GS linker; and italic letters represent the coding sequence for a 3x hemagglutinin (HA) tag.

[0464] In some embodiments, the Cas9 domain is a Cas9 domain (SaCas9) from Staphylococcus aureus. In some embodiments, the SaCas9 domain is a nuclease-active SaCas9, a SaCas9 without nuclease activity (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, SaCas9 comprises an N579A mutation, or a corresponding mutation in any amino acid sequence provided herein.

[0465] In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence with a non-classical PAM. In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence with a NNGRRT or NNNRRT PAM. In some embodiments, the SaCas9 domain comprises one or more of E781X, N967X, and R1014X mutations, or a corresponding mutation of any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of E781K, N967K, and R1014H mutations, or a corresponding mutation of any amino acid sequence provided herein. In some embodiments, the SaCas9 domain comprises E781K, N967K, or R1014H mutations, or a corresponding mutation of any amino acid sequence provided herein.

[0466] In some embodiments, the variant Cas protein can be SpCas9, SpCas9-VRQR, SspCas9-VRER, xCas9(sp), SaCas9, SaCas9-KKH, SpCas9-MQKSER, SpCas9-LRKIQK, or SpCas9-LRVSQL.

[0467] Exemplary SaCas9 sequences

[0468]

[0469] Residue N579, which is underlined and shown in bold above, can be mutated (e.g., to A579) to obtain a SaCas9 nickase.

[0470] Exemplary SaCas9n sequences

[0471] KRNYILGLLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKAL EEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQ KKKPTLKQIAKEILVNEEDIKGYRVTSTTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQK EIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEE ASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGY KHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKL KKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVI KKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG

[0472] Residue A579, which can be mutated from N579 to obtain the SaCas9 nickase, is underlined and shown in bold.

[0473] Exemplary SaKKH Cas9

[0474]

[0475] Residue A579, which can be mutated from N579 to obtain the SaCas9 nickase, is underlined and shown in bold. Residues K781, K967 and H1014, which can be mutated from E781, N967 and R1014 to obtain the SaKKH Cas9, are underlined and shown in italics.

[0476] The polynucleotide programmable nucleotide binding domain of the base editor itself may include one or more domains. For example, the polynucleotide programmable nucleotide binding domain may include one or more nuclease domains. In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain may include an endonuclease or an exonuclease. Herein, the term "exonuclease" refers to a protein or polypeptide capable of digesting a nucleic acid (e.g., RNA or DNA) from a free end, and the term "endonuclease" refers to a protein or polypeptide capable of catalyzing (e.g., cleaving) an internal region in a nucleic acid (e.g., DNA or RNA). In some embodiments, an endonuclease may cleave one strand of a double-stranded nucleic acid. In some embodiments, an endonuclease may cleave two 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.

[0477] In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain can cut zero, one or two chains of the target polynucleotide. In some embodiments, the polynucleotide programmable nucleotide binding domain can include a nickase domain. Herein, the term "nickase" refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain, which can only cleave one of the two chains in a duplex nucleic acid molecule (e.g., DNA). In some embodiments, a nickase can be derived from a polynucleotide programmable nucleotide binding domain in a fully catalytically active (e.g., natural) form by introducing one or more mutations into an active polynucleotide programmable nucleotide binding domain. For example, if a polynucleotide programmable nucleotide binding domain includes a nickase domain derived from Cas9, the nickase domain derived from Cas9 can include a D10A mutation and a histidine at position 840. In such embodiments, residue H840 maintains catalytic activity and can therefore cleave a chain of a nucleic acid duplex. In another embodiment, a nickase domain derived from Cas9 can include an H840A mutation, while the amino acid at position 10 remains D. In some embodiments, the nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide programmable nucleotide binding domain by removing all or part of the nuclease domain not required for nickase activity. For example, if the polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the nickase domain derived from Cas9 can comprise all or part of a RuvC domain or a HNH domain.

[0478] A base editor comprising a polynucleotide programmable nucleotide binding domain comprising a nickase domain can produce single-stranded DNA breaks (nicks) at a specific (e.g., determined by the complementary sequence of the bound guide nucleic acid) polynucleotide target sequence. In some embodiments, the chain of the nucleic acid duplex target polynucleotide sequence cleaved by a base editor comprising a nickase domain (e.g., a nickase domain derived from Cas9) is not edited by the base editor (i.e., the chain cleaved by the base editor is opposite to the chain comprising the methyl to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., a nickase domain derived from Cas9) can cleave the chain of a DNA molecule targeted for editing. In such embodiments, the non-targeted chain is not cleaved.

[0479] Also provided herein is a base editor comprising a polynucleotide programmable nucleotide binding domain that is catalytically dead (i.e., incapable of cleaving a target polynucleotide sequence). Herein, the terms "catalytically dead" and "nuclease dead" are used interchangeably and refer to a polynucleotide programmable nucleotide binding domain having one or more mutations and / or deletions, resulting in the loss of the ability to cleave nucleic acid chains. In some embodiments, as a result of specific point mutations in one or more nuclease domains, a catalytically dead polynucleotide programmable nucleotide binding domain base editor may lack nuclease activity. For example, in the case where a base editor comprises a Cas9 domain, the Cas9 may comprise both a D10A mutation and an H840A mutation. Such mutations inactivate two nuclease domains, resulting in the 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., a RuvC1 domain and / or an HNH domain). In further embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (eg, D10A or H840A) and a deletion of all or part of the nuclease domain.

[0480] Mutations of polynucleotide programmable nucleotide binding domains that can generate catalytically dead polynucleotide programmable nucleotide binding domains from a previously functional version of the polynucleotide programmable nucleotide binding domain are also contemplated herein. For example, in the case of a catalytically dead Cas9 ("dCas9"), variants having mutations other than D10A and H840A are provided that result in a nuclease-inactive Cas9. For example, such mutations include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain).

