Disruption of splice acceptor sites in disease-related genes using adenosine deaminase base editors, including for the treatment of genetic diseases

Abstract

The present invention features compositions and methods for treating, reducing, or ameliorating the debilitating effects of amyotrophic lateral sclerosis (ALS) and spinal-bulbar muscular atrophy (SBMA). Compositions and methods are provided that use novel, improved base editors (e.g., adenosine base editors) comprising a polynucleotide-programmable nucleotide-binding domain and a nucleobase-editing domain in conjunction with a guide polynucleotide to disrupt normal transcription of genes associated with a genetic disease or condition, such as ALS or SBMA, by modifying the target gene associated with the genetic disorder or condition with the base editor system.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 805,271 filed February 13, 2019, U.S. Provisional Application No. 62 / 852,228 filed May 23, 2019, U.S. Provisional Application No. 62 / 852,224 filed May 23, 2019, U.S. Provisional Application No. 62 / 873,140 filed July 11, 2019, U.S. Provisional Application No. 62 / 873,144 filed July 11, 2019, U.S. Provisional Application No. 62 / 931,722 filed November 6, 2019, U.S. Provisional Application No. 62 / 941,569 filed November 27, 2019, and U.S. Provisional Application No. 62 / 966,526 filed January 27, 2020, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Unless otherwise indicated, all publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety. [Background technology]

[0003] Targeted editing of nucleic acid sequences, such as targeted cleavage or modification of genomic DNA, is a promising approach for studying gene function and may provide novel therapeutics for human genetic diseases. Currently available base editors include cytidine base editors (e.g., BE4) that convert targeted C·G base pairs to T·A, and adenine base editors (e.g., ABE7.10) that convert A·T to G·C. There is a need in the art for improved base editors that can induce modifications within target sequences with greater specificity and efficiency. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention provides compositions comprising novel adenine base editors (e.g., ABE8) with increased efficiency, and methods of using base editors comprising adenosine deaminase variants to edit target sequences. [Means for solving the problem]

[0005] In certain aspects, provided herein is a method of treating a neurological disorder in a subject in need thereof, comprising administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding same; and (ii) a guide polynucleotide or a nucleic acid sequence encoding same, wherein said adenosine base editor comprises a programmable DNA-binding domain and an adenosine deaminase domain, and wherein said guide polynucleotide directs said adenosine base editor to effect a single nucleobase modification at a splice site of a target gene associated with said neurological disorder in said subject, thereby treating said neurological disorder in the subject.

[0006] In one embodiment, the adenosine deaminase comprises an amino acid substitution at amino acid position 82 or 166 in SEQ ID NO:2, or a substitution corresponding thereto. In one embodiment, the single nucleobase modification results in alternative splicing of a transcript encoded by the target gene. In one embodiment, the alternative splicing produces a truncated or non-functional protein encoded by the target gene. In one embodiment, the single nucleobase modification results in reduced expression of the target gene in the subject. In one embodiment, the target gene is the superoxide dismutase 1 (SOD1) gene and the neurological disease is amyotrophic lateral sclerosis (ALS). In one embodiment, the target gene is the androgen receptor (AR) gene and the neurological disease is spinal and bulbar muscular atrophy (SBMA).

[0007] In certain aspects, provided herein is a method of treating amyotrophic lateral sclerosis (ALS) in a subject in need thereof, comprising administering to the subject (i) a base editor or a nucleic acid sequence encoding same; and (ii) a guide polynucleotide or a nucleic acid sequence encoding same, wherein said base editor comprises a programmable DNA-binding domain and a deaminase domain, and wherein said guide polynucleotide directs said adenosine base editor to modify a single nucleobase at a splice site of a superoxide dismutase 1 (SOD1) gene in said subject, thereby treating ALS in said subject.

[0008] In certain aspects, provided herein is a method of treating amyotrophic lateral sclerosis (ALS) in a subject in need thereof, comprising administering to the subject (i) a base editor or a nucleic acid sequence encoding same; and (ii) a guide polynucleotide or a nucleic acid sequence encoding same, wherein said base editor comprises a programmable DNA-binding domain and a deaminase domain, and wherein said guide polynucleotide directs said adenosine base editor to result in a single nucleobase modification in said subject's superoxide dismutase 1 (SOD1) gene, wherein said single nucleobase modification results in a premature stop codon in the SOD1 gene, thereby treating ALS in said subject.

[0009] In one embodiment, the deaminase is an adenosine deaminase containing an amino acid substitution at amino acid position 8 or 166 in the numbering of SEQ ID NO: 2, or a substitution corresponding thereto. In one embodiment, the single nucleobase modification is an A to G modification. In one embodiment, the single nucleobase modification is in a splice acceptor site of the SOD1 gene. In one embodiment, the splice site is a splice acceptor site 5' of an exon of the SOD1 gene. In one embodiment, the exon of the SOD1 gene is exon 3 corresponding to SEQ ID NO: 3, or a variant thereof. In one embodiment, the exon 3 of the SOD1 gene is adjacent to a splice acceptor AG at nucleotide position 6828 of the SOD1 polynucleotide sequence or a variant thereof in the numbering of SEQ ID NO: 3. In one embodiment, the exon of the SOD1 gene is exon 4 corresponding to SEQ ID NO: 3, or a variant thereof. In one embodiment, the single nucleobase modification generates a transcript or a variant thereof that lacks exons 3 to 5 of the human SOD1 gene corresponding to SEQ ID NO: 3. In one embodiment, after said administration, expression of the SOD1 gene is reduced by at least 40% in said subject.

[0010] In one embodiment, the guide polynucleotide comprises a nucleic acid sequence complementary to a splice acceptor nucleic acid sequence or a splice donor nucleic acid sequence of the SOD1 gene. In one embodiment, the guide polynucleotide comprises any nucleic acid sequence selected from Table 19 or Table 23. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of 5'-UUAAAGGAAAGUAAUGGACCAGU-3', 5'-UAAAUAGGCUGUACCAGUGCAGG-3', 5'-UUCAUUAUUAGGCAUGUUGGAGA-3', 5'-AAAUAGGCUGUACCAGUGCAGGU-3', and 5'-UAUUAGGCAUGUUGGAGACUUGG-3'.

[0011] In certain aspects, provided herein is a method of treating spinal and bulbar muscular atrophy (SBMA) in a subject, comprising administering to the subject (i) a base editor or a nucleic acid sequence encoding same; and (ii) a guide polynucleotide or a nucleic acid sequence encoding same, wherein said base editor comprises a programmable DNA-binding domain and a deaminase domain, and said guide polynucleotide directs said adenosine base editor to effect a single nucleobase modification at a splice site of an androgen receptor (AR) gene in said subject, thereby treating SBMA in said subject.

[0012] In certain aspects, provided herein is a method of treating spinal and bulbar muscular atrophy (SBMA) in a subject, comprising administering to the subject (i) a base editor or a nucleic acid sequence encoding same; and (ii) a guide polynucleotide or a nucleic acid sequence encoding same, wherein the adenosine base editor comprises a programmable DNA-binding domain and a deaminase domain, and wherein the guide polynucleotide directs the adenosine base editor to result in a single nucleobase modification in an androgen receptor (AR) gene of the subject, wherein the single nucleobase modification results in a premature stop codon in the AR gene, thereby treating SBMA in the subject.

[0013] In one embodiment, the nucleobase modification results in a CAG-TAG codon change in the AR gene. In one embodiment, the codon modification is in exon 1 or exon 2 of the AR gene. In one embodiment, the deaminase is an adenosine deaminase comprising an amino acid substitution at amino acid position 82 or 166 in the numbering of SEQ ID NO: 2. In one embodiment, the single nucleobase modification is an A to G modification. In one embodiment, the A to G nucleobase modification is in a splice acceptor site of the AR gene. In one embodiment, the splice site is a splice acceptor site 5' of an exon of the AR gene. In one embodiment, the exon of the AR gene is exon 2 corresponding to SEQ ID NO: 4 or a variant thereof. In one embodiment, the splice site is a splice donor site 3' of an exon of the AR gene. In one embodiment, the exon of the AR gene is exon 1 corresponding to SEQ ID NO: 4 or a variant thereof. In one embodiment, the expression of the AR gene in the subject is reduced by at least 40% after the administration.

[0014] In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to a splice acceptor nucleic acid sequence or a splice donor nucleic acid sequence of the AR gene. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from Table 41 A or 41 B. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of 5'-ACUUACCGCAUGUCCCCGUAAGG-3', 5'-AGUGCAGUUAGGGCUGGGAAGGG-3', and 5'-AAGUGCAGUUAGGGCUGGGAAGG-3'.

[0015] In one embodiment, the subject is a mammal or a human. In one embodiment, the administration is via delivery to a cell in the central nervous system (CNS) of the subject. In one embodiment, the cell is a motor neuron.

[0016] In certain aspects, provided herein is a method of modifying a target gene or a regulatory element thereof associated with a neurological disorder, comprising contacting the target gene or a regulatory element thereof with (i) an adenosine base editor or a nucleic acid sequence encoding same, and (ii) a guide polynucleotide or a nucleic acid sequence encoding same, wherein the adenosine base editor comprises a programmable DNA-binding domain and an adenosine deaminase domain, and the guide polynucleotide directs the adenosine base editor to effect modification of a single nucleobase at a splice site of the target gene.

[0017] In one embodiment, the adenosine deaminase comprises an amino acid substitution at amino acid position 82 or 166 in SEQ ID NO:2. In one embodiment, the single nucleobase modification results in alternative splicing of a transcript encoded by the target gene, a truncated and / or non-functional protein encoded by the target gene, and / or reduced expression of the target gene when expressed in a cell. In one embodiment, the target gene is the superoxide dismutase 1 (SOD1) gene and the neurological disease is amyotrophic lateral sclerosis (ALS). In one embodiment, the target gene is the androgen receptor (AR) gene and the neurological disease is spinal and bulbar muscular atrophy (SBMA).

[0018] In certain aspects, provided herein is a method of regulating expression of the superoxide dismutase (SOD1) gene, comprising contacting the SOD1 gene or a regulatory element thereof with (i) a base editor or a nucleic acid sequence encoding same, and (ii) a guide polynucleotide or a nucleic acid sequence encoding same, wherein said base editor comprises a programmable DNA-binding domain and a deaminase domain, and wherein said guide polynucleotide directs said adenosine base editor to modify a single nucleobase at a splice site of the superoxide dismutase 1 (SOD1) gene.

[0019] In certain aspects, provided herein is a method of modifying a superoxide dismutase (SOD1) gene, the method comprising contacting the SOD1 gene or a regulatory element thereof with (i) a base editor or a nucleic acid sequence encoding same, and (ii) a guide polynucleotide or a nucleic acid sequence encoding same, wherein said guide polynucleotide directs said adenosine base editor to result in a single nucleobase modification in said subject's superoxide dismutase 1 (SOD1) gene, wherein said single nucleobase modification results in a premature stop codon in the SOD1 gene.

[0020] In one embodiment, the single nucleobase modification is an A to G modification. In one embodiment, the nucleobase modification is in a splice acceptor site of the SOD1 gene. In one embodiment, the splice site is a splice acceptor site 5' of an exon of the SOD1 gene. In one embodiment, the exon of the SOD1 gene is exon 3 corresponding to SEQ ID NO: 3 or a variant thereof. In one embodiment, the exon 3 of the SOD1 gene is adjacent to the splice acceptor at nucleotide position 6828 of the SOD1 polynucleotide sequence in the numbering of SEQ ID NO: 3. In one embodiment, the exon of the SOD1 gene is exon 4 corresponding to SEQ ID NO: 3 or a variant thereof. In one embodiment, the single nucleobase modification generates a transcript or a variant thereof lacking exons 3 to 5 of the SOD1 gene corresponding to SEQ ID NO: 3.

[0021] In one embodiment, the guide polynucleotide comprises a nucleic acid sequence complementary to a splice acceptor nucleic acid sequence or a splice donor nucleic acid sequence of the SOD1 gene. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence selected from Table 19 or Table 23. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of 5'-UUAAAGGAAAGUAAUGGACCAGU-3', 5'-UAAAUAGGCUGUACCAGUGCAGG-3', 5'-UUCAUUAUUAGGCAUGUUGGAGA-3', 5'-AAAUAGGCUGUACCAGUGCAGGU-3', and 5'-UAUUAGGCAUGUUGGAGACUUGG-3'.

[0022] In certain embodiments, provided herein are methods of modulating expression of the androgen receptor (AR) gene, comprising contacting the AR gene or a regulatory element thereof with (i) a base editor or a nucleic acid sequence encoding same, and (ii) a guide polynucleotide or a nucleic acid sequence encoding same, wherein said base editor comprises a programmable DNA-binding domain and a deaminase domain, and wherein said guide polynucleotide directs said adenosine base editor to effect a single nucleobase modification at a splice site of the androgen receptor (AR) gene.

[0023] In certain aspects, provided herein is a method of modifying an androgen receptor (AR) gene, comprising contacting the AR gene or a regulatory element thereof with (i) a base editor or a nucleic acid sequence encoding same, and (ii) a guide polynucleotide or a nucleic acid sequence encoding same, wherein said base editor comprises a programmable DNA-binding domain and a deaminase domain, and wherein said guide polynucleotide directs said adenosine base editor to result in a single nucleobase modification in an androgen receptor (AR) gene in said subject, wherein said single nucleobase modification results in a premature stop codon in said AR gene.

[0024] In one embodiment, the deaminase is an adenosine deaminase comprising an amino acid substitution at amino acid position 82 or 166 in the numbering of SEQ ID NO: 2. In one embodiment, the deaminase is a cytidine deaminase. In one embodiment, the single nucleobase modification is a C to T modification. In one embodiment, the nucleobase modification results in a CAG-TAG codon change in the AR gene. In one embodiment, the codon change is in exon 1 or exon 2 of the AR gene. In one embodiment, the single nucleobase modification is an A to G modification. In one embodiment, the A to G nucleobase modification is in the splice acceptor site of the AR gene.

[0025] In one embodiment, the splice site is a splice acceptor site located 5' of the exon of the AR gene. In one embodiment, the exon of the AR gene is exon 2 corresponding to SEQ ID NO: 4 or a variant thereof. In one embodiment, the splice site is a splice donor site located 3' of the exon of the AR gene. In one embodiment, the exon of the AR gene is exon 1 corresponding to SEQ ID NO: 4 or a variant thereof.

[0026] In one embodiment, the guide polynucleotide comprises a nucleic acid sequence selected from Table 41 A or 41 B. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence complementary to a splice acceptor nucleic acid sequence or a splice donor nucleic acid sequence of an AR gene. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence selected from Table 41 A or 41 B. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of 5'-ACUUACCGCAUGUCCCCGUAAGG-3', 5'-AGUGCAGUUAGGGCUGGGAAGGG-3', 5'-AAGUGCAGUUAGGGCUGGGAAGG-3', and complementary strands thereof.

[0027] In some embodiments, the contact is within a cell. In some embodiments, the single nucleobase modification results in less than 15% indels in the genome of the cell. In some embodiments, the single nucleobase modification results in less than 5% indels in the genome of the cell. In some embodiments, the single nucleobase modification results in less than 2% indels in the genome of the cell. In some embodiments, the cell is a mammalian cell or a human cell. In some embodiments, the cell is a central nervous system cell. In some embodiments, the cell is a motor neuron.

[0028] In some embodiments, the contacting is in a population of cells. In some embodiments, after the contacting, at least 40% of the cell population comprises a single nucleobase modification.

[0029] In some embodiments, after said contacting, at least 50% of the cell population comprises a single nucleobase modification. In some embodiments, after said contacting, at least 60% of the cell population comprises a single nucleobase modification. In some embodiments, after said contacting, at least 85% of the cell population is viable. In some embodiments, the population of cells is mammalian or human cells. In some embodiments, the population of cells is central nervous system cells. In some embodiments, the population of cells is motor neurons.

[0030] In one embodiment, the adenosine deaminase comprises TadA deaminase, hi one embodiment, the adenosine deaminase is TadA7.10.

[0031] In one embodiment, the adenosine deaminase is TadA comprising a V28S mutation or a T166R mutation, or a mutation corresponding thereto, as numbered in SEQ ID NO: 2. In one embodiment, the adenosine deaminase comprises one or more mutations selected from Y147T, Y147R, Q154S, Y123H, and Q154R, or a mutation corresponding thereto, as numbered in SEQ ID NO: 2. In one embodiment, the adenosine deaminase is selected from the group consisting of Y147T + Q154R; Y147T + Q154S; Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + 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 their corresponding mutations.

[0032] In some embodiments, the adenosine deaminase comprises a C-terminal deletion beginning at a residue selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 157. In some embodiments, the adenosine deaminase comprises a TadA dimer. In some embodiments, the adenosine deaminase comprises an adenosine deaminase monomer.

[0033] In various aspects and embodiments described above, the polynucleotide-programmable DNA-binding domain is a Cas9 domain. In some embodiments, the Cas9 domain is a Cas9 nickase domain. In some embodiments, the Cas9 domain comprises a SpCas9 domain. In some embodiments, the SpCas9 domain contains a D10A and / or H840A amino acid substitution or a corresponding amino acid substitution as numbered in SEQ ID NO: 1. In some embodiments, the Cas9 domain comprises a SaCas9 domain. In some embodiments, the Cas9 domain has specificity for an engineered PAM. In some embodiments, the Cas9 domain has specificity for a PAM sequence selected from the group consisting of NGG, NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, and NGC, where N is A, G, C, or T and R is A or G.

[0034] In some aspects, provided herein are populations of cells produced by the methods described herein.

[0035] In certain aspects, provided herein is a base editor system comprising: (i) a base editor or a nucleic acid sequence encoding the same; and (ii) a guide polynucleotide or a nucleic acid sequence encoding the same, wherein the base editor comprises a programmable DNA-binding domain and a deaminase domain, and the guide polynucleotide directs the adenosine base editor to modify a single nucleobase at a splice site of superoxide dismutase 1 (SOD1).

[0036] In certain aspects, provided herein is a base editor system comprising: (i) a base editor or a nucleic acid sequence encoding same; and (ii) a guide polynucleotide or a nucleic acid sequence encoding same, wherein said base editor comprises a programmable DNA-binding domain and a deaminase domain; wherein said guide polynucleotide directs said adenosine base editor to result in a single nucleobase modification in a superoxide dismutase 1 (SOD1) gene, wherein said single nucleobase modification results in a premature stop codon in the SOD1 gene.

[0037] In one embodiment, the deaminase is an adenosine deaminase comprising an amino acid substitution at amino acid position 82 or 166 according to the numbering in SEQ ID NO: 2. In one embodiment, the deaminase is a cytidine deaminase. In one embodiment, the single nucleobase modification is an A to G modification. In one embodiment, the A to G nucleobase modification is in a splice acceptor site of the SOD1 gene. In one embodiment, the splice site is a splice acceptor site 5' of an exon of the SOD1 gene. In one embodiment, the exon of the SOD1 gene is exon 3 corresponding to SEQ ID NO: 3 or a variant thereof. In one embodiment, the exon 3 of the SOD1 gene is adjacent to a splice acceptor AG or a variant thereof at nucleotide position 6828 of the SOD1 polynucleotide sequence according to the numbering in SEQ ID NO: 3. In one embodiment, alternative splicing of the SOD1 transcript generates a transcript or a variant thereof lacking exons 3 to 5 of the SOD1 gene corresponding to SEQ ID NO: 3. In one embodiment, the exon of the SOD1 gene is exon 4 corresponding to SEQ ID NO: 3 or a variant thereof.

[0038] In one embodiment, the guide polynucleotide comprises a nucleic acid sequence complementary to a splice acceptor nucleic acid sequence or a splice donor nucleic acid sequence of the SOD1 gene. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence selected from Table 19 or Table 23. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of 5'-UUAAAGGAAAGUAAUGGACCAGU-3', 5'-UAAAUAGGCUGUACCAGUGCAGG-3', 5'-UUCAUUAUUAGGCAUGUUGGAGA-3', 5'-AAAUAGGCUGUACCAGUGCAGGU-3', 5'-UAUUAGGCAUGUUGGAGACUUGG-3', and complementary sequences thereof.

[0039] In certain embodiments, provided herein is a base editor system comprising: (i) a base editor or a nucleic acid sequence encoding same; and (ii) a guide polynucleotide or a nucleic acid sequence encoding same, wherein said base editor comprises a programmable DNA-binding domain and a deaminase domain; and wherein said guide polynucleotide directs said adenosine base editor to effect a single nucleobase modification at a splice site of the androgen receptor (AR) gene.

[0040] In certain embodiments, provided herein is a base editor system comprising: (i) a base editor or a nucleic acid sequence encoding same; and (ii) a guide polynucleotide or a nucleic acid sequence encoding same, wherein said adenosine base editor comprises a programmable DNA-binding domain and a deaminase domain; and wherein said guide polynucleotide directs said adenosine base editor to effect a single nucleobase modification in an androgen receptor (AR) gene in a subject, wherein said single nucleobase modification results in a premature stop codon in the AR gene.

[0041] In one embodiment, the deaminase is an adenosine deaminase containing an amino acid substitution at amino acid position 82 or 166 in the numbering of SEQ ID NO: 2. In one embodiment, the deaminase is a cytidine deaminase. In one embodiment, the single nucleobase modification is a C to T modification. In one embodiment, the single nucleobase modification results in a CAG-TAG codon change in the AR gene. In one embodiment, the codon change is in exon 1 or exon 2 of the AR gene corresponding to SEQ ID NO: 4, or a variant thereof. In one embodiment, the single nucleobase modification is an A to G modification. In one embodiment, the A to G nucleobase modification is in a splice acceptor site of the AR gene. In one embodiment, the splice site is a splice acceptor site located 5' of an exon of the AR gene. In one embodiment, the exon of the AR gene is exon 2 corresponding to SEQ ID NO: 4, or a variant thereof. In one embodiment, the splice site is a splice donor site located 3' of an exon of the AR gene. In one embodiment, the exon of the AR gene is exon 1 corresponding to SEQ ID NO: 4 or a variant thereof.

[0042] In one embodiment, the guide polynucleotide comprises a nucleic acid sequence complementary to a splice acceptor nucleic acid sequence or a splice donor nucleic acid sequence of the AR gene. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence selected from Table 41 A or 41 B. In one embodiment, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of 5'-ACUUACCGCAUGUCCCCGUAAGG-3', 5'-AGUGCAGUUAGGGCUGGGAAGGG-3', 5'-AAGUGCAGUUAGGGCUGGGAAGG-3', and complementary strands thereof.

[0043] In one embodiment, the adenosine deaminase comprises TadA deaminase. In one embodiment, the adenosine deaminase is TadA7.10. In one embodiment, the adenosine deaminase is TadA comprising a V28S mutation or a T166R mutation, or a mutation corresponding thereto, as numbered in SEQ ID NO: 2. In one embodiment, the adenosine deaminase comprises one or more of Y147T, Y147R, Q154S, Y123H, and Q154R, or a mutation corresponding thereto, as numbered in SEQ ID NO: 2. In one embodiment, the adenosine deaminase is selected from the group consisting of Y147T + Q154R; Y147T + Q154S; Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + 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 a combination of mutations corresponding thereto.

[0044] In some embodiments, the adenosine deaminase comprises a C-terminal deletion beginning at a residue selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 157. In some embodiments, the adenosine deaminase comprises a TadA dimer. In some embodiments, the adenosine deaminase comprises an adenosine deaminase monomer.

[0045] In some embodiments, the polynucleotide-programmable DNA-binding domain is a Cas9 domain. In some embodiments, the Cas9 domain is a Cas9 nickase domain. In some embodiments, the Cas9 domain comprises a SpCas9 domain. In some embodiments, the SpCas9 domain contains a D10A and / or H840A amino acid substitution or a corresponding amino acid substitution in the numbering of SEQ ID NO: 1. In some embodiments, the Cas9 domain comprises a SaCas9 domain. In some embodiments, the Cas9 domain has specificity for an engineered PAM. In some embodiments, the Cas9 domain has specificity for a PAM sequence selected from the group consisting of NGG, NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, and NGC, where N is A, G, C, or T and R is A or G.