[0481] Based on the present disclosure and the knowledge in the art, other suitable dCas9 domains without nuclease activity are obvious to those skilled in the art and are within the scope of the present disclosure. Such other exemplary suitable Cas9 domains without nuclease activity include, but are not limited to, D10A / H840A, D10A / D839A / H840A and D10A / D839A / H840A / N863A mutant domains (see, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference). In some embodiments, the dCas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 99.5% identical to any one of the dCas9 domains provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of the amino acid sequences detailed herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any one of the amino acid sequences detailed herein.

[0482] Non-limiting examples of polynucleotide programmable nucleotide binding domains that can be incorporated into base editors include domains derived from CRISPR proteins, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some embodiments, the base editor comprises a polynucleotide programmable nucleotide binding domain having a natural or modified protein or part thereof, which can bind to a nucleic acid sequence via a bound guide nucleic acid during nucleic acid modification mediated by CRISPR (i.e., clustered regularly interspaced short palindromic repeats). Herein, such proteins are referred to as "CRISPR proteins". Accordingly, disclosed herein are base editors comprising polynucleotide programmable nucleotide binding domains having all or part of a CRISPR protein (i.e., a base editor comprising all or part of a CRISPR protein as a domain, the domain also being referred to as a "domain derived from a CRISPR protein" of a base editor). The domain derived from a CRISPR protein incorporated into a base editor can be modified compared to a wild-type or natural version of a CRISPR protein. For example, as described below, a domain derived from a CRISPR protein can comprise one or more mutations, insertions, deletions, rearrangements and / or recombinations relative to a wild-type or naturally occurring version of a CRISPR protein.

[0483] In some embodiments, the domain derived from the CRISPR protein incorporated into the base editor is an endonuclease (e.g., a deoxyribonuclease or a ribonuclease), which can bind to the target polynucleotide when acting in concert with the bound guide nucleic acid. In some embodiments, the domain derived from the CRISPR protein incorporated into the base editor is a nickase, which can bind to the target polynucleotide when acting in concert with the bound guide nucleic acid. In some embodiments, the domain derived from the CRISPR protein incorporated into the base editor is a catalytically dead domain, which can bind to the target polynucleotide when acting in concert with the bound guide nucleic acid. In some embodiments, the target polynucleotide bound by the CRISPR protein derived from the base editor is DNA. In some embodiments, the target polynucleotide bound by the CRISPR protein derived from the base editor is RNA.

[0484] In some embodiments, the domain derived from a CRISPR protein of a base editor may include all or part of Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense, China (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021847.1); Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningitidis (NCBI Ref: YP_002342100.1); Streptococcus pyogenes or Staphylococcus aureus.

[0485] In some embodiments, the Cas9-derived domain of the base editor is a Cas9 domain (SaCas9) from Staphylococcus aureus. In some embodiments, the SaCas9 domain is a nuclease-active SaCas9, a SaCas9 without nuclease activity (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, the SaCas9 domain comprises an N579X mutation. In some embodiments, the SaCas9 domain comprises an N579A mutation. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain may be bound to a nucleic acid sequence having a non-classical PAM. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain may be bound to a nucleic acid sequence having a NNGRRT PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of E781X, N967X, and R1014X mutations.

[0486] The base editor may include a domain derived from all or part of Cas9, which is a high-fidelity Cas9. In some embodiments, the high-fidelity Cas9 domain of the base editor is an engineered Cas9 domain, relative to the corresponding wild-type Cas9 domain, and the high-fidelity Cas9 domain includes one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of the DNA. The high-fidelity Cas9 domain with reduced electrostatic interaction with the sugar-phosphate backbone of the DNA may have a lower off-target effect. In some embodiments, the Cas9 domain (e.g., a wild-type Cas9 domain) includes one or more mutations that reduce the association between the Cas9 domain and the sugar-phosphate backbone of the DNA. In some embodiments, the Cas9 domain comprises one or more mutations that reduce the association of the Cas9 domain with the sugar-phosphate backbone of DNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or more.

[0487] 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 an ultra-high precision Cas9 variant (HypaCas9). The modified Cas9eSpCas9 (1.1) contains glutamate substitutions, which weaken the interaction between the HNH / RuvC groove and the non-target DNA strand, preventing strand separation and cutting at off-target sites. Similarly, SpCas9-HF1 reduces off-target editing by alanine substitutions, which interrupt the interaction of Cas9 with the DNA phosphate backbone. HypaCas9 contains mutations (SpCas9 N692A / M694A / Q695A / H698A) located in the REC3 domain, which increase Cas9 proofreading and target discrimination. All three high-fidelity enzymes produce less off-target editing than wild-type Cas9. Exemplary high-fidelity Cas9 is provided below.

[0488] High-fidelity Cas9 domain mutations relative to Cas9 are shown in bold and underlined.

[0489] MDKKYSIGL A IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEK YPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFK SNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT AFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG A LSRKLINGIRDKQSGKTILDFLKSDGFANRNFM A LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR A ITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0490] Guide polynucleotide

[0491] In one embodiment, the guide polynucleotide is a guide RNA. As used herein, the term "guide RNA (gRNA)" and its grammatical equivalents may refer to RNA, which may be specific for the target DNA and may form a complex with the Cas protein. The RNA / Cas complex may assist in "guiding" the Cas protein to the target DNA. Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer sequence. The target strand that is not complementary to the crRNA is first cut endonucleolytically and then trimmed by 3'-5' exonucleolytically. In essence, DNA binding and cleavage generally require proteins and two RNAs. However, a single guide RNA ("sgRNA", or simply "gRNA") may be engineered to merge various aspects of both crRNA and tracrRNA into a single RNA species. See, for example, Jinek M., et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes short motifs (PAM or protospacer adjacent motifs) in CRISPR repeats to help distinguish self from non-self. Cas9 nuclease sequences and structures are known to those skilled in the art (see, for example, "Complete genome sequence of anM1 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 each of which are incorporated herein by reference). Cas9 orthologs have been described in a variety of species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus.Based on the present disclosure, other suitable Cas9 nucleases and sequences may be apparent to those skilled in the art, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in the following documents: Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, in other words, Cas9 is a nickase.