[0046] In one aspect, provided herein is a vector comprising a nucleic acid sequence encoding a DNA-binding domain programmable by the polynucleotide in the base editor system described herein and a nucleic acid sequence encoding the adenosine deaminase domain. In one embodiment, the vector further comprises a nucleic acid sequence encoding the guide polynucleotide. In one embodiment, the vector is a viral vector.

[0047] In one aspect, provided herein is a cell comprising a base editor system or vector described herein. In some embodiments, the cell is a mammalian cell, a human cell, or a motor neuron. In some embodiments, the cell is in vivo, ex vivo, or in vitro. In some embodiments, the cell is an autologous cell isolated from a subject. In some embodiments, the cell is an allogeneic cell.

[0048] In some aspects, provided herein is a population of cells comprising a base editor system or vector described herein. In some embodiments, the population of cells is a mammalian cell, a human cell, or a motor neuron. In some embodiments, the population of cells is in vivo, ex vivo, or in vitro. In some embodiments, the cells are autologous cells isolated from a subject.

[0049] In certain aspects, provided herein are pharmaceutical compositions comprising a base editor, vector, or cell described herein and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition described herein further comprises a lipid. In another embodiment, the pharmaceutical composition described herein further comprises a virus.

[0050] In some aspects, provided herein are kits comprising the base editor systems or vectors described herein.

[0051] In various embodiments of the methods described herein, at least one nucleotide of the guide polynucleotide comprises a non-natural modification. In various embodiments of the methods described herein, at least one nucleotide of the nucleic acid sequence comprises a non-natural modification. In various embodiments of the methods described herein, at least one nucleotide of the nucleic acid sequence of the base editor system comprises a non-natural modification. In some embodiments, the non-natural modification is a chemical modification. In some embodiments, the chemical modification is 2'-O-methylation. In some embodiments, the nucleic acid sequence comprises phosphorothioate.

[0052] The description and examples herein illustrate in detail embodiments of the present disclosure. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will recognize that the present disclosure has numerous variations and modifications that fall within its scope.

[0053] The practice of the embodiments disclosed herein will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, within the skill of those in the art. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds.); the series Methods in Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0054] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0055] While various 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, while the present disclosure may be described in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment. Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0056] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description, which sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and by consideration of the accompanying drawings, which are described below.

[0057] definition The following definitions supplement those in the art and are directed to this application and are not to be construed as attributing any related or unrelated matter, such as commonly owned patents or applications. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing of the present disclosure, the preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art to which this invention belongs.The following references provide those skilled in the art with the general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The HarperCollins Dictionary of Biology (1991).

[0059] In this application, the use of the singular includes the plural unless specifically stated otherwise. It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" and is understood to be inclusive unless specifically stated otherwise. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is non-limiting.

[0060] As used in the specification and claims, the terms "comprising" (and any of its forms, such as "comprise" and "comprises"), "having" (and any of its forms, such as "have" and "has"), "including" (and any of its forms, such as "include" and "includes"), or "containing" (and any of its forms, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0061] The term "about" or "approximately" means within an acceptable error range for a particular value, as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations, according to practice in the art. 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 a value within the same order of magnitude, e.g., within 5-fold or within 2-fold. When a particular value is described in the application and claims, unless otherwise specified, the term "about" meaning within an acceptable error range for that particular value should be presumed.

[0062] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0063] References in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0064] An "abasic base editor" refers to an agent that can excise a nucleobase and insert a DNA nucleobase (A, T, C, or G). An abasic base editor comprises a nucleic acid glycosylase polypeptide or a fragment thereof. In one embodiment, the nucleic acid glycosylase is a mutant human uracil DNA glycosylase or an active fragment thereof that contains Asp at amino acid 204 of the sequence below, or the corresponding position in uracil DNA glycosylase (e.g., substituting Asn at amino acid 204), and has cytosine-DNA glycosylase activity. In one embodiment, the nucleic acid glycosylase is a mutant human uracil DNA glycosylase or an active fragment thereof that contains Ala, Gly, Cys, or Ser at amino acid 147 of the sequence below, or the corresponding position in uracil DNA glycosylase (e.g., substituting Tyr at amino acid 147), and has thymine-DNA glycosylase activity. The sequence of an exemplary human uracil-DNA glycosylase, isoform 1, is as follows: 1 mgvfclgpwg lgrklrtpgk gplqllsrlc gdhlqaipak kapagqeepg tppssplsae 61 qldriqrnka aallrlaarn vpvgfgeswk khlsgefgkp yfiklmgfva eerkhytvyp 121 pphqvftwtq mcdikdvkvv ilgqdp y hgp nqahglcfsv qrpvppppsl eniykelstd 181 iedfvhpghg dlsgwakqgv lll n avltvr ahqanshker gweqftdavv swlnqnsngl 241 vfllwgsyaq kkgsaidrkr hhvlqtahps p l svy r gffg crhfsktnel lqksgkkpid 301 wkel

[0065] The sequence of human uracil-DNA glycosylase, isoform 2 is as follows: 1 migqktlysf fspsparkrh apspepavqg tgvagvpees gdaaaipakk apagqeepgt 61 ppssplsaeq ldriqrnkaa allrlaarnv pvgfgeswkk hlsgefgkpy fiklmgfvae 121 erkhytvypp phqvftwtqm cdikdvkvvi lgqdp y hgpn qahglcfsvq rpvppppsle 181 niykelstdi edfvhpghgd lsgwakqgvl ll n avltvra hqanshkerg weqftdavvs 241 wlnqnsnglv fllwgsyaqk kgsaidrkrh hvlqtahpsp l svy r gffgc rhfsktnell 301 qksgkkpidw kel

[0066] In other embodiments, the abasic editor is any of the abasic editors described in PCT / JP205 / 080958 and US20170321210, which are incorporated herein by reference. In certain embodiments, the abasic editor comprises a mutation at the bolded, underlined position in the above sequence, or at the corresponding amino acid in any other abasic editor or uracil deglycosylase known in the art. In one embodiment, the abasic editor comprises a mutation at Y147, N204, L272, and / or R276 or a corresponding position. In another embodiment, the abasic editor comprises a Y147A or Y147G mutation or a corresponding mutation. In another embodiment, the abasic editor comprises an N204D mutation or a corresponding mutation. In another embodiment, the abasic editor comprises an L272A mutation or a corresponding mutation. In another embodiment, the abasic editor comprises an R276E or R276C mutation or a corresponding mutation.

[0067] "Adenosine deaminase" refers to a polypeptide or fragment thereof that can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine or deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., engineered adenosine deaminases, evolved adenosine deaminases) can be from any organism, such as bacteria.

[0068] In some embodiments, the adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase is a TadA variant. In some embodiments, the TadA variant is TadA*8. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is non-naturally occurring. For example, in some embodiments, the deaminase or deaminase domain has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identity to a naturally occurring deaminase. For example, deaminase domains are described in International PCT Application Nos. PCT / 2007 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is incorporated by reference herein in its entirety.Also, Komor, AC, et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, AC, et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017) ), and Rees, HA, et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 Dec;19(12):770-788. doi: See also 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0069] The wild-type TadA (wt) adenosine deaminase has the following sequence (also referred to as the TadA reference sequence): MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 2).

[0070] In some embodiments, the adenosine deaminase comprises a modification in the following sequence: MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG LHDPTAHAEI MALRQGGLVM QNYRLIDATL YVTFEPCVMC AGAMIHSRIG RVVFGVRNAK TGAAGSLMDV LHYPGMNHRV EITEGILADE CAALLCYFFR MPRQVFNAQK KAQSSTD (also known as TadA*7.10).

[0071] In some embodiments, TadA*7.10 comprises at least one modification. In some embodiments, TadA*7.10 comprises modifications at amino acids 82 and / or 166. In certain embodiments, variants of the reference sequence comprise one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. The modification Y123H is also referred to herein as H123H (the modification H123Y in TadA*7.10 reverted to Y123H(wt)). In other embodiments, the variant of the TadA*7.10 sequence 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.

[0072] In other embodiments, the invention provides adenosine deaminase variants containing deletions, e.g., TadA*8, including C-terminal deletions beginning at residue 149, 150, 151, 152, 153, 154, 155, 156, or 157. In other embodiments, the adenosine deaminase variant is a TadA (e.g., TadA*8) monomer that contains one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant is a TadA (e.g., TadA*8) monomer comprising a combination of modifications 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.

[0073] In still other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each having one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8) each having a combination of modifications 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.

[0074] In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type TadA adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type TadA adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising a combination of modifications 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.

[0075] In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8) that includes one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising a combination of the following modifications: Y147T + Q154R; Y147T + Q154S; Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y147R + Q154R + Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76Y; V82S + Y123H + Y147R + Q154R; or I76Y + V82S + Y123H + Y147R + Q154R.

[0076] In one embodiment, the adenosine deaminase is TadA*8 comprising or consisting essentially of the following sequence, or a fragment thereof having adenosine deaminase activity: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0077] In some embodiments, TadA*8 is truncated. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to full-length TadA*8. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to full-length TadA*8. In some embodiments, the adenosine deaminase variant is full-length TadA*8.

[0078] In certain embodiments, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from one of the following:

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

[0080] Escherichia coli TadA: MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD

[0081] E. coli TadA (N-terminal truncated): MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD

[0082] Staphylococcus aureus (S. aureus) TadA: MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN

[0083] Bacillus subtilis (B. subtilis) TadA: MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE

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

[0085] Shewanella putrefaciens (S. putrefaciens) TadA: MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE

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

[0087] Caulobacter crescentus (C. crescentus) TadA: MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIIAAMRAAAKLGNYRLTDLTVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI

[0088] Geobacter sulfurreducens (G. sulfurreducens) TadA: MSSLKKTPIRDDAYWMGKAIREAAKAAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP

[0089] TadA*7.10 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0090] Additional TadA7.10 or TadA7.10 variants contemplated as components of heterodimers with TadA*8 include:

[0091] GSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0092] TadA7.10 CP65 TAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDP

[0093] TadA7.10 CP83 YRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQN

[0094] TadA7.10 CP136 MNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPG

[0095] TadA7.10 C-truncated GSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFN

[0096] TadA7.10 C-truncated 2 GSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQ

[0097] TadA7.10 delta59-66+C-truncated GSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFN

[0098] TadA7.10 delta 59-66 GSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD.

[0099] In some embodiments, the adenosine deaminase variant comprises a modification in TadA7.10. In some embodiments, TadA7.10 comprises a modification at amino acid 82 or 166. In certain embodiments, the variant of the reference sequence comprises one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R. In other embodiments, the adenosine deaminase variant comprises a combination of modifications selected from the group consisting of: Y147R + Q154R + Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y147T + Q154R; Y147T + Q154S; and Y123H + Y147R + Q154R + I76Y.

[0100] In other embodiments, the invention provides adenosine deaminase variants that include deletions, for example, TadA7.10, that include C-terminal deletions beginning at residues 149, 150, 151, 152, 153, 154, 155, 156, or 157. In other embodiments, the adenosine deaminase variant is a TadA monomer that includes one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, Q154R. In other embodiments, the adenosine deaminase variant is a monomer comprising the following modifications: Y147R + Q154R + Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y147T + Q154R; Y147T + Q154S; and Y123H + Y147R + Q154R + I76Y. In yet other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains, each having one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type adenosine deaminase domain or a TadA7.10 domain and an adenosine deaminase variant domain comprising one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA7.10 domain and an adenosine deaminase variant of TadA7.10 comprising the following modifications: Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y147T + Q154R; Y147T + Q154S; and Y123H + Y147R + Q154R + I76Y.

[0101] "Administering" refers herein to providing one or more compositions described herein to a patient or subject. By way of example, and not limitation, administration, e.g., injection, of a composition can be by intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more such routes can be used. Parenteral administration can be by, for example, bolus injection or by gradual perfusion over time. In some embodiments, parenteral administration includes intravascular, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, intradermal, intraperitoneal, transtracheal, subcutaneous, subkeratinous, intraarticular, intracapsular, subarachnoid, and intrasternal infusion or injection. Alternatively, or concurrently, administration can be by the oral route.

[0102] By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0103] "Alteration" refers to a change (e.g., an increase or decrease) in the structure, expression level, or activity of a gene or polypeptide, as detected by standard art known methods, such as those described herein. As used herein, alteration includes a change in the sequence of a polynucleotide or polypeptide, or a change in expression level, e.g., a 10% change, a 25% change, a 40% change, a 50% change, or more change in expression level.

[0104] "Ameliorate" means to lessen, inhibit, attenuate, reduce, arrest, or stabilize the occurrence or progression of a disease.

[0105] "Analog" refers to a molecule that is not identical but has similar functional or structural characteristics. For example, a polynucleotide or polypeptide analog retains the biological activity of the corresponding naturally occurring polynucleotide or polypeptide while possessing certain modifications that enhance the analog's function compared to the naturally occurring polynucleotide or polypeptide. Such modifications can increase the analog's DNA affinity, efficiency, specificity, protease or nuclease resistance, membrane permeability, and / or half-life, for example, without altering ligand binding. Analogs can include non-naturally occurring polynucleotides or amino acids.

[0106] "Base editor (BE)" or "nucleobase editor (NBE)" refers to an agent that binds to a polynucleotide and has nucleobase-modifying activity. In various embodiments, the base editor comprises a nucleobase-modifying polypeptide (e.g., a deaminase) and a nucleic acid-programmable nucleotide-binding domain together with a guide polynucleotide (e.g., a guide RNA). In various embodiments, the agent is a biomolecular complex that includes a protein domain with base-editing activity, i.e., a domain that can modify bases (e.g., A, T, C, G, U) in a nucleic acid molecule (e.g., DNA). In some embodiments, the polynucleotide-programmable DNA-binding domain is fused or linked to a deaminase domain. In one embodiment, the agent is a fusion protein that includes a domain with base-editing activity. In another embodiment, the protein domain with base-editing activity is linked to a guide RNA (e.g., via an RNA-binding motif on the guide RNA and an RNA-binding domain fused to a deaminase). In certain embodiments, the domain with base-editing activity can deaminate a base in a nucleic acid molecule. In certain embodiments, the base editor can deaminate one or more bases in a DNA molecule. In some embodiments, the base editor can deaminate adenosine (A) in DNA. In some embodiments, the base editor is an adenosine base editor (ABE).

[0107] "Cytidine deaminase" refers to a polypeptide or fragment thereof capable of catalyzing a deamination reaction that converts an amino group to a carbonyl group. In some embodiments, the cytidine deaminase has at least about 85% identity to an APOBEC or AID. In one embodiment, the cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine. PmCDA1 from Petromyzon marinus (Petromyzon marinus cytosine deaminase 1, "PmCDA1"), or AID (activation-induced cytidine deaminase; AICDA) from mammals (e.g., human, pig, cow, horse, monkey, etc.), and APOBEC are exemplary cytidine deaminases.

[0108] In some embodiments, the base editor is a reprogrammable base editor fused to a deaminase (e.g., adenosine deaminase or cytidine deaminase). In some embodiments, the base editor is Cas9 fused to a deaminase (e.g., adenosine deaminase or cytidine deaminase). In some embodiments, the base editor is a nuclease-inactive Cas9 (dCas9) fused to a deaminase (e.g., adenosine deaminase or cytidine deaminase). In some embodiments, the Cas9 is a circular permutant Cas9 (e.g., spCas9 or saCas9). Circular permutant Cas9s are known in the art and are described, for example, in Oakes et al., Cell 176, 254-267, 2019. In some embodiments, the base editor is fused to a base excision repair inhibitor, such as a UGI domain or a dISN domain. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase and an inhibitor of base excision repair, such as a UGI domain or a dISN domain. In other embodiments, the base editor is an abasic base editor.

[0109] In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the adenosine deaminase is evolved from TadA. In some embodiments, the base editor of the present invention comprises a napDNAbp domain with an internally fused catalytic (e.g., deaminase) domain. In some embodiments, the napDNAbp is Cas12a (Cpf1) with an internally fused deaminase domain. In some embodiments, the napDNAbp is Cas12b (c2c1) with an internally fused deaminase domain. In some embodiments, the napDNAbp is Cas12c (c2c3) with an internally fused deaminase domain. In some embodiments, the napDNAbp is Cas12d (CasX) with an internally fused deaminase domain. In some embodiments, the napDNAbp is Cas12e (CasY) with an internally fused deaminase domain. In some embodiments, the napDNAbp is Cas12g with an internally fused deaminase domain. In some embodiments, the napDNAbp is Cas12h with an internally fused deaminase domain. In some embodiments, the napDNAbp is Cas12i with an internally fused deaminase domain. In some embodiments, the base editor is a catalytically dead Cas12 (dCas12) fused to a deaminase domain. In some embodiments, the base editor is a Cas12 nickase (nCas12) fused to a deaminase domain.

[0110] In some embodiments, base editors are generated by cloning an adenosine deaminase variant (e.g., TadA*8) into a scaffold comprising a circularly permuted Cas9 (e.g., spCAS9 or saCAS9) and a bipartite nuclear localization sequence (e.g., ABE8). Circularly permuted Cas9s are known in the art and are described, for example, in Oakes et al., Cell 176, 254-267, 2019. Exemplary circular permutations are described below, where bolded sequences indicate sequences derived from Cas9, italicized sequences indicate linker sequences, and underlined sequences indicate the bipartite nuclear localization sequence.

[0111] CP5 (with MSP “NGC=Pam Variant with mutations Regular Cas9 likes NGG” PID=Protein Interacting Domain and “D10A” nickase): TIFF0007753096000001.tif170164

[0112] In some embodiments, ABE8 is selected from a base editor from Tables 6-9, 13, or 14 below. In some embodiments, ABE8 comprises an adenosine deaminase variant evolved from TadA. In some embodiments, the adenosine deaminase variant of ABE8 is a TadA*8 variant as set forth in Tables 7, 9, 13, or 14 below. In some embodiments, the adenosine deaminase variant is a TadA*7.10 variant (e.g., TadA*8) that includes one or more modifications selected from the group of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In various embodiments, ABE8 is selected from the group consisting of Y147T + Q154R; Y147T + Q154S; Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y147R + Q154R +Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76Y; V82S + Y123H + Y147R + and I76Y + V82S + Y123H + Y147R + Q154R (e.g., TadA*7.10 (e.g., TadA*8) having a combination of modifications selected from the group consisting of I76Y + V82S + Y123H + Y147R + Q154R). In some embodiments, ABE8 is a monomeric construct. In some embodiments, ABE8 is a heterodimeric construct. In some embodiments, the ABE8 base editor comprises the following sequence: MSEVEFSHEYWMRHALTLAKRADEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0113] In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-associated (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 base excision repair inhibitor is a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the base excision repair inhibitor is an inosine base excision repair inhibitor.

[0114] Details of base editors are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US 2016 / 058344 (WO 2017 / 070632), each of which is incorporated by reference in its entirety. Komor, AC, et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, AC, et al. 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: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 Dec;19(12):770-788. doi: See also 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0115] By way of example, a cytidine base editor used in the base editing compositions, systems, and methods described herein has the following nucleic acid sequence (8877 base pairs) (Addgene, Watertown, MA.; Komor AC, et al., 2017, Sci Adv., 30;3(8):eaao4774. doi: 10.1126 / sciadv.aao4774). Polynucleotide sequences having at least 95% or greater identity to the BE4 nucleic acid sequence are also encompassed.

[0116] 1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagctcagag 421 actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 481 gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 541 gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 601 aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 661 acctggtttc tcagctggag cccatgcggc gaatgtagta gggccatcac tgaattcctg 721 tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 781 ccccgcaatc gacaaggcct gcgggatttg atctcttcag gtgtgactat ccaaattatg 841 actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 901 gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 961 atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1021 ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1081 gccaccgggt tgaaatctgg tggttcttct ggtggttcta gcggcagcga gactcccggg 1141 acctcagagt ccgccacacc cgaaagttct ggtggttctt ctggtggttc tgataaaaag 1201 tattctattg gtttagccat cggcactaat tccgttggat gggctgtcat aaccgatgaa 1261 tacaaagtac cttcaaagaa atttaaggtg ttggggaaca cagaccgtca ttcgattaaa 1321 aagaatctta tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg 1381 aaacgaaccg ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa 1441 atttttagca atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc 1501 ttccttgtcg aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat 1561 gaggtggcat atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac 1621 tcaactgata aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc 1681 cgtgggcact ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg 1741 1801 ggcgtggatg cgaaggctat tcttagcgcc cgcctctcta aatcccgacg gctagaaaac 1861 ctgatcgcac aattacccgg agagaagaaa aatgggttgt tcggtaacct tagatcgctc 1921 tcactaggcc tgacaccaaa ttttaagtcg aacttcgact tagctgaaga tgccaaattg 1981 cagcttagta aggacacgta cgatgacgat ctcgacaatc tactggcaca attggagat 2041 cagtagcgg acttatttt ggctgccaa aaccttagcg atgcaatcct cctactcgac 2101 atactgagag ttatactga gattaccaag gcgccgtta ccgctcaat gatcaaagg 2161 tacgatgaac atcaccaga cttgacactt ctcaagccc tagtccgtca gcaactgcct 2221 gagaaata aggaaatatt cttgatcag tcgaaaaacg ggtacgcagg ttatattgac 2281 ggcggagcga gtcagagga attctacaag tttatcaac ccatattaga gagatggat 2341 gggacggaag agttgcttgt aaaaccaat cgcgagatc tactgcgaaa gcagcggact 2401 ttcgacacg gtagcattcc acatcaatc cacttaggcg aattgcatgc tatacttaga 2461 aggcaggagg atttttacc gttcccaaa gatacgtg aaagattga gaaaatccta 2521 acctttcgca taccttacta tgtgggaccc ctggcccgag ggaactcg gttcgcatgg 2581 atgacagaa agtccgaaga aacgattact ccatggaatt tgaggaagt tgtcgataaa 2641 gtgcgtcag ctcaatcgtt catcgagagg atgaccact ttgacagaa tttaccgaac 2701 gaaaagtat tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc 2761 acgaaagtta agtatgtcac tgaggcatg cgtaaacccg cctttctaag cggagacag 2821 aagaaagcaa tagtagatct gttattcaag accaccgca aagtgacagt tagcattg 2881 aagaggact actttaagaa attgaatgc tcgattctg tcgagatc cggggtagaa 2941 gatcgattta atgcgtcact tgtacgtat catgacctcc taagataat taagataag 3001 gacttcctgg ataacgaaga gatgagat atcttagaag atagtgtt gactcttacc 3061 ctctttgaag atcgggaat gattgaggaa agactaaaa catacgctca cctgttcgac 3121 gataagtta tgaacagtt aaagaggcgt cgctatacgg gctgggacg attgtcgcgg 3181 aaacttatca acggataag agacaagcaa agtggtaaa ctattctcga ttttctaaag 3241 agcgacggct tcgccatag gaactttatg cagctgatcc atgatgactc tttaccttc 3301 aagaggata tacaaaggc acaggttcc ggacaagggg actcattgca cgaacatatt 3361 gcgaatcttg ctggttcgcc agccatcaa aagggcatac tccacagt caagtagtg 3421 gatgagctag ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca 3481 cgcgaaaatc aaacgactca gaagggcaa aaaaacagtc gagagcggat gaagagaata 3541 gaagggta ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc 3601 3661 3721 tccttttga aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga 3781 gggaaaagtg acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg 3841 3901 aggggtggct tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc 3961 cgccaaatca caaagcatgt tgcacagata ctagattccc gaatgaatac gaatacgac 4021 gagaacgata agctgattcg ggaagtcaaa gtaatcactt taagtcaaa attggtgtcg 4081 4141 cacgacgctt atcttaatgc cgtcgtaggg accgcactca ttaagaata cccgaagcta 4201 gaagtgagt ttgtgtatgg tgattacaaa gtttatgacg tccgtagat gatcgcgaaa 4261 agcgacagg agataggcaa ggctacagcc aaatactct tttattctaa cattatgaat 4321 ttctttaga cggaatcac tctgcaac gagagagat gcaacgacc tttattgac 4381 accaatgggg agacaggtga aatcgtatgg gataagggcc gggactcgc gacggtgaga 4441 aaagttttgt ccatgcccca agtcacata gtaagaaaa ctgaggtgca gaccggaggg 4501 tttcaagg atcgattct tccaaaagg atagtgata agctcatcgc tcgtaaaag 4561 gactgggacc cgaaaagta cggtggctc gatagcccta cagttgccta ttctgtccta 4621 gtagtggcaa aagttgagaa gggaaaatcc aagaactga agtcagtcaa agaatttg 4681 gggataacga ttatggagcg ctcgtctttt gaaaagaacc ccatcgactt ccttgaggcg 4741 aaggttaca aggaagtaa aaaggatctc ataatttaac taccaagta tagtctgttt 4801 gagttagaaa atggccgaaa acggatgttg gctagcgccg gagagcttca aaagggaac 4861 gaactcgcac taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag 4921 ttgaaaggtt cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat 4981 tatctcgacg aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat 5041 gccaatctgg acaaagtatt aagcgcatac aacaagcaca gggataaacc catacgtgag 5101 caggcggaaa atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc 5161 aagtattttg acacaacgat agatcgcaaa cgatacactt ctaccaagga ggtgctagac 5221 gcgacactga ttcaccaatc catcacggga ttatatgaaa ctcggataga tttgtcacag 5281 cttgggggtg actctggtgg ttctggagga tctggtggtt ctactaatct gtcagatatt 5341 attgaaaagg agaccggtaa gcaactggtt atccaggaat ccatcctcat gctcccagag 5401 gaggtggaag aagtcattgg gaacaagccg gaaagcgata tactcgtgca caccgcctac 5461 gacgagagca ccgacgagaa tgtcatgctt ctgactagcg acgcccctga atacaagcct 5521 tgggctctgg tcatacagga tagcaacggt gagaacaaga ttaagatgct ctctggtggt 5581 tctggaggat ctggtggttc tactaatctg tcagatatta ttgaaaagga gaccggtaag 5641 caactggtta tccaggaatc catcctcatg ctcccagagg aggtggaaga agtcattggg 5701 aacaagccgg aaagcgatat actcgtgcac accgcctacg acgagagcac cgacgagaat 5761 gtcatgcttc tgactagcga cgcccctgaa tacaagcctt gggctctggt catacaggat 5821 agcaacggtg agaacaagat taagatgctc tctggtggtt ctcccaagaa gaagaggaaa 5881 gtctaaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg 5941 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 6001 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 6061 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 6121 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 6181 ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc 6241 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 6301 taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 6361 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 6421 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 6481 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 6541 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 6601 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 6661 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 6721 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 6781 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 6841 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 6901 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 6961 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 7021 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 7081 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagt ggtagctctt 7141 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 7201 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 7261 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa agatcttca 7321 cctagatcct ttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 7381 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgctat 7441 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 7501 taccatctgg ccccagtgct gcaatgac cgcgagaccc acgctcaccg gctccagatt 7561 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 7621 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 7681 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 7741 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 7801 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 7861 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 7921 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 7981 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 8041 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 8101 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 8161 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaagg 8221 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 8281 gcattatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaata 8341 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg 8401 gagatcgatc tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat 8461 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca 8521 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg 8581 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat 8641 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt 8701 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 8761 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 8821 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatc

[0117] BE4 amino acid sequence:

[0118] By way of example, an adenine base editor (ABE) used in the base editing compositions, systems, and methods described herein has the nucleic acid sequence (8877 base pairs) provided below (Addgene, Watertown, MA.; Gaudelli NM, et al., Nature. 2017 Nov 23;551(7681):464-471. doi: 10.1038 / nature24644; Koblan LW, et al., Nat Biotechnol. 2018 Oct;36(9):843-846. doi: 10.1038 / nbt.4172.). Polynucleotide sequences having at least 95% or greater identity to the ABE nucleic acid sequence are also encompassed.

[0119] ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT GACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGATTTCCAAGTCTCCACCCCATTG ACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCC ATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGT CAGATCCGCTAGAGATCCGCGGCCGCTAATACGACTCACTATAGGGAGAGCCGCCACCATGAAACGGACA GCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTCTCTGAAGTCGAGTTTAGCCACGAGT ATTGGATGAGGCACGCACTGACCCTGGCAAAGCGAGCATGGGATGAAAGAGAAGTCCCCGTGGGCGCCGT GCTGGTGCACAACAATAGAGTGATCGGAGAGGGATGGAACAGGCCAATCGGCCGCCACGACCCTACCGCA CACGCAGAGATCATGGCACTGAGGCAGGGAGGCCTGGTCATGCAGAATTACCGCCTGATCGATGCCACCC TGTATGTGACACTGGAGCCATGCGTGATGTGCGCAGGAGCAATGATCCACAGCAGGATCGGAAGAGTGGT GTTCGGAGCACGGGACGCCAAGACCGGCGCAGCAGGCTCCCTGATGGATGTGCTGCACCACCCCGGCATG AACCACCGGGTGGAGATCACAGAGGGAATCCTGGCAGACGAGTGCGCCGCCCTGCTGAGCGATTTCTTTA GAATGCGGAGACAGGAGATCAAGGCCCAGAAGAAGGCACAGAGCTCCACCGACTCTGGAGGATCTAGCGG AGGATCCTCTGGAAGCGAGACACCAGGCACAAGCGAGTCCGCCACACCAGAGAGCTCCGGCGGCTCCTCC GGAGGATCCTCTGAGGTGGAGTTTTCCCACGAGTACTGGATGAGACATGCCCTGACCCTGGCCAAGAGGG CACGCGATGAGAGGGAGGTGCCTGTGGGAGCCGTGCTGGTGCTGAACAATAGAGTGATCGGCGAGGGCTG GAACAGAGCCATCGGCCTGCACGACCCAACAGCCCATGCCGAAATTATGGCCCTGAGACAGGGCGGCCTG GTCATGCAGAACTACAGACTGATTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTGATGTGCGCCG GCGCCATGATCCACTCTAGGATCGGCCGCGTGGTGTTTGGCGTGAGGAACGCAAAAACCGGCGCCGCAGG CTCCCTGATGGACGTGCTGCACTACCCCGGCATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCA GATGAATGTGCCGCCCTGCTGTGCTATTTCTTTCGGATGCCTAGACAGGTGTTCAATGCTCAGAAGAAGG CCCAGAGCTCCACCGACTCCGGAGGATCTAGCGGAGGCTCCTCTGGCTCTGAGACACCTGGCACAAGCGA GAGCGCAACACCTGAAAGCAGCGGGGGCAGCAGCGGGGGGTCAGACAAGAAGTACAGCATCGGCCTGGCC ATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGG TGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGA AACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGC TATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGT CCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGC CTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGAC CTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACC TGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGA GGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGA CGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGGAGAAAGAATGGCCTGTTCGGAAACCTGATTGCCC TGAGCCTGGGCCTGACCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAG CAAGGACACCTACGACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGGCCGACCTGTTTT CTGGCCGCCAAGAACCTGCTCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCA AGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGC TCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCC GGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGG ACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAA CGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTAC CCATTCCTGAAGGACAACCGGGAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCC CTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAA CTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAG AACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGC TGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGC CATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAG AAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACAT ACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGA AGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCC CACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCC GGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGG CTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAA GCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTA AGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGA ATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACA CCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGA ACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGAC TCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAG AGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTT CGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAG CTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACG ACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCG GAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAAC GCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACA AGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTT CTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGG CCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGC GGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAA AGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAG TACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGT CCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAA TCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAG TACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAA ACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGG CTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATC GAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCT ACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAA TCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAA GAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTC AGCTGGGAGGTGACTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAG GAAAGTCTAACCGGTCATCATCACCATCACCATTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTT CTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGT GGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCT CTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGATACCGTCGACCTCTAGCTAGAGCTTGGCGTA ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA AGCATAAAGTGTAAAGCCTAGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGC CCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGG TTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGA GCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCT CCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGAT ACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTA ACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTA CACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACACTCAGTGGAACGAAAACTC ACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTAC GATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCT CCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGT TGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCC CAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGA TCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTAC TGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGT ATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAA AAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGA GCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATAC TCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATG TATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGA TCGGGAGATCGATCTCCCGATCCCCTAGGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAA GCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAAC AAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGAT GTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCAT TAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCAT TGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC

[0120] "Base editing activity" refers to acting to chemically modify a base in a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, e.g., converting a target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, e.g., converting A·T to G·C. In another embodiment, the base editing activity is cytidine deaminase activity, e.g., converting a target C·G to T·A, or adenosine or adenine deaminase activity, e.g., converting A·T to G·C. In some embodiments, base editing activity is assessed by editing efficiency. Base editing efficiency can be measured by any suitable means, e.g., Sanger sequencing or next-generation sequencing. In some embodiments, base editing efficiency is measured by the percentage of all sequencing reads with a nucleobase conversion caused by a base editor, e.g., the percentage of all sequencing reads with a target AT base pair converted to a GC base pair. In some embodiments, base editing efficiency is measured by the percentage of all cells with a nucleobase conversion caused by a base editor when base editing is performed in a population of cells.

[0121] The term "base editor system" refers to a system for editing nucleobases of a target nucleotide sequence. In various embodiments, the base editor system comprises: (1) a polynucleotide-programmable nucleotide-binding domain (e.g., Cas9); (2) a deaminase domain (e.g., adenosine deaminase or cytidine deaminase) for deaminating the nucleobase; and (3) one or more guide polynucleotides (e.g., guide RNAs). In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a polynucleotide-programmable DNA-binding domain. In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor system is ABE8.

[0122] In some embodiments, a base editor system may include two or more base editing components. For example, a base editor system may include multiple deaminases. In some embodiments, a base editor system may 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 single pair of guide polynucleotides can be used to target different deaminases to a target nucleic acid sequence.

[0123] The deaminase domain and polynucleotide-programmable nucleotide-binding component of the base editor system can be bound to each other covalently or non-covalently, or by any combination of such binding and interaction. For example, in some embodiments, the deaminase domain can be targeted to a target nucleotide sequence by a polynucleotide-programmable nucleotide-binding domain. In certain embodiments, the polynucleotide-programmable nucleotide-binding domain can be fused or linked to the deaminase domain. In some embodiments, the polynucleotide-programmable nucleotide-binding domain can target the deaminase domain to a target nucleotide sequence by non-covalently interacting or binding with the deaminase domain. For example, in some embodiments, the deaminase domain can include an additional heterologous moiety or domain that can interact, associate, or form a complex with an additional heterologous moiety or domain that is part of the polynucleotide-programmable nucleotide-binding domain. In some embodiments, the additional heterologous moiety can bind, interact, associate, or form a complex with a polypeptide. In some embodiments, the additional heterologous moiety can bind, interact, associate, or form a complex with a polynucleotide. In some embodiments, an additional heterologous moiety can be attached to a guide polynucleotide. In some embodiments, an additional heterologous moiety can be attached to a polypeptide linker. In some embodiments, an additional heterologous moiety can be attached to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a steryl alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0124] The base editor system can further comprise a guide polynucleotide component. It should be understood that the components of the base editor system can be bound to each other via covalent bonds, non-covalent interactions, or any combination of such bonds and interactions. In certain embodiments, the deaminase domain can be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the deaminase domain can include an additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) that can interact, bind, or form a complex with a portion or segment (e.g., a polynucleotide motif) of the guide polynucleotide. In some embodiments, the additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or linked to the deaminase domain. In some embodiments, the additional heterologous moiety can bind, interact, associate, or form a complex with a polypeptide. In some embodiments, the additional heterologous moiety can bind, interact, associate, or form a complex with a polynucleotide. In some embodiments, the additional heterologous moiety can be bound to a guide polynucleotide. In some embodiments, the additional heterologous moiety can be attached to a polypeptide linker. In some embodiments, an additional heterologous moiety can be linked to the polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0125] In certain embodiments, the base editor system can further include a base excision repair (BER) inhibitor component. It should be understood that the components of the base editor system can be bound to each other via covalent bonds, non-covalent interactions, or any combination of these bonds and interactions. The inhibitor of the BER component can include a BER inhibitor. In certain embodiments, the BER inhibitor can be a uracil DNA glycosylase inhibitor (UGI). In certain embodiments, the BER inhibitor can be an inosine BER inhibitor. In certain embodiments, the BER inhibitor can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain. In certain embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to a BER inhibitor. In certain embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain and a BER inhibitor. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the BER inhibitor to a target nucleotide sequence by non-covalently interacting with or associating with the BER inhibitor. For example, in some embodiments, an inhibitor of a BER component may comprise an additional heterologous moiety or domain that may interact, associate, or complex with an additional heterologous moiety or domain that is part of the polynucleotide programmable nucleotide binding domain.

[0126] In certain embodiments, the BER inhibitor can be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the BER inhibitor can include an additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) that can interact, associate, or complex with a portion or segment (e.g., a polynucleotide motif) of the guide polynucleotide. In some embodiments, the additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) of the guide polynucleotide can be fused or linked to the BER inhibitor. In some embodiments, the additional heterologous moiety can bind, interact, associate, or complex with the polynucleotide. In some embodiments, the additional heterologous moiety can be linked to the guide polynucleotide. In some embodiments, the additional heterologous moiety can be linked to a polypeptide linker. In some embodiments, the additional heterologous moiety can be linked to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0127] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease containing the Cas9 protein or a fragment thereof (e.g., a protein containing an active, inactive, or partially active DNA cleavage domain of Cas9 and / or a gRNA-binding domain of Cas9). Cas9 nuclease is sometimes referred to as casnl nuclease or CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. CRISPR is an adaptive immune system that provides defense against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In the type II CRISPR system, correct processing of the pre-crRNA requires a transcoding small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. The tracrRNA guides the processing of the pre-crRNA by RNase 3. Cas9 / crRNA / tracrRNA then endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. The target strand not complementary to the crRNA is first endonucleolytically cleaved and then exonucleolytically trimmed 3'-5'. In nature, DNA binding and cleavage typically require both a protein and both RNAs. However, single guide RNAs ("sgRNAs," or simply "gRNAs") can be engineered to incorporate both crRNA and tracrRNA aspects 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.The sequence and structure of Cas9 nuclease are well known to those of skill 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 which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, including Cas9 sequences from the 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.

[0128] An exemplary Cas9 is Streptococcus pyogenes Cas9 (spCas9), the amino acid sequence of which is provided below. TIFF0007753096000002.tif166165 (single underline: HNH domain; double underline: RuvC domain)

[0129] Nuclease-inactivated Cas9 proteins may be interchangeably referred to as "dCas9" proteins (meaning nuclease-"dead" Cas9) or catalytically inactive Cas9. Methods for generating 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 contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, and the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can suppress the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28;152(5):1173-83(2013)). In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase, referred to as an "nCas9" protein (for "nickase" Cas9). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, the protein comprises one of the following two Cas9 domains: (1) the gRNA-binding domain of Cas9; (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as "Cas9 variants." Cas9 variants share homology with Cas9 or fragments thereof.For example, a Cas9 variant has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to wild-type Cas9. In some embodiments, the Cas9 mutant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) that has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of the corresponding wild-type Cas9.

[0130] In certain embodiments, the fragments are at least 100 amino acids in length, hi certain embodiments, the fragments are 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.

[0131] In one embodiment, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, nucleotide and amino acid sequences are as follows): TIFF0007753096000003.tif168163 (single underline: HNH domain; double underline: RuvC domain)

[0132] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence: TIFF0007753096000004.tif173168 (single underline: HNH domain; double underline: RuvC domain)

[0133] In some embodiments, 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). TIFF0007753096000005.tif170167 (SEQ ID NO: 1) (Single underline: HNH domain; double underline: RuvC domain)

[0134] In certain embodiments, Cas9 is Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1) or Neisseria meningitidis (NCBI Ref: YP_002342100.1), or Cas9 from any other organism.

[0135] In some embodiments, the dCas9 corresponds to, or comprises part or all of, a Cas9 amino acid sequence with one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises D10A and H840A mutations or corresponding mutations in another Cas9. In some embodiments, the dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A): TIFF0007753096000006.tif171167 (single underline: HNH domain; double underline: RuvC domain)

[0136] In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 in the amino acid sequence provided above, or the residue at the corresponding position in any of the amino acid sequences provided herein, remains a histidine.

[0137] In other embodiments, dCas9 variants are provided that have mutations other than D10A and H840A, e.g., that result in nuclease-inactivated Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, dCas9 variants or homologs are provided that have at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity. In some embodiments, variants of dCas9 are provided that have amino acid sequences that are 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.

[0138] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but rather comprise only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0139] It should be understood that additional 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 this disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is nuclease-dead Cas9 (dCas9). In some embodiments, the Cas9 protein is Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is nuclease-active Cas9.

[0140] Exemplary catalytically inactive Cas9 (dCas9):

[0141] Exemplary catalytic Cas9 nickases (nCas9):

[0142] Exemplary catalytically active Cas9:

[0143] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., nanoarchaea), which constitute the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, Cas9 refers to CasX or CasY, as described, for example, in Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genome-resolved metagenomics, many CRISPR-Cas systems have been identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was discovered as part of an active CRISPR-Cas system in the little-studied nanoarchaea. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, have been discovered, which are among the most compact systems discovered to date. In some embodiments, Cas9 represents CasX or a variant of CasX. In some embodiments, Cas9 represents CasY or a variant of CasY. It should be understood that other RNA-guided DNA binding proteins can also be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0144] In certain embodiments, napDNAbp useful in the methods of the invention comprise circular permutations, which are known and described, for example, in Oakes et al., Cell 176, 254-267, 2019. Below are exemplary circular permutations, where the bolded sequence indicates the sequence derived from Cas9, the italicized sequence represents the linker sequence, and the underlined sequence represents the bipartite nuclear localization sequence. CP5 (with MSP “NGC=Pam Variant with mutations Regular Cas9 likes NGG” PID=Protein Interacting Domain and “D10A” nickase): TIFF0007753096000007.tif171166

[0145] Non-limiting examples of polynucleotide-programmable nucleotide binding domains that can be incorporated into base editors include domains from CRISPR proteins, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs).

[0146] In some embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) of any of the fusion proteins provided herein can be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp is a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to any CasX or CasY protein described herein. It should be understood that Cas12b / C2c1, CasX, and CasY from other bacterial species may also be used in accordance with the present disclosure.

[0147] Cas12b / C2c1 (uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2|C2C1_ALIAG CRISPR-associated endo-nuclease C2c1 OS = Alicyclobacillus acido-terrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) GN=c2c1 PE=1 SV=1

[0148] CasX (uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53) >tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx protein OS = Sulfolobus islandicus (strain HVE10 / 4) GN = SiH_0402 PE=4 SV=1 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE VEPHYLIIAAAGWVLTRLGKVSEGDYVGVNVFTRPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRYIVLANIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAIVNGELIRGEG

[0149] >tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS = Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4 SV=1 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAK VSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0150] Deltaproteobacteria CasX MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQK WYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0151] CasY (ncbi.nlm.nih.gov / protein / APG80656.1) >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacterium]

[0152] The term "Cas12" or "Cas12 domain" refers to an RNA-guided nuclease comprising a Cas12 protein or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas12 and / or a gRNA-binding domain of Cas12). Cas12 belongs to the Class 2, Type V CRISPR / Cas system. Cas12 nucleases are sometimes referred to as CRISPR (clustered regularly interspaced short palindromic repeat)-associated nucleases. The sequence of an exemplary Bacillus hisashii Cas12b (BhCas12b) Cas12 domain is provided below.

[0153] Amino acid sequences having at least 85% or greater identity to the BhCas12b amino acid sequence are also useful in the methods of the invention.

[0154] "Cytidine deaminase" refers to a polypeptide or fragment thereof capable of catalyzing a deamination reaction that converts an amino group to a carbonyl group. In one embodiment, the cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine. PmCDA1 from Petromyzon marinus (Petromyzon marinus cytosine deaminase 1, "PmCDA1"), or AID (activation-induced cytidine deaminase; AICDA) from mammals (e.g., human, pig, cow, horse, monkey, etc.), and APOBEC are exemplary cytidine deaminases.

[0155] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid that shares common properties. A practical method for defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Such analysis allows for the definition of groups of amino acids in which amino acids within a group preferentially exchange with each other and are therefore most similar to each other in their effect on overall protein structure (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include amino acid substitutions such as arginine to lysine, which can maintain a positive charge; aspartic acid to glutamic acid, which can maintain a negative charge; threonine to serine, which maintains a free -OH; and asparagine to glutamine, which can maintain a free NH2.

[0156] The terms "coding sequence" or "protein-coding sequence," as used interchangeably herein, refer to a segment of a polynucleotide that encodes a protein. This region or sequence is bounded at its 5'-proximal end by a start codon and at its 3'-proximal end by a stop codon. A coding sequence is also called an open reading frame.

[0157] 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 of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., genetically engineered adenosine deaminases, evolved adenosine deaminases) can be derived from any organism, such as bacteria. In some embodiments, the adenosine deaminase is derived from a bacterium, such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Caulobacter crescentus.

[0158] In some embodiments, the adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase is a TadA variant. In some embodiments, the TadA variant is TadA*8. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is non-naturally occurring. For example, in some embodiments, the deaminase or deaminase domain has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identity to a naturally occurring deaminase. For example, deaminase domains are described in International PCT Application Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), each of which is incorporated by reference herein in its entirety.Komor, AC, et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage”Nature 551, 464-471 (2017); Komor, AC, et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity”Science Advances 3:eaao4774 (2017) and Rees, HA, et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 Dec;19(12):770-788. doi: See also 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0159] "Detection" refers to identifying the presence, absence, or amount of an analyte to be detected. In one embodiment, a sequence variation in a polynucleotide or polypeptide is detected. In another embodiment, the presence of an indel is detected.

[0160] "Detectable label" refers to 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 beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., those commonly used in enzyme-linked immunosorbent assays (ELISAs)), biotin, digoxigenin, or haptens.

[0161] By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0162] As used herein, the term "effective amount" refers to an amount of a biologically active agent sufficient to induce a desired biological response. The effective amount of an active agent(s) used to practice the present invention for the therapeutic treatment of a disease will vary depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the appropriate amount and dosage will be determined by the attending physician or veterinarian. Such an amount is referred to as an "effective" amount. In one embodiment, an effective amount is an amount of a base editor (e.g., a programmable DNA-binding protein, a nucleobase editor, and a gRNA) of the present invention sufficient to introduce a modification into a gene of interest in a cell (e.g., in vitro or in vivo). In some embodiments, an effective amount of a fusion protein provided herein, e.g., an effective amount of a nucleobase editor comprising an nCas9 domain and a deaminase domain (e.g., an adenosine deaminase or cytidine deaminase), may refer to an amount of the fusion protein sufficient to induce editing of a target site specifically bound and edited by the nucleobase editor. In one embodiment, an effective amount is the amount of a base editor necessary to achieve a therapeutic effect (e.g., reducing or controlling a disease or its symptoms or conditions). Such a therapeutic effect need not be sufficient to alter the gene of interest in all cells of a subject, tissue, or organ, but need only alter the gene of interest in about 1%, 5%, 10%, 25%, 50%, 75% or more of the cells present in the subject, tissue, or organ.

[0163] In some embodiments, an effective amount of a nucleobase editor comprising a fusion protein provided herein (e.g., an nCas9 domain and a deaminase domain (e.g., adenosine deaminase, cytidine deaminase)) refers to the amount of fusion protein sufficient to induce editing of a target site specifically bound and edited by the nucleobase editor described herein. As will be understood by one of skill in the art, an effective amount of an agent (e.g., a fusion protein, nuclease, hybrid protein, protein dimer, complex of a protein (or protein dimer) and a polynucleotide, or polynucleotide) can vary depending on various factors, such as, for example, the desired biological response, e.g., the particular allele, genome, or target site to be edited, the cell or tissue being targeted, and / or the agent used.