[0492] In some embodiments, the guide polynucleotide is at least one single guide RNA ("sgRNA" or "gRNA"). In some embodiments, the guide polynucleotide is at least one tracrRNA. In some embodiments, the guide polynucleotide does not require a PAM sequence to guide a polynucleotide programmable DNA binding domain (e.g., Cas9 or Cpf1) to a target nucleotide sequence.

[0493] The polynucleotide programmable nucleotide binding domains of the base editor disclosed herein (e.g., domains derived from CRISPR) can identify the target polynucleotide sequence by associating with the guide polynucleotide. The guide polynucleotide (e.g., gRNA) is typically single-stranded and can be programmed to site-specifically bind (i.e., via complementary base pairing) to the target sequence of the polynucleotide, thereby directing the base editor that cooperates with the guide nucleic acid to the target sequence. The guide polynucleotide can be DNA. The guide polynucleotide can be RNA. As will be appreciated by those skilled in the art, in the guide polynucleotide sequence, uracil (U) replaces thymine (T) in the sequence. In some embodiments, the guide polynucleotide comprises natural nucleotides (e.g., adenosine). In some embodiments, the guide polynucleotide comprises non-natural (non-natural) nucleotides (e.g., peptide nucleic acids or nucleotide analogs). In some embodiments, the targeting region of the guide nucleic acid sequence can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. The targeting region of the guide nucleic acid can be between 10-30 nucleotides in length, or between 15-25 nucleotides in length, or between 15-20 nucleotides in length. In some embodiments, the guide polynucleotide may be truncated by 1, 2, 3, 4, etc. nucleotides, especially at the 5' end. As a non-limiting example, a guide polynucleotide of 20 nucleotides in length may be truncated by 1, 2, 3, 4, etc. nucleotides, especially at the 5' end.

[0494] In some embodiments, the guide polynucleotide comprises two or more individual polynucleotides that can interact with each other via, for example, complementary base pairing (e.g., a dual guide polynucleotide). For example, the guide polynucleotide can comprise a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). For example, the guide polynucleotide can comprise one or more trans-activating CRISPR RNAs (tracrRNAs).

[0495] In a type II CRISPR system, targeting nucleic acids by a CRISPR protein (e.g., Cas9) typically requires complementary base pairing between a first RNA molecule (crRNA) comprising a sequence that recognizes the target sequence and a second RNA molecule (trRNA) comprising a repeat sequence, which forms a scaffold region that stabilizes the guide RNA-CRISPR protein complex. Such dual guide RNA systems can be used as guide polynucleotides to direct the base editors disclosed herein to the target polynucleotide sequence.

[0496] In some embodiments, the base editors provided herein utilize a single guide polynucleotide (e.g., sgRNA). In some embodiments, the base editors provided herein utilize dual guide polynucleotides (e.g., dual gRNA). In some embodiments, the base editors provided herein utilize one or more guide polynucleotides (e.g., multi-gRNA). In some embodiments, single guide polynucleotides are used in different base editors described herein. For example, single guide polynucleotides can be used for adenosine base editors.

[0497] In other embodiments, the guide polynucleotide can include both a polynucleotide targeting portion of a nucleic acid and a scaffold portion of a nucleic acid in a single molecule (i.e., a single molecule guide nucleic acid). For example, a single molecule guide polynucleotide can be a single guide RNA (sgRNA or gRNA). Herein, the term guide polynucleotide contemplates any single, double, or multi-molecule nucleic acid that is capable of interacting with a base editor and directing it to a target polynucleotide sequence.

[0498] Typically, a guide polynucleotide (e.g., a crRNA / trRNA complex or gRNA) includes a "polynucleotide targeting segment" that includes a sequence that can recognize and bind to a target polynucleotide sequence and a guide polynucleotide "protein binding segment" within a polynucleotide programmable nucleotide binding domain of a base editor. In some embodiments, the polynucleotide targeting segment of a guide polynucleotide recognizes and binds to a DNA polynucleotide, thereby facilitating the editing of bases in the DNA. In other embodiments, the polynucleotide targeting segment of a guide polynucleotide recognizes and binds to an RNA polynucleotide, thereby facilitating the editing of bases in the RNA. Herein, "segment" refers to a segment or region of a molecule, for example, 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 include a region of more than one molecule. For example, if a guide polynucleotide comprises a plurality of nucleic acid molecules, a protein binding segment may include all or part of a plurality of independent molecules that hybridize, for example, along a region of complementarity. In some embodiments, a protein-binding segment of a DNA-targeting RNA comprising two separate molecules may comprise: (i) base pairs 40-75 of a first RNA molecule that is 100 base pairs in length; and (ii) base pairs 10-25 of a first RNA molecule that is 50 base pairs in length. Unless specifically defined otherwise in a particular context, the definition of "segment" is not limited to a specific number of total base pairs, is not limited to any specific number of base pairs from a given RNA molecule, is not limited to a specific number of separate molecules within a complex, and may include regions of any total length of an RNA molecule and may include regions that have complementarity with other molecules.