[0164] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule, which portion comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment can comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0165] "Guide RNA" or "gRNA" refers to 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 Cpf1). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). A 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 is sometimes referred to as a single guide RNA (sgRNA), although "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 the target nucleic acid (e.g., directs binding of the Cas9 complex to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and contains a stem-loop structure. For example, in some embodiments, domain (2) is identical to or homologous to the tracrRNA as provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNAs (e.g., those comprising domain 2) can be found in U.S. Provisional Patent Application No. USSN 61 / 874,682, entitled "Switchable Cas9 Nucleases and Uses Thereof," filed September 6, 2013, and U.S. Provisional Patent Application No. USSN 61 / 874,746, entitled "Delivery System For Functional Nucleases," filed September 6, 2013, the entire contents of each of which are incorporated herein by reference. In some embodiments, the gRNA comprises two or more of domains (1) and (2) and may be referred to as an "extended gRNA." The extended gRNA binds to two or more Cas9 proteins and binds to the target nucleic acid in two or more different regions, as described herein.The gRNA contains a nucleotide sequence complementary to the target site, which mediates binding of the nuclease / RNA complex to the target site and provides sequence specificity for the nuclease:RNA complex.

[0166] "Hybridization" refers to hydrogen bonding between complementary nucleobases, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that form hydrogen bonds to pair.

[0167] The term "inhibitor of base repair" or "IBR" refers to a protein that can inhibit the activity of a nucleic acid repair enzyme, such as a base excision repair (BER) enzyme. In one embodiment, the IBR is an inhibitor of inosine base excision repair. Examples of base repair inhibitors include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4 PDG, UDG, hSMUGL, and hAAG. In one embodiment, the IBR is an inhibitor of Endo V or hAAG. In one embodiment, the IBR is catalytically inactive EndoV or catalytically inactive hAAG. In one embodiment, the base repair inhibitor is an inhibitor of Endo V or hAAG. In one embodiment, the base repair inhibitor is catalytically inactive EndoV or catalytically inactive hAAG.

[0168] In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI refers to a protein that can inhibit the base excision repair enzyme uracil-DNA glycosylase. In some embodiments, the UGI domain comprises wild-type UGI or a fragment thereof. In some embodiments, the UGI proteins provided herein comprise fragments of UGI and proteins homologous to UGI or UGI fragments. In some embodiments, the base repair inhibitor is an inhibitor of inosine base excision repair. In some embodiments, the base repair inhibitor is a "catalytically inactive inosine-specific nuclease" or "dead inosine-specific nuclease." Without wishing to be bound by any particular theory, catalytically inactive inosine glycosylases (e.g., alkyladenine glycosylases (AAG)) can bind to inosine but cannot create an abasic site or remove the inosine, thereby sterically blocking the newly formed inosine moiety from DNA damage / repair mechanisms. In some embodiments, catalytically inactive inosine-specific nucleases can bind to inosine in nucleic acids but do not cleave nucleic acids.Representative catalytically inactive inosine-specific nucleases include, but are not limited to, catalytically inactive alkyl adenosine glycosylase (AAG nuclease) (e.g., from humans) and catalytically inactive endonuclease V (EndoV nuclease) (e.g., from E. coli).In some embodiments, catalytically inactive AAG nucleases include an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0169] By "increase" is meant a positive change of at least 10%, 25%, 50%, 75%, or 100%.

[0170] An "intein" is a fragment of a protein that can excise itself and link the remaining fragment (extein) with a peptide bond in a process known as protein splicing. Inteins are also called "protein introns." The process by which an intein excises itself and links the remainder of a protein is referred to herein as "protein splicing" or "intein-mediated protein splicing." In some embodiments, the inteins of a precursor protein (the intein-containing protein prior to intein-mediated protein splicing) are derived from two genes. Such inteins are referred to herein as split inteins (e.g., split intein-N and split intein-C). For example, in cyanobacteria, DnaE, ​​the catalytic subunit a of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c. The intein encoded by the dnaE-n gene may be referred to herein as "intein N." The intein encoded by the dnaE-c gene may be referred to herein as "intein C."

[0171] Other intein systems can also be used. For example, synthetic inteins based on the dnaE intein, i.e., the intein pair Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C), have been described (e.g., Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5, incorporated herein by reference). Non-limiting examples of intein pairs that can 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. Patent No. 8,394,604, incorporated herein by reference).

[0172] Exemplary nucleotide and amino acid sequences of inteins are provided. DnaE intein-N DNA: TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTCTGCCAATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCGATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGGGGA GAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAGGGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTTCCTAAT DnaE intein-N protein: CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDR GEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN DnaE Intein-C DNA: ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGA TATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAGCTTCTAAT Intein-C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN Cfa-N DNA: TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCCTATTGGAAAGATTGTCGAAGAGGAATTGAATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGCACAATCGCGGCGAACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACGAGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTGCCA Cfa-N protein: CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGDLLKQVDGLP Cfa-C DNA: ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAGGAAAGTAAAGATAATATCTCGAAAAGTCTTGGTACCCAAAATGTCTATGATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTAGCCAGCAAC Cfa-C protein: MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN

[0173] To join the N-terminal portion of split-Cas9 with the C-terminal portion of split-Cas9, an intein-N and an intein-C can be fused to the N-terminal portion of split-Cas9 and the C-terminal portion of split-Cas9, respectively. For example, in some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of split-Cas9, i.e., forming the structure N--[N-terminal portion of split-Cas9]-[intein-N]--C. In some embodiments, intein-C is fused to the N-terminus of the C-terminal portion of split-Cas9, i.e., forming the structure N--[intein-C]--[C-terminal portion of split-Cas9]-C. The mechanism of intein-mediated protein splicing for linking proteins (e.g., split Cas9) to which an 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, for example, by WO2014004336, WO2017132580, US20150344549, and US20180127780, each of which is incorporated herein by reference in its entirety.

[0174] The terms "isolated," "purified," or "biologically pure" refer to material that has been removed, to varying degrees, from components that normally accompany it when found in its native state. "Isolated" refers to the degree of separation from the original source or surrounding environment. "Purified" refers to a greater degree of separation than isolation. A "purified" or "biologically pure" protein has been sufficiently free of other substances so that the impurities do not substantially affect the biological properties of the protein or cause other adverse effects. That is, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can mean that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For proteins that can undergo modifications, such as phosphorylation or glycosylation, different modifications can result in different isolated proteins that can be separately purified.

[0175] By "isolated polynucleotide" is meant a nucleic acid (e.g., DNA) that is free of the genes that flank the nucleic acid molecule of the invention in the naturally occurring genome of the organism from which it is derived. Thus, the term includes recombinant DNA that is present, for example, in a vector; in an autonomously replicating plasmid or virus; in the genomic DNA of a prokaryote or eukaryote; or as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments generated by PCR or restriction endonuclease digestion). Furthermore, the term includes RNA molecules transcribed from DNA molecules, as well as recombinant DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

[0176] By "isolated polypeptide" is meant a polypeptide of the invention separated from components that naturally accompany it. Typically, a polypeptide is isolated when it is at least 60% by weight free from the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75% by weight, more preferably at least 90%, and most preferably at least 99% by weight, a polypeptide of the invention. Isolated polypeptides of the invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0177] As used herein, the term "linker" can refer to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group, or a molecule that connects two molecules or moieties (e.g., two components of a protein complex or ribonucleocomplex, or two domains of a fusion protein, e.g., a polynucleotide-programmable DNA-binding domain (e.g., dCas9) and a deaminase domain (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase), or a napDNAbp domain (e.g., Cas12b) and a deaminase domain (e.g., adenosine deaminase or cytidine deaminase)). In certain embodiments, the linker flanks the deaminase domain inserted within a Cas protein or fragment thereof. The linker can connect different components or different parts of a component of a base editor system. For example, in some embodiments, the linker can connect the guide polynucleotide-binding domain of a polynucleotide-programmable nucleotide-binding domain and the catalytic domain of a deaminase. In some embodiments, a linker can connect a CRISPR polypeptide and a deaminase. In some embodiments, a linker can connect a Cas9 and a deaminase. In some embodiments, a linker can connect a dCas9 and a deaminase. In some embodiments, a linker can connect an nCas9 and a deaminase. For example, in some embodiments, a linker can connect Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, or Cas12i to a deaminase. In some embodiments, a linker can connect a guide polynucleotide and a deaminase. In some embodiments, a linker can connect a deamination component of a base editor system and a polynucleotide-programmable nucleotide-binding component. In some embodiments, a linker can connect the RNA binding portion of the deaminating component of the base editor system and the napDNAbp component.In some embodiments, a linker can connect the RNA-binding portion of a deaminating component of a base editor system to a polynucleotide-programmable nucleotide-binding component. In some embodiments, a linker can connect the RNA-binding portion of a deaminating component of a base editor system to the RNA-binding portion of a polynucleotide-programmable nucleotide-binding component. A linker can be disposed between or sandwiched between two groups, molecules, or other moieties and linked to each through covalent or non-covalent interactions, thus connecting the two. In some embodiments, a linker can be an organic molecule, group, polymer, or chemical moiety. In some embodiments, a linker can be a polynucleotide. In some embodiments, a linker can be a DNA linker. In some embodiments, a linker can be an RNA linker. In some embodiments, a linker can comprise an aptamer capable of binding to a ligand. In some embodiments, the ligand can be a carbohydrate, peptide, protein, or nucleic acid. In some embodiments, a linker can comprise an aptamer derived from a riboswitch. The riboswitch from which the aptamer is derived can be selected from a theophylline riboswitch, a thiamine pyrophosphate (TPP) riboswitch, an adenosine cobalamin (AdoCbl) riboswitch, an S-adenosylmethionine (SAM) riboswitch, an SAH riboswitch, a flavin mononucleotide (FMN) riboswitch, a tetrahydrofolate riboswitch, a lysine riboswitch, a glycine riboswitch, a purine riboswitch, a GlmS riboswitch, or a prequeosin 1 (PreQ1) riboswitch. In some embodiments, the linker can comprise an aptamer bound to a protein domain, such as a polypeptide or polypeptide ligand. In some embodiments, the polypeptide ligand can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.In some embodiments, the polypeptide ligand can be part of a base editor system component. For example, a nucleic acid base editing component can include a deaminase domain and an RNA recognition motif.

[0178] In some embodiments, the linker can be an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker can be about 5-100 amino acids in length, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids in length. In some embodiments, the linker can be about 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 amino acids in length. Longer or shorter linkers are also contemplated.

[0179] In some embodiments, a linker connects the gRNA binding domain of an RNA programmable nuclease, including a Cas9 nuclease domain, with the catalytic domain of a nucleic acid editing protein (e.g., cytidine or adenosine deaminase). In some embodiments, a linker connects dCas9 and a nucleic acid editing protein. For example, a linker is placed between two groups, molecules, or other moieties, or is flanked by two groups, molecules, or other moieties, and is linked to each other via a covalent bond, thus connecting the two. In some embodiments, a linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, a linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5 to 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.

[0180] In some embodiments, the nucleobase editor domain is fused via a linker comprising the amino acid sequence of SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS. In some embodiments, the nucleobase editor domain is fused via a linker comprising the amino acid sequence SGSETPGTSESATPES, which may be referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker is n , (GGGS) n , (GGGGS) n , (G)n、 (EAAAK) n , (GGS) n , SGSETPGTSESATPES, or (XP) n motif, or any combination thereof, where n is independently an integer from 1 to 30 and X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0181] 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 SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS. In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0182] By "marker" is meant any protein or polynucleotide having an altered expression level or activity that is associated with a disease or disorder.

[0183] As used herein, the term "mutation" refers to the substitution of a residue in a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or the deletion or insertion of one or more residues in a sequence. Mutations are typically described herein by identifying the original residue, then identifying the position of the residue in the sequence, and identifying the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art and are provided, for example, by 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 editors of the present disclosure can efficiently generate "intended mutations," e.g., point mutations, in a nucleic acid (e.g., a nucleic acid in a subject's genome) without generating a significant number of unintended mutations, e.g., unintended point mutations. In some embodiments, the intended mutation is a mutation caused by a particular base editor (e.g., a cytidine base editor or an adenosine base editor) attached to a guide polynucleotide (e.g., a gRNA) that is specifically designed to produce the intended mutation.

[0184] Generally, mutations made or identified in a sequence (e.g., an amino acid sequence described herein) are numbered relative to a reference (or wild-type) sequence, i.e., a sequence that does not contain the mutation. Those skilled in the art will readily understand how to determine the location of mutations in amino acid and nucleic acid sequences relative to a reference sequence.

[0185] The term "non-conservative mutation" refers to an amino acid substitution between different groups, such as tryptophan to lysine or serine to phenylalanine. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the wild-type protein.

[0186] The term "nuclear localization sequence," "nuclear localization signal," or "NLS" refers to an amino acid sequence that promotes the import of a protein into the cell nucleus. Nuclear localization sequences are known in the art and are described, for example, in International PCT Application PCT / EP 2000 / 011690, filed November 23, 2000, and published May 31, 2001 as WO / 2001 / 038547, by Plank et al., the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS, for example, as described by Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0187] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a compound containing a nucleobase and an acidic moiety, such as a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules containing three or more nucleotides, are linear molecules in which adjacent nucleotides are linked to each other via phosphodiester bonds. In certain embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In certain embodiments, "nucleic acid" refers to an oligonucleotide chain containing three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably to refer to a polymer of nucleotides (e.g., a chain of at least three nucleotides). In certain embodiments, "nucleic acid" encompasses RNA and single- and / or double-stranded DNA. Nucleic acids can naturally occur, for example, in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. Alternatively, a nucleic acid molecule may be, for example, a non-naturally occurring molecule, recombinant DNA or RNA, an artificial chromosome, an engineered genome, or a fragment thereof, or synthetic DNA, RNA, DNA / RNA hybrid, or a non-naturally occurring molecule containing non-naturally occurring nucleotides or nucleosides. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. In the case of chemically synthesized molecules, the nucleic acid may contain nucleoside analogs, such as, for example, chemically modified bases or sugars, and analogs with backbone modifications, where appropriate. Nucleic acid sequences are shown in the 5' to 3' direction unless otherwise indicated.In certain embodiments, nucleic acids are comprised of natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, 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-propynyl-uridine, C5-propynyl-cytidine, C5- methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanine, O(6)-methylguanine, and 2-thiocytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0188] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" may be used interchangeably with "polynucleotide programmable nucleotide binding domain" and refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA), that guides the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide-programmable nucleotide binding domain is a polynucleotide-programmable DNA binding domain. In some embodiments, the polynucleotide-programmable nucleotide binding domain is a polynucleotide-programmable RNA binding domain. In some embodiments, the polynucleotide-programmable nucleotide binding domain is a Cas9 protein. The Cas9 protein can bind to a guide RNA that guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, such as a nuclease-active Cas9, Cas9 nickase (nCas9), or nuclease-inactive Cas9 (dCas9). Non-limiting examples of nucleic acid programmable DNA binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i.Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also called Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, and Cse 1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3 , Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Type II Examples of such proteins include Cas effector proteins, type V Cas effector proteins, type VI Cas effector proteins, CARF, DinG, homologs thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, even if they may not be specifically listed herein. See, for example, Makarova et al. "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR J. 2018 Oct;1:325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., "Functionally diverse type V CRISPR-Cas systems" Science. 2019 Jan 4;363(6422):88-91. doi: 10.1126 / science.aav7271 (the entire contents of each are incorporated herein by reference).

[0189] The terms "nucleobase," "nitrogenous base," or "base" are used interchangeably herein to refer to nitrogen-containing biological compounds that form nucleosides, which are the building blocks of nucleotides. The ability of nucleobases to base pair and stack with each other directly leads to long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The five nucleobases, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), are referred to as primary 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-primary) modified bases. Non-limiting exemplary modified nucleobases include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydromethylcytosine. Hypoxanthine and xanthine can be produced in the presence of mutagens, and both are produced by deamination (replacement of an amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be produced by deamination of cytosine. A "nucleoside" consists of a nucleobase and a five-carbon sugar (ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Nucleosides with modified nucleobases include inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), pseudouridine (Ψ), etc. A nucleotide consists of a nucleobase, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group.

[0190] The term "nucleic acid programmable DNA-binding protein" or "napDNAbp" refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid, that directs the napDNAbp to a specific nucleic acid sequence. For example, a Cas12 protein can bind to a guide RNA that directs the Cas12 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas12 domain, e.g., a nuclease-active Cas12 domain. Examples of napDNAbps include Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Other napDNAbps are within the scope of this disclosure, even though they may not be specifically listed herein. 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 are incorporated herein by reference).

[0191] The term "nucleobase editing domain" or "nucleobase editing protein," as used herein, refers to a protein or enzyme that can catalyze nucleobase modifications in RNA or DNA, such as the deamination of 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 addition and insertion. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., adenine deaminase or adenosine deaminase; or cytidine deaminase or cytosine deaminase). In some embodiments, the nucleobase editing domain is a multiple deaminase domain (e.g., adenine deaminase or adenosine deaminase and cytidine or cytosine deaminase). In some embodiments, the nucleobase editing domain can be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase editing domain can be a nucleobase editing domain engineered or evolved from a naturally occurring nucleobase editing domain. The nucleobase editing domain can be derived from any organism, such as bacteria, humans, chimpanzees, gorillas, monkeys, cows, dogs, rats, or mice. For example, nucleobase editing proteins 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 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); and 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), the entire contents of which are incorporated herein by reference.

[0192] As used herein, "obtaining," as in "obtaining a drug," includes synthesizing, purchasing, or otherwise acquiring the drug.

[0193] As used herein, "patient" or "subject" refers to a mammalian subject or individual who has been diagnosed with, is at risk of developing, or is suspected of having or developing 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 may be humans, non-human primates, cats, dogs, pigs, cows, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, guinea pigs), and other mammals that may benefit from the treatments disclosed herein. Exemplary human patients may be male and / or female.

[0194] A "patient in need thereof" or "subject in need thereof" is referred to herein as a patient who has been diagnosed with, is at risk of having, has been predetermined to have, or is suspected of having a disease or disorder.

[0195] The terms "pathogenic mutation," "pathogenic variant," "disease-causing mutation," "disease-causing variant," "deleterious mutation," or "predisposing mutation" refer to a genetic change or mutation that increases an individual's susceptibility or predisposition to a particular disease or disorder. In some embodiments, a pathogenic mutation comprises the substitution of at least one wild-type amino acid in a protein encoded by a gene with at least one pathogenic amino acid.

[0196] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in carrying or transporting a compound from one site in the body (e.g., a delivery site) to another site (e.g., an organ, tissue, or body part). A pharmaceutically acceptable carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the tissues of the subject (e.g., physiological compatibility, sterility, physiological pH, etc.). Terms such as "excipient," "carrier," "pharmaceutically acceptable carrier," "vehicle," and the like are used interchangeably herein.

[0197] The term "pharmaceutical composition" may refer to a composition formulated for pharmaceutical use.

[0198] The terms "protein," "peptide," "polypeptide," and their grammatical equivalents are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The term refers to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide is at least three amino acids in length. A protein, peptide, or polypeptide can refer to an individual protein or a group of proteins. One or more amino acids in a protein, peptide, or polypeptide can be modified by the addition of chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modifications. A protein, peptide, or polypeptide can also be a single molecule or a multimolecular complex. A protein, peptide, or polypeptide can be simply a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide can be naturally occurring, recombinant, synthetic, or any combination thereof. As used herein, the term "fusion protein" refers to a hybrid polypeptide containing protein domains from at least two different proteins. One protein can be located at the amino-terminal (N-terminal) or carboxy-terminal (C-terminal) portion of the fusion protein, thus forming an amino-terminal fusion protein or a carboxy-terminal fusion protein, respectively. The protein can contain different domains, such as a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9, which guides the protein to bind to the target site) and a nucleic acid cleavage domain, or the catalytic domain of a nucleic acid editing protein. In some embodiments, the protein includes a proteinaceous portion, such as an amino acid sequence constituting the nucleic acid binding domain, and an organic compound, such as a compound that can act as a nucleic acid cleavage agent. In some embodiments, the protein is complexed with or associated with a nucleic acid (e.g., RNA or DNA).Any protein provided herein can be produced by any method known in the art.For example, the protein provided herein can be produced through recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers.The method for recombinant protein expression and purification is well known, including that described by 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.

[0199] The polypeptides and proteins (including functional portions and functional variants thereof) disclosed herein can contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, and indoline-2-carboxylic acid. , 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, aminocyclohexanecarboxylic acid, aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. Polypeptides and proteins can be associated with post-translational modifications of one or more amino acids of the polypeptide construct. Non-limiting examples of post-translational modifications include phosphorylation, acylation, including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation, including methylation and ethylation, ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation, and iodination.

[0200] The term "polynucleotide programmable nucleotide binding domain" or "nucleic acid programmable DNA binding protein (napDNAbp)" refers to a protein that binds to a nucleic acid (e.g., DNA or RNA), such as a guide polynucleotide (e.g., guide RNA), that directs the polynucleotide programmable nucleotide binding domain 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 Cas12 protein.

[0201] The term "recombinant," as used herein with respect to a protein or nucleic acid, refers to a protein or nucleic acid that does not occur in nature but is the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any naturally occurring sequence.

[0202] By "decreasing" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0203] "Reference" refers to a standard or control condition. In one embodiment, the reference is a wild-type or healthy cell. In another embodiment, without limitation, the reference is an untreated cell that is not exposed to the test condition or is exposed to a placebo or normal saline, medium, buffer, and / or a control vector that does not carry the polynucleotide of interest.

[0204] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a particular sequence; for example, a segment of a full-length cDNA or gene sequence, or the entire cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is generally at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is generally at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides, or any integer therebetween or thereabout. In some embodiments, the reference sequence is the wild-type sequence of a protein of interest. In other embodiments, the reference sequence is a polynucleotide sequence encoding the wild-type protein.

[0205] 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 targets for cleavage. In some embodiments, when an RNA-programmable nuclease is complexed with an RNA, it can be referred to as a nuclease:RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA). A 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 is sometimes referred to as a single guide RNA (sgRNA), although "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 the target nucleic acid (e.g., directs binding of the Cas9 complex to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and contains a stem-loop structure. For example, in some embodiments, domain (2) is identical to or homologous to the tracrRNA as provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNAs (e.g., those comprising domain 2) can be found in U.S. Provisional Patent Application No. USSN 61 / 874,682, entitled "Switchable Cas9 Nucleases and Uses Thereof," filed September 6, 2013, and U.S. Provisional Patent Application No. USSN 61 / 874,746, entitled "Delivery System For Functional Nucleases," filed September 6, 2013, the entire contents of each of which are incorporated herein by reference. In some embodiments, the gRNA comprises two or more of domains (1) and (2), and may be referred to as an "extended gRNA."For example, the extended gRNA can bind, e.g., to two or more Cas9 proteins and bind to a target nucleic acid in two or more different regions, as described herein. The gRNA contains a nucleotide sequence complementary to the target site, which mediates binding of the nuclease / RNA complex to the target site and provides sequence specificity for the nuclease:RNA complex.

[0206] In some embodiments, the RNA-programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 from Streptococcus pyogenes (Casnl) (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 host factor RNase III," Deltcheva E. et al., Nature 471:602-607(2011)).

[0207] Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins can, in principle, target any sequence specified by a guide RNA. Methods of using RNA-programmable nucleases such as Cas9 for site-specific cleavage (e.g., to modify genomes) are known in the art (e.g., Cong, L. et al., Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, WY et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013), the entire contents of each of which are incorporated herein by reference.