[0499] The guide RNA or guide polynucleotide may comprise two or more RNAs, for example, CRISPR RNA (crRNA) and reverse activation crRNA (tracrRNA). The guide RNA or guide polynucleotide may sometimes comprise single-stranded RNA, or a single guide RNA (sgRNA) formed by fusing a portion (e.g., a functional portion) of crRNA and tracrRNA. The guide RNA or guide polynucleotide may also be a double RNA comprising crRNA and tracrRNA. In addition, crRNA may hybridize with a target DNA.

[0500] As described above, the guide RNA or guide polynucleotide can be an expression product. For example, the DNA encoding the guide RNA can be a vector comprising a sequence encoding the guide RNA. The guide RNA or guide polynucleotide can be transferred into the cell by transfecting the cell with an isolated guide RNA or a plasmid DNA comprising a sequence encoding the guide RNA and a promoter. The guide RNA or guide polynucleotide can also be transferred into the cell by other means such as using virus-mediated gene delivery.

[0501] The guide RNA or guide polynucleotide can be isolated. For example, the guide RNA can be transferred into a cell or organism in the form of isolated RNA. The guide RNA can be prepared by in vitro transcription using an in vitro transcription system known in the art. The guide RNA can be transferred into a cell in the form of isolated RNA rather than in the form of a plasmid containing the coding sequence of the guide RNA.

[0502] The guide RNA or guide polynucleotide can include three regions: a first region at the 5' end that can be complementary to a target site in a chromosomal sequence, a second internal region that can form a stem-loop structure, and a third 3' region that can be single-stranded. The first region of each guide RNA can also be different, so that each guide RNA guides the fusion protein to a specific target site. Furthermore, in all guide RNAs, the second region and the third region of each guide RNA can be the same.

[0503] The first region of the guide RNA or guide polynucleotide can be complementary to the sequence at the target site in the chromosome sequence, and the first region of the appropriate guide RNA can be base paired with the target site. In some embodiments, the first region of the guide RNA may include or include about 10 nucleotides to 25 nucleotides (i.e., 10 nucleotides to nucleotides; or about 10 nucleotides to about 25 nucleotides; or 10 nucleotides to about 25 nucleotides; or about 10 nucleotides to 25 nucleotides) or more. For example, the region in which base pairing is performed between the first region of the guide RNA and the target site of the chromosome sequence can be or can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25 or more nucleotides in length. In some embodiments, the first region of the guide RNA can be or can be about 19, 20 or 21 nucleotides in length.

[0504] The guide RNA or guide polynucleotide can also include a second region that forms a secondary structure. For example, the secondary structure formed by the guide RNA can include a stem (or hairpin) and a loop. The length of the loop and the stem can be variable. For example, the length range of the loop can be or can be about 3 to 10 nucleotides, and the length range of the stem can be or can be about 6 to 20 base pairs. The stem can include one or more 1 to 10 or about 10 nucleotide protuberances. The overall length of the second region can be or can be in the length range of about 16 to 60 nucleotides. For example, the loop can be or can be a length of about 4 nucleotides, and the stem can be or can be about 12 base pairs.

[0505] The guide RNA or guide polynucleotide can also include a third region 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 remainder of the guide RNA. Furthermore, the length of the third region can be variable. The third region can be more than or more than about 4 nucleotides in length. For example, the overall length of the third region can be or can be in the range of about 5 to 60 nucleotides in length.

[0506] The guide RNA or guide polynucleotide can target any exon or intron of a gene target. In some embodiments, the guide can target exon 1 or 2 of a gene; in other embodiments, the guide can target exon 3 or 4 of a gene. The composition can include multiple RNAs that all target the same exon, or in some embodiments, multiple RNAs that can target different exons. Exons and introns of a gene can be targeted.

[0507] The guide RNA or guide polynucleotide can target a nucleic acid sequence of about 20 nucleotides. The target nucleic acid can be less than about 20 nucleotides. The target nucleic acid can be at least (about) 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 nucleotides or a length anywhere between 1-100 nucleotides. The target nucleic acid can be at most (about) 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50 nucleotides or a length anywhere between 1-100 nucleotides. The target nucleic acid sequence can be (about) 20 bases of the first nucleotide of the 5' next to the PAM. The guide RNA can target a nucleic acid sequence. 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.

[0508] A guide polynucleotide, e.g., a guide RNA, can refer to a nucleic acid that can hybridize to another nucleic acid in the genome of a cell (e.g., a target nucleic acid or a pre-spacer sequence). A guide polynucleotide can be RNA. A guide polynucleotide can be DNA. A guide polynucleotide can be programmed or designed to site-specifically bind to a nucleic acid sequence. A guide polynucleotide can comprise one polynucleotide chain and can be referred to as a single guide polynucleotide. A guide polynucleotide can comprise two polynucleotide chains and can be referred to as a dual guide polynucleotide. A guide RNA can be introduced into a cell as an RNA molecule. For example, an RNA molecule can be transcribed and / or chemically synthesized in vitro. RNA can be derived from a synthetic DNA molecule (e.g., Gene fragments) are transcribed. Then, the guide RNA can be introduced into the cell as an RNA molecule. The guide RNA can also be introduced into the cell in the form of a non-RNA nucleic acid molecule (e.g., a DNA molecule). For example, the DNA encoding the guide RNA can be operably linked to a promoter control sequence to express the guide RNA in the cell of interest. The RNA coding sequence can be operably linked to a promoter sequence recognized by RNA polymerase III (Pol III). Plasmid vectors that can be used to express guide RNA include, but are not limited to, px330 vectors and px333 vectors. In some embodiments, the plasmid vector (e.g., px333 vector) may contain at least two DNA sequences encoding the guide RNA.