[0208] The term "single nucleotide polymorphism (SNP)" refers to a single nucleotide variation occurring at a specific position in the genome, where each variation is present to a noticeable degree in a population (e.g., >1%). For example, at a particular base position in the human genome, a C nucleotide can occur in most individuals, but in a minority of individuals, that position is occupied by an A. This means that there is an SNP at this specific position, and the two nucleotide variations, C or A, are alleles at this position. SNPs underlie differences in susceptibility to disease. Disease severity and the body's response to treatment are also manifestations of genetic variation. SNPs can occur in the coding region of a gene, the noncoding region of a gene, or in intergenic regions (regions between genes). In some embodiments, SNPs within a coding sequence do not necessarily change the amino acid sequence of the resulting protein due to the degeneracy of the genetic code. SNPs in coding regions are of two types: synonymous and nonsynonymous SNPs. Synonymous SNPs do not affect the protein sequence, while nonsynonymous SNPs do alter the amino acid sequence of a protein. There are two types of nonsynonymous SNPs: missense and nonsense. SNPs that are not located in protein-coding regions can affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNA. Gene expression affected by this type of SNP is called an eSNP (expressed SNP) and can be upstream or downstream of the gene. Single-nucleotide variants (SNVs) are single-nucleotide variations with unlimited frequency that can occur somatically. Somatic single-nucleotide variations can also be called single-nucleotide modifications.

[0209] By "specifically binds" is meant a nucleic acid molecule, polypeptide, or complex thereof (e.g., a nucleic acid programmable DNA binding domain and a guide nucleic acid), compound, or molecule that recognizes and binds to a polypeptide and / or nucleic acid molecule of the invention, but does not substantially recognize or bind to other molecules in a sample (e.g., a biological sample).

[0210] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to pairing to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof under various stringency conditions. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0211] For example, stringent salt concentrations are typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions will typically include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, detergent (e.g., sodium dodecyl sulfate (SDS)) concentration, and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In one embodiment, hybridization occurs at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization occurs at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In another embodiment, hybridization occurs at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be readily apparent to those of skill in the art.

[0212] In most applications, the washing steps following hybridization also vary in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As described above, washing stringency can be increased by decreasing salt concentration or increasing temperature. For example, stringent salt concentrations for washing steps are preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for washing steps typically include temperatures of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In one embodiment, washing steps are performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washing steps are performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps are performed in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 68° C. Further variations of these conditions will be readily apparent to those of skill in the art.Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0213] "Split" means divided into two or more pieces.

[0214] "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 separate nucleotide sequences. Polypeptides corresponding to the N-terminal and C-terminal portions of the Cas9 protein can be spliced ​​to form a "reconstituted" Cas9 protein. In certain embodiments, the Cas9 protein is split into two fragments within a disordered region of the protein, e.g., 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 incorporated herein by reference). In some embodiments, the protein is split into two fragments at any C, T, A, or S within the region between approximately amino acids A292-G364, F445-K483, or E565-T637 of SpCas9, or at the corresponding position in any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other napDNAbp. In certain embodiments, the protein is split into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of splitting a protein into two fragments is referred to as "splitting" the protein.

[0215] In other embodiments, the N-terminal portion of the Cas9 protein comprises amino acids 1-573 or 1-637 of wild-type S. pyogenes Cas9 (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 wild-type SpCas9.

[0216] The C-terminal portion of a split Cas9 can be joined with the N-terminal portion of a 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. Thus, in some embodiments, the C-terminal portion of the split Cas9 comprises amino acids (551-651)-1368 of spCas9. "(551-651)-1368" means beginning with an amino acid between amino acids 551 and 651 (inclusive) and ending at amino acid 1368.For example, the C-terminal portion of a split-Cas9 consists of amino acids 551-1368, 552-1368, 553-1368, 554-1368, 555-1368, 556-1368, 557-1368, 558-1368, 559-1368, 560-1368, 561-1368, 562-1368, 563-1368, 564-1368, 565-1368, 566-1368, 567-1368, 568-1368, 569-1368, 570-1368, 571-1368, 572-1368, 573-1368, 574 -1368, 575-1368, 576-1368, 577-1368, 578-1368, 579-1368, 580-1368, 581-1368, 582-1368, 583-1368, 584-1368, 585-1368, 586-1368, 587-1368, 588-1368, 589-1368, 590-1368, 591-1368, 592-1368, 593-1368, 594-1368, 595-1368, 596-1368, 597-1368, 598-1368, 599-1368, 600-1368 , 601-1368, 602-1368, 603-1368, 604-1368, 605-1368, 606-1368, 607-1368, 608-1368, 609-1368, 610-1368, 611-1368, 612-1368, 613-1368, 614-1368, 615-1368, 616-1368, 617-1368, 618-1368, 619-1368, 620-1368, 621-1368, 622-1368, 623-1368, 624-1368, 625-1368, 626-1368, 627- 1368, 628-1368, 629-1368, 630-1368, 631-1368, 632-1368, 633-1368, 634-1368, 635-1368, 636-1368, 637-1368, 638-1368, 639-1368, 640-1368, 641-1368, 642-1368, 643-1368, 644-1368, 645-1368, 646-1368, 647-1368, 648-1368, 649-1368, 650-1368, or 651-1368.In some embodiments, the C-terminal portion of the split Cas9 protein comprises a portion of amino acids 574-1368 or 638-1368 of SpCas9.

[0217] "Subject" means a mammal, including, but not limited to, a human or a non-human mammal such as a cow, horse, dog, sheep, or cat. Subjects include livestock, domestic animals raised to produce labor and provide goods such as food, including, but not limited to, cows, goats, chickens, horses, pigs, rabbits, and sheep.

[0218] By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). In one embodiment, such a sequence has at least 60%, 80%, or even 85%, 90%, 95%, or even 99% identity at the amino acid level or nucleic acid to the sequence used for comparison.

[0219] Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program can be used, e.g., -3 and e -100 A probability score between indicates closely related sequences. COBALT can be used, for example, with the following parameters: a) Alignment parameters: Gap penalties -11, -1 and End-Gap penalties -5, -1 b) CDD parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved columns and Recompute on c) Query clustering parameters: Use query clusters on; Word Size 4; Max cluster distance 0.8; Alphabet Regular. The EMBOSS Needle is used, for example, with the following parameters: a) Matrix: BLOSUM62; b) GAP OPEN: 10; c) GAP EXTEND: 0.5; d) OUTPUT FORMAT: pair; e) END GAP PENALTY: false; f) END GAP OPEN: 10; and g) END GAP EXTEND: 0.5.

[0220] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a nucleobase editor. In one embodiment, the target site is deaminated by a deaminase (e.g., a cytidine or adenine deaminase) or a fusion protein comprising same.

[0221] As used herein, the terms "treat," "treating," "treatment," and the like refer to alleviating or ameliorating a disorder and / or its associated symptoms, or achieving a desired pharmacological and / or physiological effect. It will be understood that treating a disorder or condition does not require (nor does complete elimination preclude) the complete elimination of the associated disorder, condition, or symptoms. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, diminishes, eliminates, alleviates, alleviates, reduces the intensity of, or cures, the disease and / or adverse symptoms resulting therefrom. In certain embodiments, the effect is prophylactic, i.e., the effect protects against or prevents the occurrence or recurrence of the disease or condition. To this end, the methods of the present disclosure comprise administering a therapeutically effective amount of a composition as described herein.

[0222] "Uracil glycosylase inhibitor," or alternatively "UGI," refers to an agent that inhibits the uracil excision repair system. In one embodiment, the agent is a protein or fragment thereof that binds to host uracil-DNA glycosylase and prevents the removal of uracil residues from DNA. In one embodiment, UGI is a protein, fragment, or domain thereof that can inhibit the uracil-DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain comprises wild-type UGI or a modified version thereof. In some embodiments, the UGI domain comprises a fragment of an exemplary amino acid sequence provided below. In some embodiments, the UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the exemplary UGI sequence provided below. In some embodiments, the UGI comprises an amino acid sequence homologous to an exemplary UGI amino acid sequence or fragment thereof, as described below. In some embodiments, UGI or a portion thereof has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identity to wild-type UGI or a UGI sequence or a portion thereof, as described below. Exemplary UGIs include the following amino acid sequences: >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD APEYKPWALVIQDSNGENKIKML.

[0223] The term "vector" refers to a means for introducing a nucleic acid sequence into a cell, resulting in a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, and episomes. An "expression vector" is a nucleic acid sequence that comprises a nucleotide sequence that is expressed in a recipient cell. Expression vectors may contain additional nucleic acid sequences to enhance and / or facilitate expression of the introduced sequence, such as initiation sequences, termination sequences, enhancer sequences, promoter sequences, and secretion sequences.

[0224] Any composition or method provided herein can be combined with any one or more of the other compositions and methods provided herein.

[0225] DNA editing has emerged as a viable means to correct disease states by correcting pathogenic mutations at the gene level. Until recently, all DNA editing platforms functioned by inducing DNA double-strand breaks (DSBs) at specific genomic sites and relying on endogenous DNA repair pathways to determine product outcomes in a semi-stochastic manner, resulting in a complex population of genetic products. While precise and user-defined repair outcomes can be achieved via the homology-directed repair (HDR) pathway, numerous challenges have prevented highly efficient repair using HDR in therapeutically relevant cell types. In practice, this pathway is less efficient than the competing, error-prone non-homologous end-joining pathway. Furthermore, HDR is strictly restricted to the G1 and S phases of the cell cycle, preventing accurate repair of DSBs in post-mitotic cells. As a result, it has proven difficult or impossible to modify genome sequences with high efficiency in a user-defined and programmable manner in these populations.

[0226] Superoxide dismutase 1 (SOD1) is an enzyme protein encoded by the SOD1 gene. SOD1 enzymes are abundant in cells throughout the body. The SOD1 enzyme binds copper (Cu) and zinc (Zn) to decompose toxic charged oxygen molecules called superoxide radicals or reactive oxygen species (ROS). These are byproducts of normal cellular processes and must be periodically decomposed to avoid cell damage, transformation, or death. At least 200 mutations in the SOD1 gene have been found to cause amyotrophic lateral sclerosis (ALS), a condition characterized by progressive muscle weakness, loss of muscle mass, and loss of motor control. Most of these mutations alter a single amino acid in the superoxide dismutase enzyme. Worldwide, mutations in the SOD1 gene cause 15 to 20 percent of familial ALS cases. Approximately half of all Americans with ALS caused by mutations in the SOD1 gene have a specific mutation—Ala5Val or A5V—that replaces the amino acid residue alanine (A) at position 5 of the enzyme with the amino acid residue valine (V). ALS caused by the A5V mutation generally results in a shorter life expectancy than ALS caused by other gene mutations.

[0227] ALS is caused by the death of nerve cells (motor neurons) that control muscle movement. The reason why motor neurons are particularly susceptible to SOD1 gene mutations is currently unknown, but the large size of these types of neurons may contribute to their greater susceptibility to disruption of normal SOD1 enzyme function. Possible ways in which altered SOD1 enzymes can cause motor neuron death include: (i) increased production of harmful superoxide radicals in cells, particularly lysosomes and / or proteosomes; (ii) increased production of other types of toxic radicals and increased cell death; and / or (iii) the accumulation of aggregates of misfolded superoxide dismutase, which can be toxic to cells.

[0228] By "SOD1 protein" is meant a polypeptide or fragment thereof having at least about 95% amino acid sequence identity to NCBI Accession No. NP_000445. An exemplary human SOD1 amino acid sequence is provided below. 1 MATKAVCVLK GDGPVQGIIN FEQKESNGPV KVWGSIKGLT EGLHGFHVHE FGDNTAGCTS 61 AGPHFNPLSR KHGGPKDEER HVGDLGNVTA DKDGVADVSI EDSVISLSGD HCIIGRTLVV 121 HEKADDLGKG GNEESTKTGN AGSRLACGVI GIAQ

[0229] "SOD1 polynucleotide" refers to a nucleic acid molecule encoding an SOD1 protein or a fragment thereof. The genomic sequence of an exemplary human SOD1 polynucleotide, available at NCBI accession numbers NC_000021.9:31659622-31668931 (Homo sapiens chromosome 21, GRCh38.p13 primary assembly), is provided below (SEQ ID NO: 3). TIFF0007753096000008.tif245159TIFF0007753096000009.tif249159TIFF0007753096000010.tif249160TIFF0007753096000011.tif208159

[0230] In the above SOD1 genomic nucleic acid sequence, the "ag" splice acceptor site at the 5' end of exon 3 of the SOD1 gene is shown in bold. The nucleic acid sequence of exon 3 of the SOD1 gene is shown in italics and double underlined in the above SOD1 genomic sequence.

[0231] An exemplary human SOD1 polynucleotide mRNA / cDNA sequence available at NCBI Reference Sequence: NM_000454.4 is provided below: gcgtcgtag tctcctgcag cgtctggggt ttccgttgca gtcctcggaa ccaggacctc ggcgtggcct agcgagttat ggcgacgaag gccgtgtgcg tgctgaaggg cgacggcca gtgcagggca tcatcaattt cgagcagaag gaagtaatg gaccagtgaa ggtgtggga aggattaaag gactgactga aggcctgcat ggattccatg ttcatgagtt tggagataat acagcaggct gtaccagtgc aggtcctcac tttaatcctc tatccagaaa acacggtggg ccaaaggatg aagagaggca tgttggagac ttgggcaatg tgactgctga caaagatggt gtggccgatg tgtctattga agattctgtg atctcactct caggagacca ttgcatcatt ggccgcacac tggtggtcca tgaaaaagca gatgacttgg gcaaaggtgg aaatgaagaa agtacaaaga caggaaacgc tggaagtcgt ttggcttgtg gtgtaattgg gatcgcccaa taaacattcc cttggatgta gtctgaggcc ccttaactca tctgttatcc tgctagctgt agaaatgtat cctgataaac attaaacact gtaatcttaa aagtgtaatt gtgtgacttt ttcagagttg ctttaaagta cctgtagtga gaactgatt tatgatcact tggaagaattt gtatagttt ataaactca gttaaaatgt ctgtttcaat gacctgtatt ttgccagact taaatcacag atgggtatta aacttgtcag aatttctttg tcattcaagc ctgtgaataa aaaccctgta tggcacttat tatgaggcta ttaaaagaat ccaaattcaa actaaaaaaa aaaaaaaaaa a

[0232] By "androgen receptor (AR) polynucleotide" is meant any polynucleotide that encodes an androgen receptor polypeptide. An exemplary androgen receptor polynucleotide is provided below (SEQ ID NO: 4): Homo sapiens androgen receptor (AR), transcript variant 1, mRNA (NM_000044.6) [Brief explanation of the drawings]

[0233] [Figure 1] Figures 1A-1C show plasmids. Figure 1A is an expression vector encoding the TadA7.10-dCas9 base editor. Figure 1B is a plasmid containing a nucleic acid molecule encoding a protein that confers chloramphenicol resistance (CamR) and spectinomycin resistance (SpectR). This plasmid also contains a kanamycin resistance gene disabled by two point mutations. Figure 1C is a plasmid containing a nucleic acid molecule encoding a protein that confers chloramphenicol resistance (CamR) and spectinomycin resistance (SpectR). This plasmid also contains a kanamycin resistance gene disabled by three point mutations. [Figure 2] Figure 2 shows an image of a bacterial colony transduced with the expression vector shown in Figure 1, which contains a nonfunctional kanamycin resistance gene. The vector contained an ABE7.10 variant generated using error-prone PCR. Bacterial cells expressing these "evolved" ABE7.10 variants were selected for kanamycin resistance using increasing concentrations of kanamycin. Bacteria expressing ABE7.10 variants with adenosine deaminase activity were able to correct the mutation introduced into the kanamycin resistance gene and restore kanamycin resistance. Kanamycin-resistant cells were selected for further analysis. [Figure 3]Figures 3A and 3B show editing of the regulatory region of the hemoglobin subunit gamma (HGB1) locus, a therapeutically relevant site for upregulation of fetal hemoglobin. Figure 3A is a diagram of a portion of the regulatory region of the HGB1 gene. Figure 3B quantifies the efficiency and specificity of adenosine deaminase variants. Editing was assayed at the hemoglobin subunit gamma 1 (HGB1) locus in HEK293T cells, which is a therapeutically relevant site for upregulation of fetal hemoglobin. The top panel shows nucleotide residues in the targeted region of the regulatory sequence of the HGB1 gene. A5, A8, A9, and A11 indicate the edited adenosine residues in HGB1. [Figure 4] Figure 4 shows the relative efficacy of adenosine base editors, including dCas9, that recognize non-canonical PAM sequences. The top panel shows the coding sequence for a hemoglobin subunit. The bottom panel shows the efficiency of adenosine deaminase variant base editors with guide RNAs of various lengths. [Figure 5] Figure 5 is a graph showing the efficiency and specificity of the ABE8 base editor, quantitating the percent editing at the intended target nucleotide and at unintended target nucleotides (bystanders). [Figure 6] Figure 6 is a graph showing the efficiency and specificity of the ABE8 base editor, quantitating the percent editing at intended target nucleotides and unintended target nucleotides (bystanders). [Figure 7]Figures 7A–7D show that eighth-generation adenine base editors mediate superior A·T to G·C conversion in human cells. Figure 7A shows an overview of adenine base editing: i) ABE8 creates an R-loop at the sgRNA targeting site in the genome; ii) TadA* deaminase chemically converts adenine to inosine through hydrolytic deamination of the ss-DNA portion of the R-loop; iii) Cas9 D10A nickase nicks the strand opposite the inosine-containing strand; iv) The inosine-containing strand can be used as a template during DNA replication; v) In the case of DNA polymerase, inosine preferentially base pairs with cytosine; and vi) After replication, inosine is replaced with guanosine. Figure 7B shows the architecture of ABE8.xm and ABE8.xd. Figure 7C shows three perspective views of E. coli TadA deaminase (PDB 1Z3A) aligned with S. aureus TadA (not shown) in complex with tRNA Arg2 (PDB 2B3J). Mutations identified over eight rounds of evolution are highlighted. Figure 7D is a graph showing the A·T to G·C base editing efficiency of the core ABE8 construct relative to the ABE7.10 construct across eight genomic sites in Hek293T cells. Values ​​and error bars reflect the mean and SD of three independent biological replicates performed on different days.

[0234] [Figure 8]Figures 8A–8C show that the Cas9 PAM variant ABE8 and the catalytically inactive Cas9 ABE8 variant mediate higher A·T to G·C conversion in human cells than the corresponding ABE7.10 variant. Values ​​and error bars reflect the mean and standard deviation (SD) of three independent biological replicates performed on different days. Figure 8A shows A·T to G·C conversion in Hek293T cells harboring NG-Cas9 ABE8s (-NG PAM). Figure 8B shows A·T to G·C conversion in Hek293T cells harboring Sa-Cas9 ABE8s (-NNGRRT PAM). Figure 8C shows A·T to G·C conversion in Hek293T cells harboring catalytically inactive dCas9-ABE8s (S. pyogenes Cas9 D10A, H840A). [Figure 9] Figures 9A-9E show a comparison of on-target and off-target editing frequencies between ABE7.10, ABEmax, and ABEmax with one BPNLS in Hek293T cells. Individual data points are shown for n=3 independent biological replicates performed on different days, and error bars represent standard deviation (SD). Figures 9A and 9B are graphs showing on-target DNA editing frequencies. Figures 9B and 9C are graphs showing the frequency of sgRNA-induced DNA off-target editing. Figure 9E is a graph showing RNA off-target editing frequencies. [Figure 10] Figures 10A-10B show the median A·T to G·C conversions and corresponding indel formation of TadA, C-terminal α-helical truncated ABE constructs in HEK293T cells. Figure 10A is a heatmap showing the median A·T to G·C editing conversions across eight genomic sites. Figure 10B is a heatmap showing indel formation. Delta residue values ​​correspond to the deletion position in TadA. Median values ​​generated from n=3 biological replicates. [Figure 11]Figure 11 is a heatmap showing the median A·T to G·C conversions of 40 ABE8 constructs across eight genomic sites in HEK293T cells. Medians were determined from two or more biological replicates. [Figure 12] Figure 12 is a heatmap showing the median % indels of 40 ABE8 constructs across eight genomic sites in HEK293T cells. Medians were determined from two or more biological replicates. [Figure 13] Figure 13 is a graph showing fold change in editing (ABE8:ABE7). Representation of average ABE8:ABE7 A·T→G·C editing across all A positions within targets at eight different genomic sites in Hek293T cells. Positions 2-12 indicate the location of the target adenine within the 20 nt protospacer, with position 20 immediately 5' to the -NGG PAM. [Figure 14] Figure 14 shows the ABE8 dendrogram, with the core ABE8 constructs selected for further study highlighted in black. [Figure 15] Figure 15 is a heatmap showing the median A⋅T to G⋅C conversions of the eight core ABE8 constructs across eight genomic sites in HEK293T cells. Medians were determined from three or more biological replicates.

[0235] [Figure 16] FIG. 16 is a heatmap showing the median indel frequencies of the eight core ABE8s tested at eight genomic sites in HEK293T cells. [Figure 17] Figure 17 is a heatmap showing the median A·T to G·C conversion of core NG-ABE8 construct 9 (-NG PAM) at six genomic sites in HEK293T cells. Median values ​​generated from n=3 biological replicates. [Figure 18]Figure 18 is a heatmap showing the median indel frequencies of core NG-ABE8 tested at six genomic sites in HEK293T cells. Median values ​​generated from n=3 biological replicates. [Figure 19] Figure 19 is a heatmap showing the median A·T to G·C conversion of the core Sa-ABE8 construct (-NNGRRT PAM) at six genomic sites in HEK293T cells. Site positions within the 22 nt protospacer are numbered from -2 to 20 (5' to 3'). Position 20 is 5' to the NNGRRT PAM. Median values ​​derived from n=3 biological replicates. [Figure 20] Figure 20 is a heatmap showing the median indel frequencies of core sa-ABE8 tested at 8 genomic sites in HEK293T cells. Median values ​​generated from n=3 biological replicates. [Figure 21] Figure 21 is a heatmap showing median A T to G C conversions of the core dC 9-ABE8-m construct at eight genomic sites in HEK293T cells. Dead Cas9 (dC 9) is defined as the D10A and H840A mutations in S. pyogenes Cas9. Median across three or more biological replicates. [Figure 22] Figure 22 is a heatmap showing median A T to G C conversions of the core dC9-ABE8-d construct at eight genomic sites in HEK293T cells. Dead Cas9 (dC9) is defined as the D10A and H840A mutations in S. pyogenes Cas9. Median values ​​generated from n≧3 biological replicates. [Figure 23]Figures 23A and 23B show the median indel frequencies of core dC9-ABE8 tested at eight genomic sites in HEK293T cells. Median values ​​generated from n≧3 biological replicates. Figure 23A is a heatmap showing indels indicated for the dC9-ABE8-m variant compared to ABE7.10. Figure 23B is a heatmap showing indels indicated for the dC9-ABE8-d variant compared to ABE7.10. [Figure 24] Figure 24 shows C·G to T·A editing by Hek293T cells treated with ABE8 and ABE7.10. Editing frequency for each site averaged across all C positions within the target. Cytosines within the protospacer are shaded. [Figure 25] Figures 25A-25H show DNA on-target editing and sgRNA-mediated DNA off-target editing by the ABE8 construct and an ABE8 construct with TadA mutations to improve DNA specificity. Individual data points are shown, and error bars represent the standard deviation (sd) for n=3 independent biological replicates performed on different days. Figures 25A and 25B are graphs showing the on-target DNA editing frequency for the core ABE8 construct compared to ABE7. Figures 25C and 25D are graphs showing the on-target DNA editing frequency for ABE8 with mutations that improve RNA off-target editing. Figures 25E and 25F are graphs showing the sgRNA-guided DNA off-target editing frequency for the core ABE8 construct compared to ABE7. Figures 25G and 25H are graphs showing the gRNA-guided DNA off-target editing frequency for ABE8 constructs with mutations that improve RNA off-target editing.