[0509] Methods for selecting, designing and verifying guide polynucleotides (e.g., guide RNA and targeting sequences) are described herein and are known to those skilled in the art. For example, in order to minimize the impact of potential substrate promiscuity of the deaminase domain (e.g., AID domain) in the nucleobase editor system, the number of residues that may be inadvertently targeted by the deamination reaction (e.g., off-target C residues that may potentially reside on ssDNA in the target nucleic acid locus) can be minimized. In addition, software tools can be used to optimize the gRNA corresponding to the target nucleic acid sequence, for example, to minimize the total off-target activity across the locus. For example, for each possible targeting domain selection using Streptococcus pyogenes Cas9, all off-target sequences (previously selected PAM, e.g., NAG or NGG) can be identified across the genome, and the sequence contains up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) of mismatched base pairs. The first region of the gRNA complementary to the target site can be identified, and all first regions (e.g., crRNA) can be sorted according to the predicted total off-target score; the top-ranked targeting domains represent those that may have the greatest on-target activity and the least off-target activity. The candidate targeting gRNA can be functionally evaluated using methods known in the art and / or as described in detail herein.

[0510] As a non-limiting example, the target DNA hybridization sequence in the crRNA of the guide RNA used with Cas9 can be identified using a DNA sequence search algorithm. Custom gRNA design software can be used to complete gRNA design based on the public tool cas-offinder, as described in Bae S., Park J., & Kim J.-S.Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guide dendonucleases. Bioinformatics 30, 1473-1475 (2014). This software scores the guide after calculating the whole genome off-target tendency of the guide. Typically, for a guide range of 17 to 24 lengths, a matching range from perfect match to the presence of 7 mismatches is considered. Once the off-target site is determined by calculation, the aggregation score of each guide is calculated and summarized into a table output using a web interface. In addition to identifying potential target sites adjacent to PAM sequences, the software also identifies all PAM adjacent sequences, which are different from the selected target site by 1, 2, 3 or more than 3 nucleotides. The genomic DNA sequence and the repeat elements of the target nucleic acid sequence (e.g., target gene) can be obtained using a publicly available summary such as the RepeatMasker program. RepeatMasker retrieves the repeat elements and low complexity regions of the input DNA sequence. The output is a detailed annotation of the repeat sequences present in a given query sequence.

[0511] After identification, the first region of the guide RNA (e.g., crRNA) can be ranked into several levels based on their distance from the target site, their orthogonality, and the presence of a 5' nucleotide that is closely matched to the relevant PAM sequence (e.g., a 5'G based on the identification of a close match in the human genome containing the relevant PAM (e.g., NGG PAM for S. pyogenes, NNGRRT or NNGRRV PAM for S. aureus). As used herein, orthogonality refers to the number of sequences in the human genome that contain the lowest number of mismatches with the target sequence. For example, "high level of orthogonality" or "good orthogonality" can refer to a 20-mer targeting domain that has no identical sequences in the human genome other than the intended target, nor any sequences that contain one or two mismatches in the target sequence. Targeting domains with good orthogonality can be selected to minimize off-target DNA cleavage.

[0512] In some embodiments, a reporter system can be used to detect base editing activity and test candidate guide polynucleotides. In some embodiments, the reporter system may include a reporter gene-based assay, wherein the base editing activity results in the expression of the reporter gene. For example, the reporter system may include a reporter gene comprising an inactivated start codon (e.g., a mutation of 3'-TAC-5' to 3'-CAC-5' on the template strand). After the target C is successfully deaminated, the corresponding mRNA will be transcribed as 5'-AUG-3' instead of 5'-GUG-3', so that the reporter gene can be translated. Suitable reporter genes will be obvious to those skilled in the art. Non-limiting examples of reporter genes include genes encoding green fluorescent protein (GFP), red fluorescent protein (RFP), luciferase, secreted alkaline phosphatase (SEAP), or any other gene whose expression is detectable and obvious to those skilled in the art. The reporter system can be used to test a number of different gRNAs, for example, to determine which nucleotide residues / some nucleotides of the target DNA sequence the corresponding deaminase will target. sgRNAs targeting non-template chain nucleotide residues can also be tested to evaluate the off-target effects of specific base editing proteins (e.g., Cas9 deaminase fusion proteins). In some embodiments, such gRNAs can be designed so that the mutated start codon will not base pair with the gRNA. The guide polynucleotides may include standard nucleotides, modified nucleotides (e.g., pseudouridine), nucleotide isomers, and / or nucleotide analogs. In some embodiments, the guide polynucleotides may include at least one detectable marker. The detectable marker may be a fluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluors, Halo tags, or any other suitable fluorescent dye), a detection tag (e.g., biotin, digoxin, etc.), a quantum dot, or a gold particle.

[0513] The guide polynucleotide can be chemically synthesized and / or enzymatically synthesized. For example, the guide RNA can be synthesized using a standard solid phase synthesis method based on phosphoramidite. As another alternative, the guide RNA can be synthesized in vitro by operably linking the DNA encoding the guide RNA to a promoter control sequence recognized by a bacteriophage RNA polymerase. Suitable examples of specific promoter sequences include T7, T3, SP6 promoter sequences or variants thereof. In embodiments where the guide RNA comprises two independent molecules (e.g., crRNA and tracrRNA), crRNA can be chemically synthesized, and tracrRNA can be enzymatically synthesized.

[0514] In some embodiments, the base editor system can include multiple guide polynucleotides, such as gRNAs. For example, the gRNA can target one or more target loci (e.g., at least one (1) gRNA, at least 2 gRNAs, at least 5 gRNAs, at least 10 gRNAs, at least 20 gRNAs, at least 30 g RNAs, or at least 50 gRNAs). In some embodiments, multiple gRNA sequences can be randomly arranged and preferably separated by direct repeat sequences.

[0515] The DNA sequence encoding the guide RNA or guide polynucleotide can also be part of the vector. In some embodiments, the vector contains additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selectable marker sequences (e.g., GFP or antibiotic resistance genes such as puromycin), replication origins, etc. The DNA molecule encoding the guide RNA or guide polynucleotide can also be linear or circular.

[0516] In some embodiments, one or more components of the base editor system may be encoded by a DNA sequence. Such DNA sequences may be introduced into an expression system (e.g., a cell) together or individually. For example, a DNA sequence encoding a polynucleotide programmable nucleotide binding domain and a guide RNA may be introduced into a cell, and each DNA sequence may be part of an independent molecule (e.g., a vector containing a polynucleotide programmable nucleotide binding domain encoding sequence and a second vector containing a guide RNA encoding sequence), or both may be part of the same molecule (e.g., a vector containing coding (and regulatory) sequences for both a polynucleotide programmable nucleotide binding domain and a guide RNA).

[0517] The guide polynucleotide may comprise one or more modifications to provide new or enhanced characteristics to the nucleic acid. The guide polynucleotide may comprise a nucleic acid affinity tag. The guide polynucleotide may comprise synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides.

[0518] In some embodiments, the gRNA or guide polynucleotide may comprise a modification. The modification may be made at any position of 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.

[0519] Modification of the gRNA or guide polynucleotide can be substitution, insertion, deletion, chemical modification, physical modification, stabilization, purification, or any combination thereof.

[0520] The gRNA or guide polynucleotide can also be modified by: 5' adenylation, 5' guanosine-triphosphate blocking, 5' N7-methylguanosine-triphosphate blocking, 5' triphosphate blocking, 3' phosphate, 3' phosphorothioate, 5' thiocyanate, 5' phosphorothioate, Cis-Syn thymine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9, 3'-3' modification, 5'-5' modification, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, bibiotin, PC biotin, psoralen C2...

Claims

1. A use of (i) an adenosine base editor or a polynucleotide encoding the adenosine base editor and (ii) a guide polynucleotide or a polynucleotide encoding the guide polynucleotide for the preparation of a medicament for treating Rett syndrome, wherein: a) The adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain is composed of the following amino acid sequence with a changed combination or a fragment thereof lacking only the N-terminal methionine: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, wherein the fragment lacking only the N-terminal methionine is composed of the following amino acid sequence: and b) the guide polynucleotide directs the adenosine base editor to generate an A to G nucleobase change within a methyl CpG binding protein 2 (MECP2) gene comprising a single nucleotide polymorphism associated with Rett syndrome; wherein the A to G nucleobase change is located at the single nucleotide polymorphism associated with Rett syndrome, the single nucleotide polymorphism results in an R106W amino acid mutation in a MECP2 polypeptide encoded by the MECP2 gene, and wherein: i) the combination of changes is selected from the group consisting of: Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the guide polynucleotide consists of the sequence 5′-gCCAUGUCCAGCCUUCAGGCA-3′; or ii) the combination of changes is selected from the group consisting of: V82S + Q154S; V82S + Y147R; V82S + Y123H; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the guide polynucleotide consists of the sequence 5′-gUCCAUGUCCAGCCUUCAGGC-3′; or iii) the combination of changes is selected from the group consisting of: V82S + Q154S; V82S + Y123H; V82S + Y123H + Y147T; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the guide polynucleotide consists of a sequence of 5′-gAGCUUCCAUGUCCAGCCUUC-3′.

2. The use according to claim 1, wherein The A to G nucleobase change converts the single nucleotide polymorphism associated with Rett syndrome to the wild-type nucleobase.

3. A base editor system, comprising: (a) an adenosine base editor or a polynucleotide encoding the adenosine base editor, wherein: The adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain is composed of the following amino acid sequence with a changed combination or a fragment thereof lacking only the N-terminal methionine: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, wherein the fragment lacking only the N-terminal methionine is composed of the following amino acid sequence: and (b) a guide polynucleotide or a polynucleotide encoding the guide polynucleotide, wherein the guide polynucleotide directs the adenosine base editor to generate an A to G nucleobase change within a methyl CpG binding protein 2 (MECP2) gene comprising a single nucleotide polymorphism associated with Rett syndrome; wherein the A to G nucleobase change is located at the single nucleotide polymorphism associated with Rett syndrome, wherein the single nucleotide polymorphism associated with Rett syndrome results in an R106W amino acid mutation in a MECP2 polypeptide encoded by the MECP2 gene, and wherein: i) the combination of changes is selected from the group consisting of: Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the guide polynucleotide consists of the sequence 5′-gCCAUGUCCAGCCUUCAGGCA-3′; or ii) the combination of changes is selected from the group consisting of: V82S + Q154S; V82S + Y147R; V82S + Y123H; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the guide polynucleotide consists of the sequence 5′-gUCCAUGUCCAGCCUUCAGGC-3′; or iii) the combination of changes is selected from the group consisting of: V82S + Q154S; V82S + Y123H; V82S + Y123H + Y147T; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the guide polynucleotide consists of a sequence of 5′-gAGCUUCCAUGUCCAGCCUUC-3′.

4. The base editor system according to claim 3, wherein: The A to G nucleobase change converts the single nucleotide polymorphism associated with Rett syndrome to the wild-type nucleobase.