[0236] [Figure 26]Figure 26 is a graph showing indel frequencies at 12 previously identified sgRNA-dependent Cas9 off-target loci in human cells; individual data points are shown and error bars represent the s.d. for n=3 independent biological replicates performed on different days. [Figure 27] Figures 27A and 27B show A·T to G·C conversion in primary cells and the phenotypic results. Figure 27A is a graph showing A·T to G·C conversion at the -198 HBG1 / 2 site in ABE-treated CD34+ cells from two separate donors. NGS analysis performed 48 and 144 hours after treatment. The -198 HBG1 / 2 target sequence is shown with A7 highlighted. Percent A·T→G·C plotted for A7. Figure 27B is a graph showing the percentage of gamma globin formed as a percentage of alpha globin. Values ​​shown are from two different donors after ABE treatment and erythroid differentiation. [Figure 28] Figures 28A and 28B show A·T→G·C transversions at the −198 promoter site upstream of HBG1 / 2 in CD34+ cells treated with ABE8. Figure 28A is a heatmap showing the frequency of ABE8 A→G edits at 48 and 144 hours after editor treatment in CD34+ cells from two donors, where donor 2 is heterozygous for sickle cell disease. Figure 28B is a graphical representation of the distribution of total sequencing reads containing either the A7 edit alone or the (A7+A8) combined edit. [Figure 29] Figure 29 is a heat map showing indel frequencies at -198 in the gamma globin promoter in ABE8-treated CD34+ cells. Frequencies shown are from two donors at 48 and 144 hours. [Figure 30] FIG. 30 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of untreated differentiated CD34+ cells (donor 1). [Figure 31] FIG. 31 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE7.10-m. [Figure 32] FIG. 32 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE7.10-d. [Figure 33] Figure 33 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.8-m.

[0237] [Figure 34] FIG. 34 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.8-d. [Figure 35] FIG. 35 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.13-m. [Figure 36] FIG. 36 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.13-d. [Figure 37] FIG. 37 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.17-m. [Figure 38] FIG. 38 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.17-d. [Figure 39] FIG. 39 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.20-m. [Figure 40]FIG. 40 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 1) treated with ABE8.20-d. [Figure 41] Figure 41 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of untreated differentiated CD34+ cells (donor 2). Note: Donor 2 is heterozygous for sickle cell disease. [Figure 42] Figure 42 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE7.10-m. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 43] Figure 43 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE7.10-d. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 44] Figure 44 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE8.8-m. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 45] Figure 45 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE8.8-d. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 46] Figure 46 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE8.13-m. Note: Donor 2 is heterozygous for sickle cell disease.

[0238] [Figure 47]Figure 47 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE8.13-d. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 48] Figure 48 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE8.17-m. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 49] Figure 49 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE8.17-d. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 50] Figure 50 shows the UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells (donor 2) treated with ABE8.20-m. Note: Donor 2 is heterozygous for sickle cell disease. [Figure 51] Figures 51A-51E show that editing by ABE8.8 at two independent sites reached over 90% editing before enucleation on day 11 after erythroid differentiation and approximately 60% gamma globin relative to alpha globin or total beta family globins on day 18 after erythroid differentiation. Figure 51A is a graph showing the average of ABE8.8 editing in two healthy donors in two independent experiments. Editing efficiency was measured with primers that distinguish between HBG1 and HBG2. Figure 51B is a graph showing the average of one healthy donor in two independent experiments. Editing efficiency was measured with primers that recognize both HBG1 and HBG2. Figure 51C is a graph showing ABE8.8 editing in a donor with a heterozygous E6V mutation. Figures 51D and 51E are graphs showing the increase in gamma globin in ABE8.8-edited cells. [Figure 52]Figures 52A and 52B show the percent editing (%Editing) using ABE variants to correct sickle cell mutations. Figure 52A is a graph showing screening of different editor variants with approximately 70% editing in SCD patient fibroblasts. Figure 52B is a graph showing CD34 cells from a healthy donor edited with the lead ABE variant, targeting a synonymous mutation A13 at the adjacent proline that lies within the editing window and serves as a proxy for editing of the SCD mutation. The ABE8 variant showed an average editing frequency of approximately 40% at proxy A13. [Figure 53] Figures 53A and 53B show RNA amplicon sequencing to detect intracellular A→I editing in RNA associated with ABE treatment. Individual data points are shown, and error bars represent the standard deviation (sd) for n=3 independent biological replicates performed on different days. Figure 53A is a graph showing the frequency of A→I editing in target RNA amplicons for the core ABE8 construct compared to ABE7 and Cas9 (D10A) nickase controls. Figure 53B is a graph showing the frequency of A→I editing in target RNA amplicons for ABE8 with mutations reported to improve RNA off-target editing.

[0239] [Figure 54] Figure 54 is a bar graph showing the total percent of A to G base editing at splice acceptor target sites (AG nucleic acid sites) achieved by the assayed adenosine base editors (ABE8 variants) compared to the control. The protospacer PAM sequence of the ABE evaluated was NGG ABE. [Figure 55]Figure 55 shows a schematic diagram of the exon 3 splice acceptor of the SOD1 genomic nucleic acid sequence as a target for an A-to-G nucleotide change that causes splice disruption of the SOD1 exon 3 transcript. Shown in the diagram are the genomic DNA location number (top) in the exon 3 region of the SOD1 nucleic acid sequence; the SOD1 genomic nucleic acid sequence in the exon 3 region; the intronic nucleic acid sequence 5' of exon 3; the target splice acceptor nucleic acid sequence 5' of exon 3; and the nucleic acid sequence of the corresponding guide RNA (gRNA), with the splice acceptor ("AG") nucleic acid immediately 5' of SOD1 exon 3 indicated by an upward arrow. Also shown are bystander adenosine (A) nucleobases (boxed) in the intronic sequence near the AG splice acceptor 5' of SOD1 exon 3. [Figure 56] Figure 56 is a table showing the efficiency of target site nucleobase modification in a target nucleic acid sequence observed after PCR and deep sequencing of DNA using the base editor system and methods described herein. Shown is the percentage efficiency of precise A-to-G nucleotide changes (modifications) in the target splice acceptor (AG) nucleic acid sequence located 5' of exon 3 of the SOD1 genomic nucleic acid sequence, as detected by PCR of genomic DNA in cells where base editing occurred, followed by deep sequencing (MySeq). Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid (in base pairs (bp)) is shown along the bottom of the table. The table shows an efficiency of approximately 81% (80.77%) of A-to-G conversion at the splice acceptor target nucleic acid site (position 6 along the target site) using the ABE8 base editor variant and the systems and methods described herein. Modification of bystander A nucleobases to G nucleobases (positions 2, 3, and 4 shown boxed to the left of the target A nucleobase in the splice acceptor) was minimal compared to editing of the target A in the splice acceptor nucleic acid sequence.

[0240] [Figure 57] Figures 57A-57L are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 58] Figure 58 is a bar graph showing the percentage of A to G base editing at Target Position 6 of the splice acceptor target site (AG nucleic acid site) achieved by an adenosine base editor (ABE8 variant) with assayed guide 20 compared to a control. The protospacer PAM sequence of the ABE evaluated was NGG ABE. [Figure 59] Figure 59 is a bar graph showing the percentage of A to G base editing at target position 5 (left) and the total percentage of A to G base editing at positions 2, 5, and 9 (right) in the splice acceptor target site (AG nucleic acid site) achieved by an adenosine base editor (ABE8 variant) using assayed guide 42 compared to a control. The protospacer PAM sequence of the ABE evaluated was NGG ABE. [Figure 60]Figure 60A is a bar graph showing the percentage of A to G base editing at target position 5 in a splice acceptor target site (AG nucleic acid site) achieved by an adenosine base editor (PV variant) with assayed guide 41 compared to a control. The protospacer PAM sequence for the ABE evaluated was NGT ABE. Figure 60B is a table showing the efficiency of target site nucleobase modification in a target nucleic acid sequence observed after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56 above. [Figure 61] Figure 61 is a bar graph showing the total percent of A to G base editing at splice acceptor target sites (AG nucleic acid sites) achieved by adenosine base editors (PV variants) with assayed guide 24 compared to controls. The protospacer PAM sequence of the ABE evaluated was NGA ABE. [Figure 62] Figure 62 shows SOD1 protein levels (left: Western blot, right: quantification) in HEK297T cells edited with ABE8.8 or ABE7.10 using guide 20. Beta actin was used as a control. [Figure 63] Figure 63 is a bar graph showing the percentage of C→T base editing at target position 8 in a splice acceptor target site (AG nucleic acid site) achieved by a cytidine base editor (PV variant) with assayed guide 19 compared to a control. The protospacer PAM sequence of the ABE evaluated was NGC CBE.

[0241] [Figure 64]Figure 64A is a bar graph (left) showing the percentage of A to G base editing at target position 5 in a splice acceptor target site (AG nucleic acid site) achieved by an adenosine base editor (PV variant) with assayed guide 41, compared to a control. The protospacer PAM sequence of the ABE evaluated was NGT ABE. On the right is a diagram showing the numerical genomic DNA positions in the region of exon 3 of the SOD1 nucleic acid sequence to which guides 41, 20, 40, and 21 are designed to bind. See also Figure 55 above. Figure 64B is a table showing the efficiency of target site nucleobase modification in a target nucleic acid sequence after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in DNA (in base pairs (bp)) along the target site nucleic acid is shown along the bottom of the table. See also Figure 56 above. [Figure 65] Figure 65 is a bar graph (left) showing the total percent A to G base editing at splice acceptor target sites (AG nucleic acid sites) achieved by adenosine base editors (ABE variants) with assayed guide 42 compared to a control. The protospacer PAM sequence of the ABE evaluated was NGG ABE. On the right is a diagram showing the numerical genomic DNA positions in the region of exon 3 of the SOD1 nucleic acid sequence to which guides 42, 24, and 25 are designed to bind. See also Figure 55 above. [Figure 66]Figure 66A is a bar graph showing the total percent of C to T base editing achieved by the cytidine base editor (BE4 VRQR) with assayed guide 40 compared to a control. The protospacer PAM sequence for the CBE evaluated was NGA CBE. Figure 66B is a table showing the efficiency of target site nucleobase modification in a target nucleic acid sequence observed after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 67] Figure 67 A is a bar graph showing the percentage of A to G base editing at target position 5 in a splice acceptor target site (AG nucleic acid site) achieved by an adenosine base editor (PV variant) with assayed guide 18 compared to a control. The protospacer PAM sequence of the ABE evaluated was NGT ABE. [Figure 68] Figure 68 is a table showing the efficiency of target site nucleobase modification in a target nucleic acid sequence after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above.

[0242] [Figure 69] Figure 69 is a bar graph showing the total percent of C to T base editing achieved by the cytidine base editor (BE4 VRQR) with guide 16 assayed compared to the control. The protospacer PAM sequence of the CBE evaluated was NGA CBE. [Figure 70]Figure 70 is a bar graph showing the percentage of C to T base editing at target position 4 achieved by a cytidine base editor (BE4 VRQR) with assayed guide 17 compared to a control. The protospacer PAM sequence of the CBE evaluated was an NGA CBE. [Figure 71] Figure 71 is a bar graph showing the percentage of C to T base editing at target position 5 achieved by a cytidine base editor (BE4 VRQR) with assayed guide 21 compared to a control. The protospacer PAM sequence of the CBE evaluated was an NGA CBE. [Figure 72] Figures 72A-72L are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences observed after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 73] Figures 73A-73C are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 74] Figures 74A-74C are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 75]Figures 75A-75D are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above.

[0243] [Figure 76] Figures 76A-76L are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 77] Figures 77A-77L are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 78] Figures 78A-78D are tables showing the efficiency of target site nucleobase modifications in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 79]Figures 79A-79L are tables showing the efficiency of target site nucleobase modification in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Each nucleobase, A, C, G, T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. See also Figure 56, above. [Figure 80] Figure 80 shows that a stop codon was introduced into exon 1 of the androgen receptor when "CAG" was targeted using a cytidine base editor (CBE). [Figure 81] Figure 81 provides two graphs. The graph on the left shows the percent C to T editing by the specified cytidine base editor. The graph on the right shows the percent indel rate for each cytidine base editor. [Figure 82] Figures 82A-82I are tables showing the percentage efficiency of C→T editing in AR nucleic acid sequences using CBE base editor variants, as detected by PCR of genomic DNA in cells in which base editing occurred, followed by deep sequencing (MySeq). Figures 82A-82I show the percent C→T base editing at position 6 of the AR nucleic acid target site. Control reactions using water instead of CBE are shown.

[0244] [Figure 83]Figure 83A provides two graphs. The graph on the left shows that the introduction of a premature stop codon in exon 1 resulted in functional knockout of the androgen receptor in the majority of cells. The middle graph shows the percent indel rate for each cytidine base editor. The right is a diagram showing the numerical genomic DNA position in the region of exon 1 of the AR nucleic acid sequence where guide 8 is designed to bind. See also Figure 80 above. Figure 83B is a table showing the percentage efficiency of C→T editing in the AR nucleic acid sequence using CBE base editor variants, as detected by PCR followed by deep sequencing (MySeq) of genomic DNA in cells where base editing occurred. Figure 83B shows the percent C→T base editing at the target position in the AR nucleic acid target site. Control reactions using water instead of CBE are shown. [Figure 84] Figure 84A is a bar graph showing the percentage of C to T base editing at target position 6 achieved by cytidine base editors (BGX5, BGX27, BGX29, and BTX448) using guide 10 compared to controls (left) and the percentage indel rate (center). The protospacer PAM sequence of the CBE evaluated was NGG CBE. On the right is a diagram showing the numerical genomic DNA position in the region of exon 1 of the AR nucleic acid sequence to which guides 9 and 10 are designed to bind. See also Figure 80 above. Figure 84B is a table showing the efficiency of the percentage of C to T editing in AR nucleic acid sequences using CBE base editor variants, as detected by PCR followed by deep sequencing (MySeq) of genomic DNA in cells in which base editing occurred. Figure 83B shows the percent of C to T base editing at position 6 of the AR nucleic acid target site. Control reactions are shown in which water was used instead of CBE. [Figure 85]Figure 85 is a bar graph showing the total percent A to G base editing at splice acceptor target sites (AG nucleic acid sites) achieved by adenosine base editors (ABE8 variants) with assayed guide 8 (left) or guide 14 (right) compared to controls. The protospacer PAM sequence of the ABE evaluated was NGG ABE. [Figure 86] Figure 86A is a bar graph (top) and summary table (bottom) showing the percentage of C to T base editing at target position 8 achieved by a cytidine base editor (BE4 VRQR) with assayed guide 11 compared to a control. The protospacer PAM sequence for the CBE evaluated was NGA CBE. Figure 86B is a table showing the percentage efficiency of C to T editing in AR nucleic acid sequences using CBE base editor variants, as detected by PCR of genomic DNA in cells in which base editing occurred followed by deep sequencing (MySeq). Figure 86B shows the percent C to T base editing at position 8 in the AR nucleic acid target site. A control reaction is shown in which water was used instead of CBE.

[0245] [Figure 87] Figure 87A is a bar graph (top) and summary table (bottom) showing the percentage of C to T base editing at target position 5 achieved by a cytidine base editor (BE4 VRQR) with assayed guide 12 compared to a control. The protospacer PAM sequence for the CBE evaluated was NGA CBE. Figure 87B is a table showing the percentage efficiency of C to T editing in AR nucleic acid sequences using CBE base editor variants, as detected by PCR of genomic DNA in cells in which base editing occurred followed by deep sequencing (MySeq). Figure 87B shows the percent C to T base editing at position 5 of the AR nucleic acid target site. A control reaction is shown in which water was used instead of CBE. [Figure 88]Figure 88 is a bar graph showing the total percent of C to T base editing achieved by the cytidine base editor (BE4 VRQR) with assayed guide 15 compared to the control. The protospacer PAM sequence of the CBE evaluated was NGA CBE. Each nucleobase A, C, G, and T is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. [Figure 89] Figures 89A-89D are tables showing the efficiency of target site nucleobase modification in a target nucleic acid sequence after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Shown are the percentage efficiencies of precise C→T or A→G nucleotide changes (modifications) in the target nucleic acid sequence in exon 1 of the AR genomic nucleic acid sequence, as detected by PCR of genomic DNA in cells in which base editing occurred, followed by deep sequencing (MySeq). Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. [Figure 90] Figures 90A-90C are tables showing the efficiency of target site nucleobase modification in a target nucleic acid sequence after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Shown are the percentage efficiencies of precise C→T or A→G nucleotide changes (modifications) in the target nucleic acid sequence in exon 1 of the AR genomic nucleic acid sequence, as detected by PCR of genomic DNA in cells in which base editing occurred, followed by deep sequencing (MySeq). Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. [Figure 91]Figures 91A-91C are tables showing the efficiency of target site nucleobase modifications in target nucleic acid sequences after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Shown are the percentage efficiencies of precise C→T or A→G nucleotide changes (modifications) in target nucleic acid sequences in exon 1 of the AR genomic nucleic acid sequence, as detected by PCR of genomic DNA in cells in which base editing occurred, followed by deep sequencing (MySeq). Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid (in base pairs (bp)) is shown along the bottom of the table. [Figure 92] Figures 92A and 92B are tables showing the efficiency of target site nucleobase modifications in target nucleic acid sequences observed after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Shown are the percentage efficiencies of precise C→T or A→G nucleotide changes (modifications) in target nucleic acid sequences in exon 1 of the AR genomic nucleic acid sequence, as detected by PCR of genomic DNA in cells in which base editing occurred, followed by deep sequencing (MySeq). Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. [Figure 93] Figures 93A-93X are tables showing the efficiency of target site nucleobase modification in a target nucleic acid sequence after PCR and deep sequencing of DNA using the base editor systems and methods described herein. Shown are the percentage efficiencies of precise C→T or A→G nucleotide changes (modifications) in the target nucleic acid sequence in exon 1 of the AR genomic nucleic acid sequence, as detected by PCR of genomic DNA in cells in which base editing occurred, followed by deep sequencing (MySeq). Each nucleobase, A, C, G, and T, is listed vertically along the left edge of the table, and its position in the DNA along the target site nucleic acid in base pairs (bp) is shown along the bottom of the table. DETAILED DESCRIPTION OF THE INVENTION

[0246] The present invention provides compositions comprising novel adenine base editors (e.g., ABE8) with increased efficiency for generating targeted nucleobase sequence modifications, and methods of using them.

[0247] [Nucleobase Editor] Disclosed herein are base editors or nucleobase editors for editing, modifying, or altering a target nucleotide sequence of a polynucleotide. In certain embodiments, the base editors of the present invention modify an SOD1 polynucleotide. In certain embodiments, the base editors of the present invention introduce a stop codon or disrupt a splice site in an AR polynucleotide. Described herein are nucleobase editors or base editors comprising a polynucleotide-programmable nucleotide-binding domain (e.g., Cas9) and a nucleobase-editing domain (e.g., adenosine deaminase). The polynucleotide-programmable nucleotide-binding domain (e.g., Cas9), when combined with a bound guide polynucleotide (e.g., gRNA), can specifically bind to the target polynucleotide sequence (via complementary base pairing between the bases of the bound guide nucleic acid and the bases of the target polynucleotide sequence), thereby localizing the base editor to the target nucleic acid sequence 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.

[0248] Polynucleotide-programmable nucleotide-binding domains It should be understood that polynucleotide programmable nucleotide binding domain can also comprise nucleic acid programmable protein that binds to RNA.For example, polynucleotide programmable nucleotide binding domain can be bound to nucleic acid that guides 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.

[0249] The polynucleotide-programmable nucleotide-binding domain of a base editor can itself comprise one or more domains. For example, a polynucleotide-programmable nucleotide-binding domain can comprise one or more nuclease domains. In certain embodiments, the nuclease domain of a polynucleotide-programmable nucleotide-binding domain can comprise an endonuclease or exonuclease. As used herein, the term "exonuclease" refers to a protein or polypeptide capable of digesting nucleic acids (e.g., RNA or DNA) from free ends, and the term "endonuclease" refers to a protein or polypeptide capable of catalyzing (e.g., cleaving) an internal region of a nucleic acid (e.g., DNA or RNA). In certain embodiments, an endonuclease can cleave one strand of a double-stranded nucleic acid. In certain embodiments, an endonuclease can cleave both strands of a double-stranded nucleic acid molecule. In certain embodiments, a polynucleotide-programmable nucleotide-binding domain can be a deoxyribonuclease. In certain embodiments, a polynucleotide-programmable nucleotide-binding domain can be a ribonuclease.

[0250] In certain embodiments, the nuclease domain of a polynucleotide-programmable nucleotide-binding domain can cleave zero, one, or two strands of a target polynucleotide. In certain embodiments, the polynucleotide-programmable nucleotide-binding domain can comprise a nickase domain. As used herein, the term "nickase" refers to a polynucleotide-programmable nucleotide-binding domain that comprises a nuclease domain that can cleave only one strand of a double-stranded nucleic acid molecule (e.g., DNA). In certain embodiments, a nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide-programmable nucleotide-binding domain by introducing one or more mutations into the active polynucleotide-programmable nucleotide-binding domain. For example, if the polynucleotide-programmable nucleotide-binding domain comprises a nickase domain derived from Cas9, the nickase domain derived from Cas9 can comprise a D10A mutation and a histidine at position 840. In such embodiments, residue H840 retains catalytic activity, thereby enabling single-strand cleavage of a nucleic acid duplex. In another example, a Cas9-derived nickase domain can include an H840A mutation, while the amino acid residue at position 10 remains D. In some embodiments, a nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide-programmable nucleotide-binding domain by removing all or a portion of a nuclease domain that is not required for nickase activity. For example, if a polynucleotide-programmable nucleotide-binding domain includes a nickase domain from Cas9, the nickase domain from Cas9 can include a deletion of all or a portion of the RuvC domain or the HNH domain.

[0251] The amino acid sequence of an exemplary catalytically active Cas9 is as follows:

[0252] Thus, a base editor comprising a polynucleotide-programmable nucleotide-binding domain comprising a nickase domain can generate a single-stranded DNA break (nick) in a specific polynucleotide target sequence (e.g., as determined by the complementary sequence of a bound guide nucleic acid). In some embodiments, the strand of a nucleic acid double-stranded target polynucleotide sequence cleaved by a base editor comprising a nickase domain (e.g., a nickase domain derived from Cas9) is the strand not edited by the base editor (i.e., the strand cleaved by the base editor is the strand opposite the strand containing the base to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., a nickase domain derived from Cas9) can cleave the strand of a DNA molecule targeted for editing. In such embodiments, the non-target strand is not cleaved.

[0253] Also provided herein are base editors comprising a catalytically dead (i.e., incapable of cleaving a target polynucleotide sequence) polynucleotide-programmable nucleotide-binding domain. As used herein, the terms "catalytically dead" and "nuclease-inactive" are used interchangeably to refer to a polynucleotide-programmable nucleotide-binding domain having one or more mutations and / or deletions that result in an inability to cleave a strand of nucleic acid. In some embodiments, a catalytically dead polynucleotide-programmable nucleotide-binding domain base editor can lack nuclease activity as a result of specific point mutations in one or more nuclease domains. For example, in the case of a base editor comprising a Cas9 domain, Cas9 can contain both the D10A and H840A mutations. Such mutations inactivate both nuclease domains, resulting in the loss of nuclease activity. In other embodiments, a catalytically dead polynucleotide-programmable nucleotide-binding domain can contain one or more deletions of all or part of a catalytic domain (e.g., the RuvC1 and / or HNH domain). In further embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (e.g., D10A or H840A) as well as a deletion of all or part of the nuclease domain.

[0254] Also contemplated herein are mutations that can generate catalytically dead polynucleotide-programmable nucleotide-binding domains from previously functional versions of the polynucleotide-programmable nucleotide-binding domain. For example, in the case of catalytically dead Cas9 ("dCas9"), variants are provided that have mutations other than D10A and H840A, resulting in nuclease-inactivated Cas9. Such mutations include, for example, other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or RuvC1 subdomain). Additional suitable nuclease-inactive dCas9 domains will be apparent to those skilled in the art based on this disclosure and knowledge in the art, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, 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).

[0255] 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 comprising a natural or modified protein, or a portion thereof, that can bind to a nucleic acid sequence via a binding guide nucleic acid during CRISPR (i.e., Clustered Regularly Interspaced Short Palindromic Repeats)-mediated modification of the nucleic acid. Such proteins are referred to herein as "CRISPR proteins." Accordingly, disclosed herein are base editors that comprise a polynucleotide-programmable nucleotide-binding domain comprising all or a portion of a CRISPR protein (i.e., a base editor that comprises all or a portion of a CRISPR protein as a domain (which is also referred to as the "CRISPR protein-derived domain" of the base editor). The CRISPR protein-derived domain incorporated into the base editor can be modified compared to a wild-type or natural CRISPR protein. For example, as described below, a domain derived from a CRISPR protein can contain one or more mutations, insertions, deletions, rearrangements, and / or recombinations compared to a wild-type or naturally occurring CRISPR protein.

[0256] CRISPR is an adaptive immune system that provides defense against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of the pre-crRNA requires a transcoding small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. tracrRNA guides ribonuclease 3 processing of the pre-crRNA. Cas9 / crRNA / tracrRNA then endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. Target strands not complementary to the crRNA are first endonucleolytically cleaved and then exonucleolytically trimmed 3'-5'. In nature, both proteins and RNAs are required for DNA binding and cleavage. However, a single guide RNA ("sgRNA," or simply "gRNA") can be engineered to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif (the PAM or protospacer adjacent motif) in the CRISPR repeat sequence to help distinguish between "self" and "non-self."

[0257] In some embodiments, the methods described herein can utilize engineered Cas proteins. Guide RNAs (gRNAs) are short synthetic RNAs consisting of a scaffold sequence required for Cas binding and a user-defined approximately 20-base spacer that defines the genomic target to be modified. Thus, one skilled in the art can vary the genomic target of Cas protein specificity, which is determined in part by how specific the gRNA targeting sequence is for the genomic target relative to other parts of the genome.

[0258] In some embodiments, the gRNA scaffold sequence is: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU.

[0259] In some embodiments, the domain derived from a CRISPR protein incorporated into the base editor is an endonuclease (e.g., a deoxyribonuclease or ribonuclease) capable of binding to a target polynucleotide when combined with a bound guide nucleic acid. In some embodiments, the domain derived from a CRISPR protein incorporated into the base editor is a nickase capable of binding to a target polynucleotide when combined with a bound guide nucleic acid. In some embodiments, the domain derived from a CRISPR protein incorporated into the base editor is a catalytically dead domain capable of binding to a target polynucleotide when combined with a bound guide nucleic acid. In some embodiments, the target polynucleotide that binds to the CRISPR protein-derived domain of the base editor is DNA, and in some embodiments, the target polynucleotide that binds to the CRISPR protein-derived domain of the base editor is RNA.

[0260] CAs proteins that can be used herein include class 1 and class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also called Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Cssx16, Cx16, Cx, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i, CARF, DinG, their homologs, or their variants.Unmodified CRISPR enzyme, like Cas9, can have the DNA cleavage activity with two functional endonuclease regions, RuvC and HNH. CRISPR enzymes can induce cleavage of one or both strands of a target sequence, such as within the target sequence and / or within the complementary strand of the target sequence. For example, CRISPR enzymes can induce cleavage of one or both strands at 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.

[0261] Vectors can be used that encode CRISPR enzymes that are mutated relative to the corresponding wild-type enzyme so that they lack the ability to cleave one or both strands of a target polynucleotide containing the target sequence. Cas9 can refer to a polypeptide that has at least, or at least about, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from S. pyogenes). Cas9 can refer to a polypeptide having at most, or at most, about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., from S. pyogenes). Cas9 can refer to wild-type or modified forms of the Cas9 protein, which can include amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0262] In some embodiments, the base editor CRISPR protein-derived domain is derived 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 (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningitidis (NCBI Ref: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus.

[0263] [Cas9 domain, a nucleobase editor] The sequence and structure of Cas9 nuclease are well 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 which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, including Cas9 sequences from the 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.

[0264] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting exemplary Cas9 domains are provided herein. The Cas9 domain can be a nuclease-active Cas9 domain, a nuclease-inactive Cas9 domain (dCas9), or a Cas9 nickase (nCas9). In some embodiments, the Cas9 domain is a nuclease-active domain. For example, the Cas9 domain can be a Cas9 domain that cleaves both strands of a double-stranded nucleic acid (e.g., both strands of a double-stranded DNA molecule). In some embodiments, the Cas9 domain comprises any one of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences described herein.In some embodiments, the Cas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical stretches of amino acid residues compared to any one of the amino acid sequences described herein.

[0265] In some embodiments, proteins comprising a fragment of Cas9 are provided. For example, in some embodiments, the protein comprises one of the following two Cas9 domains: (1) the gRNA binding domain of Cas9; (2) the DNA cleavage domain of Cas9. In some embodiments, 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, Cas9 variants are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 mutant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In certain embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of the corresponding wild-type Cas9.In some embodiments, the fragments are at least 100 amino acids in length. In some embodiments, the fragments are 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.

[0266] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but rather comprise only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0267] The Cas9 protein can bind to a guide RNA, which guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a Cas9 domain, such as a nuclease-active Cas9, Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Examples of nucleic acid-programmable DNA-binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b / C2C1, and Cas12c / C2C3. 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): TIFF0007753096000012.tif171169 (single underline: HNH domain; double underline: RuvC domain)

[0268] In some embodiments, wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence: TIFF0007753096000013.tif167167 (single underline: HNH domain; double underline: RuvC domain)

[0269] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2 (nucleotide sequence is as follows) and Uniprot Reference Sequence: Q99ZW2 (amino acid sequence is as follows): TIFF0007753096000014.tif170166 (single underline: HNH domain; double underline: RuvC domain)

[0270] In some embodiments, Cas9 is Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1) or Neisseria meningitidis (NCBI Ref: YP_002342100.1), or represents Cas9 from any other organism.

[0271] It is understood that additional 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 this disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is nuclease-dead Cas9 (dCas9). In some embodiments, the Cas9 protein is Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is nuclease-active Cas9.

[0272] In some embodiments, the Cas9 domain is a nuclease-inactive Cas9 domain (dCas9). For example, the dCas9 domain can bind to a double-stranded nucleic acid molecule (e.g., via a gRNA molecule) without cleaving either strand of the double-stranded nucleic acid molecule. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10X mutation and a H840X mutation in the amino acid sequence described herein, or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid change. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10A mutation and a H840A mutation in the amino acid sequence described herein, or a corresponding mutation in any of the amino acid sequences described herein. As an example, the nuclease-inactive Cas9 domain comprises the following amino acid sequence provided in the cloning vector pPlatTET-gRNA 2 (Accession No. BAV54124):

[0273] The amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows: (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.)

[0274] Additional suitable nuclease-inactive dCas9 domains will be apparent to those skilled in the art based on this disclosure and knowledge in the art, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, 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).

[0275] In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase, referred to as a "nCas9" protein (for "nickase" Cas9). A nuclease-inactivated Cas9 protein may also be interchangeably referred to as a "dCas9" protein (for nuclease-"dead" Cas9) or catalytically inactive Cas9. Methods for generating 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 contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can suppress the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28;152(5):1173-83 (2013)).

[0276] In some embodiments, the dCas9 domain comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to any of the dCas9 domains provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any of the amino acid sequences described herein.

[0277] In some embodiments, the dCas9 corresponds to, or comprises part or all of, a Cas9 amino acid sequence with one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises D10A and H840A mutations or corresponding mutations in another Cas9.

[0278] In some embodiments, the dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A): TIFF0007753096000015.tif170167 (single underline: HNH domain; double underline: RuvC domain)

[0279] In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 in the amino acid sequence provided above, or the residue at the corresponding position in any of the amino acid sequences provided herein, remains a histidine.

[0280] In other embodiments, dCas9 variants are provided that have mutations other than D10A and H840A, e.g., that result in nuclease-inactivated Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, dCas9 variants or homologs are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants of dCas9 are provided that have amino acid sequences that are 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.

[0281] 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 double-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, the Cas9 nickase cleaves the target strand of a double-stranded nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is base-paired (complementary) to a gRNA (e.g., an sgRNA) bound to the Cas9. In some embodiments, the Cas9 nickase comprises a D10A mutation and has a histidine at position 840. In some embodiments, the Cas9 nickase cleaves the non-target, non-base-edited strand of a double-stranded nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is not base-paired to a gRNA (e.g., an sgRNA) bound to the Cas9. In some embodiments, the Cas9 nickase comprises a H840A mutation and has an aspartic acid residue at position 10, or a corresponding mutation. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the Cas9 nickases provided herein. Additional suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the art, and are within the scope of this disclosure.

[0282] The amino acid sequence of an exemplary catalytic Cas9 nickase (nCas9) is as follows:

[0283] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., nanoarchaea), which constitute the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, the programmable nucleotide-binding protein can be a CasX or CasY protein, as described, for example, in Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genome-resolved metagenomics, many CRISPR-Cas systems have been identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was discovered as part of an active CRISPR-Cas system in the little-studied nanoarchaea. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, have been discovered, which are among the most compact systems discovered to date. In some embodiments, in the base editor systems described herein, Cas9 is replaced by CasX or a variant of CasX. In some embodiments, in the base editor systems described herein, Cas9 is replaced by CasY or a variant of CasY. It should be understood that other RNA-guided DNA-binding proteins can also be used as nucleic acid programmable DNA-binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0284] In some embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) of any of the fusion proteins provided herein can be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide-binding protein is a naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide-binding protein comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to any CasX or CasY protein described herein. It should be understood that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.

[0285] An exemplary CasX ((uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53) tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx protein OS = Sulfolobus islandicus (strain HVE10 / 4) GN = SiH_0402 PE=4 SV=1) amino acid sequence is as follows: MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTSNIILPLSGNDKNPWTETLKCYNFPTTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0286] Exemplary CasX (>tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS = Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4 SV=1) amino acid sequence is the following: MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTSNIILPLSGNDKNPWTETLKCYNFPTTTVALSEVFKNFSQVKECEEVSAPSFVKPFEYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0287] Deltaproteobacteria CasX MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQ KWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0288] An exemplary CasY ((ncbi.nlm.nih.gov / protein / APG80656.1) >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacterium]) amino acid sequence is as follows:

[0289] In some embodiments, the Cas9 is Neisseria menigitidis Cas9 (NmeCas9) or a variant thereof. The characteristics and PAM sequences of NmeCas9 described in Edraki et al. Mol. Cell. (2019) 73(4): 714-726 are incorporated herein by reference in their entirety.

[0290] An exemplary amino acid sequence of Nme1Cas9 is provided below: type II CRISPR RNA-guided endonuclease Cas9 [Neisseria meningitidis] WP_002235162.1 1 maafkpnpin yilgldigia svgwamveid edenpiclid lgvrvferae vpktgdslam 61 arrlarsvrr ltrrrahrll rarrllkreg vlqaadfden glikslpntp wqlraaaldr 121 kltplewsav llhlikhrgy lsqrkneget adkelgallk gvadnahalq tgdfrtpael 181 alnkfekesg hirnqrgdys htfsrkdlqa elillfekqk efgnphvsgg lkegietllm 241 tqrpalsgda vqkmlghctf epaepkaakn tytaerfiwl tklnnlrile qgserpltdt 301 eratlmdepy rkskltyaqa rkllgledta ffkglrygkd naeastlmem kayhaisral 361 ekeglkdkks plnlspelqd eigtafslfk tdeditgrlk driqpeilea llkhisfdkf 421 vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee kiylppipad eirnpvvlra 481 lsqarkving vvrrygspar ihietarevg ksfkdrkeie krqeenrkdr ekaaakfrey 541 fpnfvgepks kdilklrlye qqhgkclysg keinlgrlne kgyveidhal pfsrtwddsf 601 nnkvlvlgse nqnkgnqtpy ​​eyfngkdnsr ewqefkarve tsrfprskkq rillqkfded 661 gfkernlndt ryvnrflcqf vadrmrltgk gkkrvfasng qitnllrgfw glrkvraend 721 rhhaldavvv acstvamqqk itrfvrykem nafdgktidk etgevlhqkt hfpqpweffa 781 qevmirvfgk pdgkpefeea dtpeklrtll aeklssrpea vheyvtplfv srapnrkmsg 841 qghmetvksa krldegvsvl rvpltqlklk dlekmvnrer epklyealka rleahkddpa 901 kafaepfyky dkagnrtqqv kavrveqvqk tgvwvrnhng iadnatmvrv dvfekgdkyy 961 lvpiyswqva kgilpdravv qgkdeedwql iddsfnfkfs lhpndlvevi tkkarmfgyf 1021 aschrgtgni nirihdldhk igkngilegi gvktalsfqk yqidelgkei rpcrlkkrpp 1081vr

[0291] An exemplary amino acid sequence of Nme2Cas9 is provided below: type II CRISPR RNA-guided endonuclease Cas9 [Neisseria meningitidis] WP_002230835.1 1 maafkpnpin yilgldigia svgwamveid eeenpirlid lgvrvferae vpktgdslam 61 arrlarsvrr ltrrrahrll rarrllkreg vlqaadfden glikslpntp wqlraaaldr 121 kltplewsav llhlikhrgy lsqrkneget adkelgallk gvannahalq tgdfrtpael 181 alnkfekesg hirnqrgdys htfsrkdlqa elillfekqk efgnphvsgg lkegietllm 241 tqrpalsgda vqkmlghctf epaepkaakn tytaerfiwl tklnnlrile qgserpltdt 301 eratlmdepy rkskltyaqa rkllgledta ffkglrygkd naeastlmem kayhaisral 361 ekeglkdkks plnlsselqd eigtafslfk tdeditgrlk drvqpeilea llkhisfdkf 421 vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee kiylppipad eirnpvvlra 481 lsqarkving vvrrygspar ihietarevg ksfkdrkeie krqeenrkdr ekaaakfrey 541 fpnfvgepks kdilklrlye qqhgkclysg keinlvrlne kgyveidhal pfsrtwddsf 601 nnkvlvlgse nqnkgnqtpy eyfngkdnsr ewqefkarve tsrfprskkq rillqkfded 661 gfkecnlndt ryvnrflcqf vadhilltgk gkrrvfasng qitnllrgfw glrkvraend 721 rhhaldavvv acstvamqqk itrfvrykem nafdgktidk etgkvlhqkt hfpqpweffa 781 qevmirvfgk pdgkpefeea dtpeklrtll aeklssrpea vheyvtplfv srapnrkmsg 841 ahkdtlrsak rfvkhnekis vkrvwlteik ladlenmvny kngreielye alkarleayg 901 gnakqafdpk dnpfykkggq lvkavrvekt qesgvllnkk naytiadngd mvrvdvfckv 961 dkkgknqyfi vpiyawqvae nilpdidckg yriddsytfc fslhkydlia fqkdekskve 1021 fayyincdss ngrfylawhd kgskeqqfri stqnlvliqk yqvnelgkei rpcrlkkrpp 1081vr

[0292] The Cas9 nuclease contains two functional endonuclease domains, RuvC and HNH. Upon binding to target DNA, Cas9 undergoes a conformational change that positions 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 can be repaired by one of two general repair pathways: (1) the efficient but error-prone non-homologous end joining (NHEJ) pathway; or (2) the less efficient but high-fidelity homology-directed repair (HDR) pathway.

[0293] The "efficiency" of non-homologous end joining (NHEJ) and / or homology-directed repair (HDR) can be calculated by any convenient method. For example, in certain embodiments, efficiency can be expressed as the percentage of successful HDR. For example, a test nuclease assay can be used to generate cleavage products, and the percentage can be calculated using the ratio of product to substrate. For example, a test nuclease enzyme can be used that directly cleaves DNA containing the newly incorporated restriction sequence as a result of successful HDR. The more substrate cleaved, the higher the rate of HDR (the more efficient the HDR). As an illustrative example, the rate (percentage) of HDR can be calculated using the following formula: [(cleavage product) / (substrate + cleavage product)] (e.g., (b + c) / (a ​​+ b + c), where "a" is the band intensity of the DNA substrate, and "b" and "c" are the cleavage products).

[0294] In one embodiment, efficiency can be expressed as the success rate of NHEJ. For example, a T7 endonuclease I assay can be used to generate cleavage products, and the ratio of product to substrate can be used to calculate the percentage of NHEJ. T7 endonuclease I cleaves mismatched heteroduplex DNA resulting from hybridization of wild-type and mutant DNA strands (NHEJ generates small random insertions or deletions (indels) at the initial cleavage site). More cleavages indicate a higher rate of NHEJ (more efficient NHEJ). As an illustrative example, the rate (percentage) of NHEJ can be calculated using the formula (1-(1-(b+c) / (a+b+c)) 1 / 2 ) × 100, where "a" is the band intensity of the DNA substrate, and "b" and "c" are the cleavage products (Ran et al., Cell. 2013 Sep. 12; 154(6):1380-9; and Ran et al., Nat Protoc. 2013 Nov.; 8(11): 2281-2308).

[0295] The NHEJ repair pathway is the most active repair mechanism, frequently resulting in small nucleotide insertions or deletions (indels) at DSB sites. The random nature of NHEJ-mediated DSB repair has important practical implications, as cell populations expressing Cas9 and gRNA or guide polynucleotides can result in a diverse array of mutations. In most embodiments, NHEJ generates small indels in the target DNA, resulting in amino acid deletions, insertions, or frameshift mutations that result in premature stop codons within the open reading frame (ORF) of the target gene. The ideal end result is a loss-of-function mutation within the target gene.

[0296] While 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. To utilize HDR for gene editing, a DNA repair template containing the desired sequence can be delivered to the cell type of interest along with a gRNA and Cas9 or Cas9 nickase. The repair template can contain additional homologous sequences immediately upstream and downstream of the desired edit and its target (called left and right homologous arms). The length of each homologous arm can depend on the magnitude of the introduced change, with larger insertions requiring longer homologous arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. HDR efficiency is generally low (less than 10% corrected alleles), even in cells expressing Cas9, gRNA, and an exogenous repair template. Because HDR occurs between the S and G2 phases of the cell cycle, HDR efficiency can be increased by synchronizing cells. Chemically or genetically inhibiting genes involved in NHEJ can also increase HDR frequency.

[0297] In some embodiments, Cas9 is a modified (altered) Cas9. A given gRNA target sequence may have additional sites of partial homology throughout the genome. These sites are called off-targets and need to be considered when designing gRNA. In addition to optimizing gRNA design, CRISPR specificity can also be improved by modifying Cas9. Cas9 generates double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase, a D10A mutant of SpCas9, retains one nuclease domain and generates DNA nicks instead of DSBs. Nickases can also be combined with HDR-mediated gene editing for specific gene editing.

[0298] In some embodiments, the Cas9 is a variant Cas9 protein. A variant Cas9 polypeptide has an amino acid sequence that differs by a single amino acid (e.g., by a deletion, insertion, substitution, or fusion) compared to the amino acid sequence of a wild-type Cas9 protein. In some instances, 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 instances, the variant Cas9 polypeptide has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 protein. In some embodiments, the variant Cas9 protein does not have substantial nuclease activity. When a subject Cas9 protein is a variant Cas9 protein that does not have substantial nuclease activity, it may be referred to as "dCas9."

[0299] In some embodiments, the variant Cas9 protein has reduced nuclease activity, e.g., 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 a wild-type Cas9 protein (e.g., a wild-type Cas9 protein).

[0300] In some embodiments, a variant Cas9 protein can cleave the complementary strand of a guide target sequence but has a reduced ability to cleave the non-complementary strand of a double-stranded guide target sequence. 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 to alanine at amino acid position 10), and thus can cleave the complementary strand of a double-stranded guide target sequence but has a reduced ability to cleave the non-complementary strand of a double-stranded guide target sequence (thus, when this variant Cas9 protein cleaves a double-stranded target nucleic acid, it generates a single-strand break (SSB) instead of a double-strand break (DSB)) (see, e.g., Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21).

[0301] In some embodiments, a variant Cas9 protein can cleave the non-complementary strand of a double-stranded guide target sequence, but has a reduced ability to cleave the complementary strand of the guide target sequence. For example, the variant Cas9 protein 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 to alanine at amino acid position 840) mutation, and thus can cleave the non-complementary strand of a guide target sequence, but has a reduced ability to cleave the complementary strand of a guide target sequence (thus, when this variant Cas9 protein cleaves a double-stranded guide target sequence, an SSB occurs instead of a DSB). Such a Cas9 protein has a reduced ability to cleave a guide target sequence (e.g., a single-stranded guide target sequence), but retains the ability to bind to the guide target sequence (e.g., a single-stranded guide target sequence).

[0302] In some embodiments, the variant Cas9 protein has a reduced ability to cleave both the complementary and non-complementary strands of double-stranded target DNA. As a non-limiting example, in some embodiments, the variant Cas9 protein has both the D10A and H840A mutations, resulting in a polypeptide with a reduced ability to cleave both the complementary and non-complementary strands of 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).

[0303] As another non-limiting example, in some embodiments, a variant Cas9 protein has a W476A and a W1126A mutation such that the polypeptide has a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0304] As another non-limiting example, in some embodiments, a variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations such that the polypeptide has a reduced ability to cleave 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).

[0305] As another non-limiting example, in some embodiments, a variant Cas9 protein has H840A, W476A, and W1126A mutations, resulting in a polypeptide with reduced ability to cleave target DNA (e.g., single-stranded target DNA) while retaining the ability to bind to target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, a variant Cas9 protein has H840A, D10A, W476A, and W1126A mutations, resulting in a polypeptide with reduced ability to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA) while retaining the ability to bind to target DNA (e.g., single-stranded target DNA). In some embodiments, a variant Cas9 has a restored catalytic His residue at position 840 of the Cas9 HNH domain (A840H).

[0306] As another non-limiting example, in some embodiments, a variant Cas9 protein has H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, such that the polypeptide has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind to target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, a variant Cas9 protein has D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, such that the polypeptide has a reduced ability to cleave target DNA (e.g., single-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). In some embodiments, when a variant Cas9 protein has the W476A and W1126A mutations, or when a variant Cas9 protein has the P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, the variant Cas9 protein does not efficiently bind to a PAM sequence. Thus, in some such embodiments, when such a variant Cas9 protein is used in a binding method, the method does not require a PAM sequence. In other words, in some embodiments, when such a variant Cas9 protein is used in a binding method, the method may include a guide RNA, but the method can be performed in the absence of a PAM sequence (thus, binding specificity is provided by the target segment of the guide RNA). To achieve the above effect, other residues may be mutated (i.e., inactivating one or other nuclease moiety). By way of non-limiting example, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 can be altered (i.e., substituted). Mutations other than alanine substitutions are also suitable.

[0307] In some embodiments, a variant Cas9 protein with reduced catalytic activity (e.g., when the Cas9 protein has a D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 mutation, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A) can bind to target DNA in a site-specific manner (because it is guided to the target DNA sequence by the guide RNA), so long as it retains the ability to interact with the guide RNA.

[0308] 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.

[0309] In some embodiments, a modified SpCas9 was used that contained the amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and has specificity for a modified PAM5'-NGC-3'.

[0310] Alternatives to S. pyogenes Cas9 include RNA-guided endonucleases from the Cpf1 family that exhibit cleavage activity in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease in a class II CRISPR / Cas system. This adaptive immune mechanism is found in Prevotella and Francisella bacteria. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the limitations of the CRISPR / Cas9 system. Unlike Cas9 nuclease, Cpf1-mediated DNA cleavage results in a double-strand break with a short 3' overhang. The staggered cleavage pattern of Cpf1 opens up the possibility of directional gene transfer, similar to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Like the Cas9 variants and orthologs described above, Cpf1 can also expand the number of CRISPR targetable sites to AT-rich regions or AT-rich genomes lacking the NGG PAM site preferred by SpCas9. The Cpf1 locus contains an α / β mixed domain, a RuvC-I region followed by a helical region, a RuvC-II region, and a zinc finger-like domain. The Cpf1 protein possesses a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, Cpf1 lacks the HNH endonuclease domain, and the N-terminus of Cpf1 lacks the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain organization demonstrated that Cpf1 is functionally unique and classified as a class 2, type V CRISPR system. The Cpf1 locus encoded Cas1, Cas2, and Cas4 proteins that were more similar to type I and type III systems than to type II systems. Functional Cpf1 does not require trans-activating CRISPR RNA (tracrRNA); therefore, it requires only CRISPR (crRNA).Cpf1 is not only smaller than Cas9, but also possesses a smaller sgRNA molecule (approximately half the number of nucleotides of Cas9), which is beneficial for genome editing. In contrast to the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex cleaves target DNA or RNA by identifying a protospacer adjacent to the motif 5'-YTN-3'. After identifying the PAM, Cpf1 introduces a sticky-end-like DNA double-strand break with a 4- or 5-nucleotide overhang.