5. Use of a base editor system for preparing a drug for treating Rett syndrome, wherein: The base editor system comprises a base editor complexed with one or more guide polynucleotides, wherein: a) The base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain is composed of the following amino acid sequence with a changed combination or a fragment thereof lacking only the N-terminal methionine: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, wherein the fragment lacking only the N-terminal methionine is composed of the following amino acid sequence: and b) one or more of the guide polynucleotides targets the base editor to cause a single nucleotide polymorphism associated with Rett syndrome in a MECP2 polynucleotide to change from A•T to G•C, wherein the single nucleotide polymorphism results in an amino acid mutation of R106W in a MECP2 polypeptide encoded by the MECP2 polynucleotide, and wherein: i) the combination of changes is selected from the group consisting of: Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the one or more guide polynucleotides consists of the sequence 5′-gCCAUGUCCAGCCUUCAGGCA-3′; or ii) the combination of changes is selected from the group consisting of: V82S + Q154S; V82S + Y147R; V82S + Y123H; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the one or more guide polynucleotides consists of the sequence 5′-gUCCAUGUCCAGCCUUCAGGC-3′; or iii) the combination of changes is selected from the group consisting of: V82S + Q154S; V82S + Y123H; V82S + Y123H + Y147T; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the one or more guide polynucleotides consists of a sequence of 5′-gAGCUUCCAUGUCCAGCCUUC-3′.

6. The use according to claim 5, wherein The MECP2 polynucleotide is in a cell.

7. The use according to claim 6, wherein The cells are in vivo or ex vivo.

8. The use according to claim 5, wherein The polynucleotide programmable DNA binding domain is a Cas9 selected from the group consisting of: Streptococcus pyogenes Cas9, Staphylococcus aureus Cas9, Streptococcus thermophilus 1 Cas9, and Streptococcus canis Cas9.

9. The use according to claim 5, wherein The polynucleotide programmable DNA binding domain is SpCas9 that binds to a non-5'-NGG-3' protospacer adjacent motif (PAM).

10. The use according to claim 5, wherein: The polynucleotide programmable DNA binding domain is SpCas9 bound to a PAM consisting of a 5'-NGG-3' nucleic acid sequence.

11. The use according to claim 5, wherein: The polynucleotide programmable DNA binding domain has no nuclease activity or has nickase activity.

12. The use according to claim 5, wherein: The base editor further comprises a wild-type adenosine deaminase domain.

13. A cell produced by introducing into said cell or a progenitor thereof: a) a base editor or a polynucleotide encoding the base editor, wherein: The base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain is composed of the following amino acid sequence with a changed combination or a fragment thereof lacking only the N-terminal methionine: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, wherein the fragment lacking only the N-terminal methionine is composed of the following amino acid sequence: AND b) one or more guide polynucleotides that target the base editor to cause an A•T to G•C change in a single nucleotide polymorphism in a MECP2 polynucleotide associated with Rett syndrome, wherein the single nucleotide polymorphism results in an amino acid mutation of R106W in a MECP2 polypeptide encoded by the MECP2 polynucleotide, and wherein: i) the combination of changes is selected from the group consisting of: Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the one or more guide polynucleotides consists of the sequence 5′-gCCAUGUCCAGCCUUCAGGCA-3′; or ii) the combination of changes is selected from the group consisting of: V82S + Q154S; V82S + Y147R; V82S + Y123H; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the one or more guide polynucleotides consists of the sequence 5′-gUCCAUGUCCAGCCUUCAGGC-3′; or iii) the combination of changes is selected from the group consisting of: V82S + Q154S; V82S + Y123H; V82S + Y123H + Y147T; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the one or more guide polynucleotides consists of a sequence of 5′-gAGCUUCCAUGUCCAGCCUUC-3′.

14. The cell according to claim 13, wherein The cells are neurons.

15. The cell according to claim 14, wherein The neurons express MECP2 polypeptide.

16. The cell according to claim 13, wherein The cells are from a subject suffering from Rett syndrome.

17. The cell according to claim 13, wherein The cells are mammalian cells.

18. The cell according to claim 13, wherein The polynucleotide programmable DNA binding domain is a Cas9 selected from the group consisting of: Streptococcus pyogenes Cas9, Staphylococcus aureus Cas9, Streptococcus thermophilus 1 Cas9, and Streptococcus canis Cas9.

19. The cell according to claim 13, wherein The polynucleotide programmable DNA binding domain is SpCas9 that binds to a non-5'-NGG-3' protospacer adjacent motif (PAM).

20. The cell according to claim 13, wherein The polynucleotide programmable DNA binding domain is SpCas9 bound to a PAM consisting of a 5'-NGG-3' nucleic acid sequence.

21. The cell according to claim 13, wherein The polynucleotide programmable DNA binding domain has no nuclease activity or has nickase activity.

22. The cell according to claim 13, wherein The base editor further comprises a wild-type adenosine deaminase domain.

23. The cell according to claim 13, wherein The scaffold of the guide polynucleotide consists of the following sequence: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT.

24. A base editor or a polynucleotide encoding the base editor and one or more guide polynucleotides targeting the base editor for preparing a drug for treating Rett syndrome in a subject, wherein: The base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain is composed of the following amino acid sequence with a changed combination or a fragment thereof lacking only the N-terminal methionine: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, wherein the fragment lacking only the N-terminal methionine is composed of the following amino acid sequence: SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD; wherein the one or more guide polynucleotides cause a single nucleotide polymorphism associated with Rett syndrome in a MECP2 polynucleotide to change from A•T to G•C, wherein the single nucleotide polymorphism results in an amino acid mutation of R106W in a MECP2 polypeptide encoded by the MECP2 polynucleotide, and wherein: i) the combination of changes is selected from the group consisting of: Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the one or more guide polynucleotides consists of the sequence 5′-gCCAUGUCCAGCCUUCAGGCA-3′; or ii) the combination of changes is selected from the group consisting of: V82S + Q154S; V82S + Y147R; V82S + Y123H; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the one or more guide polynucleotides consists of the sequence 5′-gUCCAUGUCCAGCCUUCAGGC-3′; or iii) the combination of changes is selected from the group consisting of: V82S + Q154S; V82S + Y123H; V82S + Y123H + Y147T; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the one or more guide polynucleotides consists of a sequence of 5′-gAGCUUCCAUGUCCAGCCUUC-3′.

25. The use according to claim 24, wherein The subject is a mammal.

26. The use according to claim 24 or 25, comprising delivering the base editor or the polynucleotide encoding the base editor, and the one or more guide polynucleotides to the cells of the subject.

27. The use according to claim 24, wherein The polynucleotide programmable DNA binding domain is a Cas9 selected from the group consisting of: Streptococcus pyogenes Cas9, Staphylococcus aureus Cas9, Streptococcus thermophilus 1 Cas9, and Streptococcus canis Cas9.

28. The use according to claim 24, wherein The polynucleotide programmable DNA binding domain is SpCas9 that binds to a non-5'-NGG-3' protospacer adjacent motif (PAM).

29. The use according to claim 24, wherein The polynucleotide programmable DNA binding domain is SpCas9 bound to a PAM consisting of a 5'-NGG-3' nucleic acid sequence.

30. The use according to claim 24, wherein The polynucleotide programmable DNA binding domain has no nuclease activity or has nickase activity.

31. The use according to claim 24, wherein The base editor further comprises a wild-type adenosine deaminase domain.

32. A composition of a base editor and a guide polynucleotide, wherein: The base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, wherein: a) The adenosine deaminase domain is composed of the following amino acid sequence with a changed combination or a fragment thereof lacking only the N-terminal methionine: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, wherein the fragment lacking only the N-terminal methionine is composed of the following amino acid sequence: AND b) the guide polynucleotide targets the base editor to cause an A•T to G•C change in a single nucleotide polymorphism in a MECP2 polynucleotide associated with Rett syndrome, wherein the single nucleotide polymorphism results in an amino acid mutation of R106W in a MECP2 polypeptide encoded by the MECP2 polynucleotide, and wherein: i) the combination of changes is selected from the group consisting of: Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the guide polynucleotide consists of the sequence 5′-gCCAUGUCCAGCCUUCAGGCA-3′; or ii) the combination of changes is selected from the group consisting of: V82S + Q154S; V82S + Y147R; V82S + Y123H; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the guide polynucleotide consists of the sequence 5′-gUCCAUGUCCAGCCUUCAGGC-3′; or iii) the combination of changes is selected from the group consisting of: V82S + Q154S; V82S + Y123H; V82S + Y123H + Y147T; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the guide polynucleotide consists of a sequence of 5′-gAGCUUCCAUGUCCAGCCUUC-3′.

33. The composition according to claim 32, wherein The polynucleotide programmable DNA binding domain is a Cas9 selected from the group consisting of: Streptococcus pyogenes Cas9, Staphylococcus aureus Cas9, Streptococcus thermophilus 1 Cas9, and Streptococcus canis Cas9.

34. The composition according to claim 32, wherein The polynucleotide programmable DNA binding domain is SpCas9 that binds to a non-5'-NGG-3' protospacer adjacent motif (PAM).

35. The composition according to claim 32, wherein The polynucleotide programmable DNA binding domain has no nuclease activity or has nickase activity.

36. The composition according to claim 32, wherein The base editor further comprises a wild-type adenosine deaminase domain.

37. The composition according to claim 32, further comprising a lipid, wherein The lipid is a cationic lipid.

38. The composition according to claim 32, which is a pharmaceutical composition comprising a pharmaceutically acceptable excipient or diluent.

39. A composition according to claim 38 for use in the treatment of Rett syndrome.

40. The composition according to claim 38, wherein The guide polynucleotide and the base editor are formulated together or separately.

41. The composition according to any one of claims 37 to 40, further comprising a vector suitable for expression in mammalian cells, wherein The vector comprises a polynucleotide encoding the base editor.

42. The composition according to claim 41, wherein The vector is a viral vector.

43. The composition according to claim 42, wherein The viral vector is a retroviral vector, an adenoviral vector, a lentiviral vector, a herpes virus vector or an adeno-associated virus vector.

44. Use of a composition according to any one of claims 37 to 43 in the preparation of a medicament for treating Rett syndrome in a subject.

45. The use according to claim 44, wherein The subject is a mammal.

46. ​​A composition comprising the cells of any one of claims 13 to 23.

47. The composition of claim 46, further comprising a pharmaceutically acceptable carrier or diluent.

48. A pharmaceutical composition comprising: (i) a nucleic acid encoding a base editor, wherein: The base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain is composed of the following amino acid sequence with a changed combination or a fragment thereof lacking only the N-terminal methionine: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, wherein the fragment lacking only the N-terminal methionine is composed of the following amino acid sequence: and (ii) a guide polynucleotide, wherein: a) The combination of changes is selected from the group consisting of: Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the guide polynucleotide consists of the sequence 5′-gCCAUGUCCAGCCUUCAGGCA-3′; or b) the combination of changes is selected from the group consisting of: V82S + Q154S; V82S + Y147R; V82S + Y123H; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the guide polynucleotide consists of the sequence 5′-gUCCAUGUCCAGCCUUCAGGC-3′; or c) the combination of changes is selected from the group consisting of: V82S + Q154S; V82S + Y123H; V82S + Y123H + Y147T; V82S + Y123H + Q154R; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R; and The spacer sequence of the guide polynucleotide consists of a sequence of 5′-gAGCUUCCAUGUCCAGCCUUC-3′.

49. The pharmaceutical composition of claim 48, further comprising a lipid.

50. The pharmaceutical composition according to claim 49, wherein The lipid is a cationic lipid.

51. The pharmaceutical composition according to claim 48 or 49, wherein The nucleic acid encoding the base editor is mRNA.

Images