[0311] [Cas12 domain, a nucleobase editor] Microbial CRISPR-Cas systems are typically divided into class 1 and class 2 systems. Class 1 systems have multisubunit effector complexes, while class 2 systems have single protein effectors. For example, Cas9 and Cpf1 are class 2 effectors, albeit of different types (type II and type V, respectively). In addition to Cpf1, class 2, type V CRISPR-Cas systems also contain Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. See, for example, Shmakov et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems," Mol. Cell, 2015 Nov. 5; 60(3): 385-397; Makarova et al., "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR Journal, 2018, 1(5): 325-336; and Yan et al., "Functionally Diverse Type V CRISPR-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) endonuclease domain. While production of mature CRISPR RNA (crRNA) is generally independent of tracrRNA, for example, Cas12b / C2c1 requires tracrRNA for crRNA production. Cas12b / C2c1 depends on both crRNA and tracrRNA for DNA cleavage.

[0312] Nucleic acid programmable DNA-binding proteins contemplated by the present invention include Cas proteins classified as Class 2, Type V (Cas12 proteins). Non-limiting examples of Cas Class 2, Type V proteins include Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i, their homologs, or modified versions. As used herein, Cas12 proteins may also be referred to as Cas12 nucleases, Cas12 domains, or Cas12 protein domains. In some embodiments, the Cas12 proteins of the present invention comprise an amino acid sequence interrupted by an internally fused protein domain, such as a deaminase domain.

[0313] In some embodiments, the Cas12 domain is a nuclease-inactive Cas12 domain or a Cas12 nickase. In some embodiments, the Cas12 domain is a nuclease-active domain. For example, the Cas12 domain can be a Cas12 domain that nicks one strand of a double-stranded nucleic acid (e.g., a double-stranded DNA molecule). In some embodiments, the Cas12 domain comprises any one of the amino acid sequences described herein. In some embodiments, the Cas12 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences described herein. In some embodiments, the Cas12 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of the amino acid sequences described herein. In some embodiments, the Cas12 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any one of the amino acid sequences described herein.

[0314] In some embodiments, proteins comprising a fragment of Cas12 are provided. For example, in some embodiments, the protein comprises one of the following two Cas12 domains: (1) the gRNA binding domain of Cas12, or (2) the DNA cleavage domain of Cas12. In some embodiments, proteins comprising Cas12 or a fragment thereof are referred to as "Cas12 variants." Cas12 variants share homology with Cas12 or a fragment thereof. For example, a Cas12 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas12. In some embodiments, a 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, a Cas12 variant comprises a fragment of Cas12 (e.g., the gRNA binding domain or the DNA cleavage domain) such that the fragment has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to the corresponding fragment of wild-type Cas12.In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 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, fragments are 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.

[0315] In some embodiments, the Cas12 corresponds to, or comprises a portion or all of, a Cas12 amino acid sequence with one or more mutations that alter Cas12 nuclease activity. Such mutations include, by way of example, amino acid substitutions within the RuvC nuclease domain of Cas12. In some embodiments, variants or homologs of Cas12 are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas12. In some embodiments, variants of Cas12 are provided that have amino acid sequences that are about 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, or more amino acids shorter or longer.

[0316] In some embodiments, the Cas12 fusion proteins provided herein comprise the full-length amino acid sequence of a Cas12 protein, e.g., one of the Cas12 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas12 sequence, but only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas12 domains are provided herein, and additional suitable sequences of Cas12 domains and fragments will be apparent to those of skill in the art.

[0317] Generally, Class 2, Type V 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 Cas12b protein (see, e.g., Strecker et al., Nature Communications, 2019, 10(1): Art. No.: 212). In one embodiment, a variant Cas12 polypeptide has an amino acid sequence that differs by one, two, three, four, five, or more amino acids (e.g., has a deletion, insertion, substitution, or fusion) compared to the amino acid sequence of a wild-type Cas12 protein. In some cases, 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 cases, a variant Cas12b is a Cas12b polypeptide that has less than 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the nickase activity of a corresponding wild-type Cas12b protein. In some cases, the variant Cas12b protein has substantially no nickase activity.

[0318] In some cases, the variant Cas12b protein has reduced nickase activity, e.g., the variant Cas12b protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the nickase activity of a wild-type Cas12b protein.

[0319] In some embodiments, the Cas12 protein comprises an RNA-guided endonuclease from the Cas12a / Cpf1 family that is active in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of the Class II CRISPR / Cas system. This adaptive immune mechanism is found in Prevotella and Francisella bacteria. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses guide RNA to locate and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the limitations of the CRISPR / Cas9 system. Unlike Cas9 nuclease, Cpf1-mediated DNA cleavage results in a double-strand break with a short 3' overhang. The zigzag cleavage pattern of Cpf1 opens up the possibility of directional gene transfer, which can enhance the efficiency of gene editing, similar to conventional restriction enzyme cloning. Like the Cas9 variants and orthologs mentioned above, Cpf1 can also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes lacking the NGGPAM sites preferred by SpCas9. The Cpf1 locus contains a mixed alpha / beta domain, RuvC-I followed by a helical region, RuvC-II, and a zinc finger-like domain. The Cpf1 protein contains a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, unlike Cas9, Cpf1 does not possess an HNH endonuclease domain, and the N-terminus of Cpf1 lacks the alpha-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain architecture indicates that Cpf1 is functionally unique and is classified as a class 2, type V CRISPR system. The Cpf1 locus encodes the Cas1, Cas2, and Cas4 proteins and is more similar to types I and III than to type II systems.Functional Cpf1 does not require trans-activating CRISPR RNA (tracrRNA), so only CRISPR (crRNA) is required. Cpf1 is not only smaller than Cas9, but its sgRNA molecule is also smaller (approximately half the number of nucleotides as Cas9), which is advantageous for genome editing. The Cpf1-crRNA complex cleaves target DNA or RNA by identifying the protospacer-adjacent motif 5'-YTN-3' or 5'-TTTN-3', in contrast to the G-rich PAM targeted by Cas9. After identifying the PAM, Cpf1 introduces a sticky-end-like DNA double-strand break with a 4- or 5-nucleotide overhang.

[0320] In some aspects of the invention, the vector encodes a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the 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 hisashii). Cas12 can refer to a polypeptide with at most, or at most, about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas12 polypeptide (e.g., from Bacillus hisashii (BhCas12b), Bacillus sp. V3-13 (BvCas12b), and Alicyclobacillus acidiphilus (AaCas12b)). Cas12 can refer to wild-type or modified forms of the Cas12 protein, which may include amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0321] [Nucleic acid-programmable DNA-binding protein] Certain aspects of the present disclosure provide fusion proteins comprising a domain that acts as a nucleic acid-programmable DNA-binding protein, which can be used to target proteins such as base editors to specific nucleic acid (e.g., DNA or RNA) sequences. In certain embodiments, the fusion protein comprises a nucleic acid-programmable DNA-binding protein domain and a deaminase domain. Non-limiting examples of nucleic acid-programmable DNA-binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also called Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, and Cse 1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3 , Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins include Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, homologs thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, even though they may not be specifically listed herein.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 are incorporated herein by reference).

[0322] An example of a nucleic acid programmable DNA-binding protein with PAM specificity distinct from Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats (Cpf1) from Prevotella and Francisella1. Like Cas9, Cpf1 is also a class 2 CRISPR effector. Cpf1 has been shown to mediate robust DNA interference with characteristics distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA and utilizing T-rich protospacer adjacent motifs (TTN, TTTN, or YTN). Furthermore, Cpf1 cleaves DNA with staggered double-strand breaks. Of the 16 Cpf1 family proteins, two enzymes from Acidaminococcus and Lachnospiraceae have been shown to have efficient genome editing activity in human cells. The Cpf1 protein is known in the art and has been previously described, for example, in Yamano et al., "Crystal structure of Cpf1 in complex with guide RNA and target DNA." Cell (165) 2016, pp. 949-962, the entire contents of which are incorporated herein by reference.

[0323] Nuclease-inactive Cpf1 (dCpf1) variants, which can be used as guide nucleotide sequence-programmable DNA-binding protein domains, are also useful in the compositions and methods of the present invention. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9, but does not have the HNH endonuclease domain, and the N-terminus of Cpf1 does not have the alpha-helix recognition lobe of Cas9. Zetsche et al., Cell, 163, 759-771, 2015 (incorporated herein by reference) showed that the RuvC-like domain of Cpf1 is responsible for cleaving both DNA strands, and inactivating the RuvC-like domain inactivates Cpf1 nuclease activity. For example, mutations corresponding to D917A, E1006A, or D1255A in Francisella novicida Cpf1 inactivate Cpf1 nuclease activity. In some embodiments, dCpf1 of the present disclosure includes mutations corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It is understood that any mutation that inactivates the RuvC domain of Cpf1, e.g., a substitution mutation, deletion, or insertion, can be used in accordance with the present disclosure.

[0324] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein can be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In certain aspects, the Cpf1 protein is a nuclease-inactive Cpf1 (dCpf1). In some embodiments, the Cpf1, nCpf1, or dCpf1 comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a Cpf1 sequence disclosed herein. In some embodiments, the dCpfl comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a Cpfl sequence disclosed herein, and includes mutations corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It should be understood that Cpfl from other bacterial species may also be used in accordance with the present disclosure.

[0325] Wild-type Francisella novicida Cpf1 (D917, E1006, and D1255 are bold and underlined). MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKL ALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDKAIKENKGEGYKKIVYKKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIE...

Claims

1. 1. A pharmaceutical composition for treating a neurological disorder in a subject, the pharmaceutical composition comprising: (i) an adenosine base editor or a nucleic acid molecule encoding the same; and (ii) a guide polynucleotide or a nucleic acid molecule encoding the same, wherein the adenosine base editor comprises a programmable DNA-binding domain and an adenosine deaminase domain, and the adenosine deaminase domain has the following amino acid sequence: TadA*7.10: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD And, or having a single alteration selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R; Y147R, Q154R, and Y123H; Y147R, Q154R, and I76Y; Y147R, Q154R, and T166R; Y147T, Q154R; Y147T, Q154S; Y123H, Y147R, Q154R, and I76Y; I76Y and V82S; V82S and Y147R; V82S, Y123H, and Y147R; V82S and Q154R; V82S, Y123H, and Q154R; V82S, Y123H, Y147R, and Q154R; I76Y, V82S, Y123H, Y147R, and Q154R; Y147R and Q154S; V82S and Q154S; V82S and Y123H; and V82S, Y123H, and Y147T having a single modification combination selected from the group consisting of: The amino acid sequence or a fragment thereof lacking only the N-terminal methionine Including, The pharmaceutical composition, wherein the guide polynucleotide induces the adenosine base editor to result in a modification of a nucleobase at a splice site of a target gene associated with a neurological disorder in the subject, and the target gene is a superoxide dismutase 1 (SOD1) gene or an androgen receptor (AR) gene.

2. 2. The pharmaceutical composition of claim 1, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 in the TadA*7.10 numbering or a corresponding substitution.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the modification of the nucleobase results in alternative splicing of a transcript encoded by the target gene.

4. The pharmaceutical composition of claim 3 , wherein the alternative splicing produces a truncated or non-functional protein encoded by the target gene.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the modification of the nucleic acid base results in reduced expression of the target gene in the subject.

6. A pharmaceutical composition for treating amyotrophic lateral sclerosis (ALS) in a subject, the pharmaceutical composition comprising: (i) a base editor or a nucleic acid molecule encoding the same; and (ii) a guide polynucleotide or a nucleic acid molecule encoding the same; The base editor comprises a programmable DNA binding domain and TadA*7.10 amino acid sequence below: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD And, or having a single alteration selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R; Y147R, Q154R, and Y123H; Y147R, Q154R, and I76Y; Y147R, Q154R, and T166R; Y147T, Q154R; Y147T, Q154S; Y123H, Y147R, Q154R, and I76Y; I76Y and V82S; V82S and Y147R; V82S, Y123H, and Y147R; V82S and Q154R; V82S, Y123H, and Q154R; V82S, Y123H, Y147R, and Q154R; I76Y, V82S, Y123H, Y147R, and Q154R; Y147R and Q154S; V82S and Q154S; V82S and Y123H; and V82S, Y123H, and Y147T having a single modification combination selected from the group consisting of: The amino acid sequence or a fragment thereof lacking only the N-terminal methionine adenosine deaminase domain, including Including, The pharmaceutical composition, wherein the guide polynucleotide directs the base editor to modify a nucleobase at a splice site of the subject's superoxide dismutase 1 (SOD1) gene.

7. 7. The pharmaceutical composition of claim 6, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 in the numbering in TadA*7.10 or a substitution corresponding thereto.

8. The pharmaceutical composition according to claim 6 or 7, wherein the modification of the nucleic acid base is in the splice acceptor site of the SOD1 gene.

9. The pharmaceutical composition of any one of claims 6 to 8, wherein the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of 5'-UUAAAGGAAAGUAAUGGACCAGU-3', 5'-UAAAUAGGCUGUACCAGUGCAGG-3', 5'-UUCAUUAUUAGGCAUGUUGGAGA-3', 5'-AAAUAGGCUGUACCAGUGCAGGU-3', and 5'-UAUUAGGCAUGUUGGAGACUUGG-3'.

10. The pharmaceutical composition according to any one of claims 1 to 5, wherein the target gene is the androgen receptor (AR) gene and the neurological disorder is spinal-bulbar muscular atrophy (SBMA).

11. A pharmaceutical composition for treating spinal and bulbar muscular atrophy (SBMA) in a subject, the pharmaceutical composition comprising: (i) a base editor or a nucleic acid molecule encoding the same; and (ii) a guide polynucleotide or a nucleic acid molecule encoding the same; The base editor comprises a programmable DNA binding domain and TadA*7.10 amino acid sequence below: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD And, or having a single alteration selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R; Y147R, Q154R, and Y123H; Y147R, Q154R, and I76Y; Y147R, Q154R, and T166R; Y147T, Q154R; Y147T, Q154S; Y123H, Y147R, Q154R, and I76Y; I76Y and V82S; V82S and Y147R; V82S, Y123H, and Y147R; V82S and Q154R; V82S, Y123H, and Q154R; V82S, Y123H, Y147R, and Q154R; I76Y, V82S, Y123H, Y147R, and Q154R; Y147R and Q154S; V82S and Q154S; V82S and Y123H; and V82S, Y123H, and Y147T having a single modification combination selected from the group consisting of: The amino acid sequence or a fragment thereof lacking only the N-terminal methionine adenosine deaminase domain, including Including, the guide polynucleotide directs the base editor to effect a nucleobase modification at a splice site of the androgen receptor (AR) gene of the subject.

12. 12. The pharmaceutical composition of claim 11, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 in the TadA*7.10 numbering.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the subject is a non-human mammal or a human.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the administration of the pharmaceutical composition is carried out via delivery to cells or motor neurons of the central nervous system (CNS) of the subject.

15. 1. An in vitro or ex vivo method for modifying a target gene or a regulatory element thereof associated with a neurological disorder, comprising contacting the target gene or a regulatory element thereof with (i) an adenosine base editor or a nucleic acid molecule encoding same, and (ii) a guide polynucleotide or a nucleic acid molecule encoding same, wherein the adenosine base editor comprises a programmable DNA-binding domain and an adenosine deaminase domain, and wherein the adenosine deaminase domain has the following amino acid sequence: TadA*7.10: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD And, or having a single alteration selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R; Y147R, Q154R, and Y123H; Y147R, Q154R, and I76Y; Y147R, Q154R, and T166R; Y147T, Q154R; Y147T, Q154S; Y123H, Y147R, Q154R, and I76Y; I76Y and V82S; V82S and Y147R; V82S, Y123H, and Y147R; V82S and Q154R; V82S, Y123H, and Q154R; V82S, Y123H, Y147R, and Q154R; I76Y, V82S, Y123H, Y147R, and Q154R; Y147R and Q154S; V82S and Q154S; V82S and Y123H; and V82S, Y123H, and Y147T having a single modification combination selected from the group consisting of: The amino acid sequence or a fragment thereof lacking only the N-terminal methionine wherein the guide polynucleotide directs the base editor to effect a nucleobase modification at a splice site of the target gene, and the target gene is superoxide dismutase 1 (SOD1) gene or the androgen receptor (AR) gene.

16. The method of claim 15, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 in the TadA*7.10 numbering.

17. 17. The method of claim 15 or 16, wherein the nucleobase modification results in alternative splicing of a transcript encoded by the target gene, a truncated and / or non-functional protein encoded by the target gene, and / or reduced expression of the target gene when expressed in a cell.

18. The method according to any one of claims 15 to 17, wherein the target gene is superoxide dismutase 1 (SOD1) gene and the neurological disorder is amyotrophic lateral sclerosis (ALS).

19. An in vitro or ex vivo method for regulating expression of a superoxide dismutase (SOD1) gene, comprising contacting the SOD1 gene or a regulatory element thereof with (i) a base editor or a nucleic acid molecule encoding the same, and (ii) a guide polynucleotide or a nucleic acid molecule encoding the same; the base editor comprising a programmable DNA binding domain and TadA*7.10 amino acid sequence below: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD And, or having a single alteration selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R; Y147R, Q154R, and Y123H; Y147R, Q154R, and I76Y; Y147R, Q154R, and T166R; Y147T, Q154R; Y147T, Q154S; Y123H, Y147R, Q154R, and I76Y; I76Y and V82S; V82S and Y147R; V82S, Y123H, and Y147R; V82S and Q154R; V82S, Y123H, and Q154R; V82S, Y123H, Y147R, and Q154R; I76Y, V82S, Y123H, Y147R, and Q154R; Y147R and Q154S; V82S and Q154S; V82S and Y123H; and V82S, Y123H, and Y147T having a single modification combination selected from the group consisting of: The amino acid sequence or a fragment thereof lacking only the N-terminal methionine adenosine deaminase domain, including wherein said guide polynucleotide directs said base editor to effect a nucleobase modification at a splice site of a superoxide dismutase 1 (SOD1) gene.

20. 1. An in vitro or ex vivo method for modulating expression of an androgen receptor (AR) gene, comprising contacting the AR gene or a regulatory element thereof with (i) a base editor or a nucleic acid molecule encoding same, and (ii) a guide polynucleotide or a nucleic acid molecule encoding same; The base editor comprises a programmable DNA binding domain and TadA*7.10 amino acid sequence below: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD And, or having a single alteration selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R; Y147R, Q154R, and Y123H; Y147R, Q154R, and I76Y; Y147R, Q154R, and T166R; Y147T, Q154R; Y147T, Q154S; Y123H, Y147R, Q154R, and I76Y; I76Y and V82S; V82S and Y147R; V82S, Y123H, and Y147R; V82S and Q154R; V82S, Y123H, and Q154R; V82S, Y123H, Y147R, and Q154R; I76Y, V82S, Y123H, Y147R, and Q154R; Y147R and Q154S; V82S and Q154S; V82S and Y123H; and V82S, Y123H, and Y147T having a single modification combination selected from the group consisting of: The amino acid sequence or a fragment thereof lacking only the N-terminal methionine adenosine deaminase domain, including wherein said guide polynucleotide directs said base editor to effect a nucleobase modification at a splice site of an androgen receptor (AR) gene.

21. 21. The method of claim 20, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 in the TadA*7.10 numbering.

22. 22. The method of claim 20 or 21, wherein the adenosine deaminase domain comprises a V28S mutation or a T166R mutation or a corresponding mutation in the numbering in TadA*7.

10.

23. A base editor system comprising: (i) a base editor or a nucleic acid molecule encoding the same; and (ii) a guide polynucleotide or a nucleic acid molecule encoding the same, The base editor comprises a programmable DNA binding domain and TadA*7.10 amino acid sequence below: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD And, or having a single alteration selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R; Y147R, Q154R, and Y123H; Y147R, Q154R, and I76Y; Y147R, Q154R, and T166R; Y147T, Q154R; Y147T, Q154S; Y123H, Y147R, Q154R, and I76Y; I76Y and V82S; V82S and Y147R; V82S, Y123H, and Y147R; V82S and Q154R; V82S, Y123H, and Q154R; V82S, Y123H, Y147R, and Q154R; I76Y, V82S, Y123H, Y147R, and Q154R; Y147R and Q154S; V82S and Q154S; V82S and Y123H; and V82S, Y123H, and Y147T having a single modification combination selected from the group consisting of: The amino acid sequence or a fragment thereof lacking only the N-terminal methionine adenosine deaminase domain, including Including, the guide polynucleotide directs the base editor to modify a nucleobase at a splice site of the superoxide dismutase 1 (SOD1) gene. Base editor system.

24. 24. The base editor system of claim 23, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 in the TadA*7.10 numbering.

25. A base editor system comprising: (i) a base editor or a nucleic acid molecule encoding the same; and (ii) a guide polynucleotide or a nucleic acid molecule encoding the same, The base editor comprises a programmable DNA binding domain and TadA*7.10 amino acid sequence below: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD And, or having a single alteration selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R; Y147R, Q154R, and Y123H; Y147R, Q154R, and I76Y; Y147R, Q154R, and T166R; Y147T, Q154R; Y147T, Q154S; Y123H, Y147R, Q154R, and I76Y; I76Y and V82S; V82S and Y147R; V82S, Y123H, and Y147R; V82S and Q154R; V82S, Y123H, and Q154R; V82S, Y123H, Y147R, and Q154R; I76Y, V82S, Y123H, Y147R, and Q154R; Y147R and Q154S; V82S and Q154S; V82S and Y123H; and V82S, Y123H, and Y147T having a single modification combination selected from the group consisting of: The amino acid sequence or a fragment thereof lacking only the N-terminal methionine adenosine deaminase domain, including Including, A base editor system, wherein the guide polynucleotide directs the base editor to effect a nucleobase modification at a splice site of the androgen receptor (AR) gene.

26. 26. The base editor system of claim 25, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 in the TadA*7.10 numbering.

27. 27. The base editor system of claim 25 or 26, wherein the adenosine deaminase domain comprises a V28S mutation or a T166R mutation or a corresponding mutation in the numbering in TadA*7.

10.

28. A vector comprising a nucleic acid molecule encoding a DNA binding domain programmable by the polynucleotide in the base editor system of any one of claims 23 to 27, and a nucleic acid molecule encoding the deaminase domain.

29. the vector further comprises a nucleic acid molecule encoding the guide polynucleotide; and / or The vector is a viral vector. The vector of claim 28.

30. 30. An ex vivo or in vitro cell comprising the base editor system of any one of claims 23 to 27 or the vector of claim 28 or 29, wherein the cell is not a cell of a human embryo.

31. The cell of claim 30 , wherein the cell is a mammalian cell.

32. the cell is a motor neuron; and / or the cells are autologous cells isolated from the subject or are allogeneic cells; A cell according to claim 30 or 31.

33. A pharmaceutical composition comprising the base editor system of any one of claims 23 to 27, the vector of claim 28 or 29, or the cell of any one of claims 30 to 32, and a pharmaceutically acceptable carrier.

34. 34. The pharmaceutical composition of claim 33, further comprising a lipid.

35. A kit comprising the base editor system of any one of claims 23 to 27 or the vector of claim 28 or 29.

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