Use of adenylate deaminase base editor to disrupt shear receptors of disease-related genes, including for treatment of genetic diseases

Abstract

The invention features the use of an adenylate deaminase base editor to destroy the scissor acceptor site of a disease-related gene, including compositions and methods for treating genetic diseases, particularly for treating, alleviating or ameliorating the asthenic effect of amyotrophic lateral sclerosis (ALS) and spinal bulbar muscular atrophy (SBMA). Provided herein are compositions and methods for using improved new base editors (e.g., adenosine base editors) comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain that cooperate with a guide polynucleotide, e.g., adenosine base editors, for example, adenosine base editors, for example, adenosine base editors, for example, adenosine base editors, for example, adenosine base editors, for example, adenosine base editors. The present invention relates to a method for modifying a target gene associated with a hereditary disease or disorder, such as ALS or SBMA, by modifying the target gene using a base editor system provided herein to disrupt normal transcription of a gene associated with the hereditary disease or disorder.

Description

[0001] This invention is a divisional application of the PCT patent application entered into China with Chinese patent application number 2020800286799, entitled “Using adenylate deaminase base editor to destroy splice acceptor sites of disease-related genes, including for the treatment of genetic diseases”, and the international application date is February 13, 2020.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] 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.

[0004] Incorporated by Reference

[0005] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Unless otherwise indicated, the publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety. Background Art

[0006] Targeted editing of nucleic acid sequences, for example, targeted cleavage or targeted modification of genomic DNA, is a promising method for studying gene function and also has the potential to provide new therapies for human genetic diseases. Currently available base editors include cytidine base editors that convert target C·G base pairs to T·A (e.g., BE4) and adenosine base editors that convert A·T to G·C (e.g., ABE7.10). There is a need in the art for improved base editors that can induce modifications in target sequences with greater specificity and efficiency. Summary of the Invention

[0007] The present invention provides compositions with increased efficiency comprising novel adenosine base editors (e.g., ABE8) and methods of editing target sequences using base editors comprising adenosine deaminase variants.

[0008] In some aspects, provided herein is a method for treating a neurological disorder in a subject in need thereof, the method comprising administering to the subject: (i) an adenosine base editor or a nucleic acid sequence encoding an adenosine base editor, and (ii) a guide polynucleotide or a nucleic acid sequence encoding a guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, and wherein the guide polynucleotide guides the adenosine base editor to effect a single nucleobase modification at a splice site of a target gene associated with the neurological disorder in the subject, thereby treating the neurological disorder in the subject.

[0009] In some embodiments, the adenosine deaminase comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2, or a corresponding substitution thereof. In some embodiments, the single nucleobase modification causes alternative splicing of the transcript encoded by the target gene. In some embodiments, the alternative splicing results in a truncated or non-functional protein encoded by the target gene. In some embodiments, the single nucleobase modification results in reduced expression of the target gene in a subject. In some embodiments, the target gene is the superoxide dismutase 1 (SOD1) gene, and wherein the neurological disease is amyotrophic lateral sclerosis (ALS). In some embodiments, the target gene is the androgen receptor (RR) gene, and wherein the neurological disease is spinal bulbar muscular atrophy (SBMA).

[0010] In some aspects, provided herein is a method of treating amyotrophic lateral sclerosis (ALS) in a subject in need thereof, the method comprising administering to the subject: (i) a base editor or a nucleic acid sequence encoding a base editor, and (ii) a guide polynucleotide or a nucleic acid sequence encoding a guide polynucleotide, wherein the base editor comprises a programmable DNA binding domain and a deaminase domain, and wherein the guide polynucleotide guides the adenosine base editor to effect a single nucleobase modification at a splice site of a superoxide dismutase 1 (SOD1) gene in the subject, thereby treating ALS in the subject.

[0011] In some aspects, provided herein is a method of treating amyotrophic lateral sclerosis (ALS) in a subject in need thereof, the method comprising administering to the subject: (i) a base editor or a nucleic acid sequence encoding a base editor, and (ii) a guide polynucleotide or a nucleic acid sequence encoding a guide polynucleotide, wherein the base editor comprises a programmable DNA binding domain and a deaminase domain, wherein the guide polynucleotide guides the adenosine base editor to effect a single nucleobase modification located in a superoxide dismutase 1 (SOD1) gene of the subject, and wherein the single nucleobase modification results in a premature stop codon in the SOD1 gene, thereby treating ALS in the subject.

[0012] In some embodiments, the deaminase is an adenosine deaminase comprising an amino acid substitution at amino acid position 82 or 166, as numbered in SEQ ID NO: 2, or a corresponding substitution thereof. In some embodiments, the single nucleobase modification is a modification in which A is replaced with G. In some embodiments, the single nucleobase modification is located at a splice acceptor site of the SOD1 gene. In some embodiments, the splice site is 5' to a splice acceptor site of an exon of the SOD1 gene. In some embodiments, the exon of the SOD1 gene is exon 3 corresponding to SEQ ID NO: 3, or a variant thereof. In some embodiments, exon 3 of the SOD1 gene is adjacent to a splice acceptor AG located at nucleotide position 6828 in the SOD1 polynucleotide sequence, as numbered in SEQ ID NO: 3, or is a variant thereof. In some embodiments, the exon of the SOD1 gene is exon 4 corresponding to SEQ ID NO: 3, or a variant thereof. In some embodiments, the single nucleobase modification produces a transcription product that lacks exons 3-5 of the human SOD1 gene corresponding to SEQ ID NO: 3, or a variant thereof. In some embodiments, after administration, the expression of the SOD1 gene in the subject is reduced by at least 40%.

[0013] 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 SOD1 gene. In some embodiments, the guide polynucleotide comprises any one of the nucleic acid sequences selected from Table 19 or Table 23. In some embodiments, 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 their complements.

[0014] In some aspects, provided herein is a method of treating spinal bulbar muscular atrophy (SBMA) in a subject, the method comprising administering to the subject: (i) a base editor or a nucleic acid sequence encoding a base editor, and (ii) a guide polynucleotide or a nucleic acid sequence encoding a guide polynucleotide, wherein the base editor comprises a programmable DNA binding domain and a deaminase domain, and wherein the guide polynucleotide guides the adenosine base editor to effect a single nucleobase modification at a splice site of an androgen receptor (AR) gene in the subject, thereby treating SBMA in the subject.

[0015] In some aspects, provided herein is a method of treating spinal bulbar muscular atrophy (SBMA) in a subject, the method comprising administering to the subject: (i) a base editor or a nucleic acid sequence encoding a base editor, and (ii) a guide polynucleotide or a nucleic acid sequence encoding a guide polynucleotide, 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 effect a single nucleobase modification located in an androgen receptor (AR) gene in the subject, and wherein the single nucleobase modification results in a premature stop codon in the AR gene, thereby treating SBMA in the subject.

[0016] In some embodiments, the nucleobase modification causes a CAG-TAG codon change in the AR gene. In some embodiments, the codon change is in exon 1 or exon 2 of the AR gene. In some embodiments, the deaminase is an adenosine deaminase comprising an amino acid replacement at amino acid position 82 or 166 as numbered in SEQ ID NO: 2. In some embodiments, the single nucleobase modification is a modification in which A is replaced by G. In some embodiments, the nucleobase modification in which A is replaced by G is located at the splice acceptor site of the AR gene. In some embodiments, the splice site is 5′ of the splice acceptor site of the exon of the AR gene. In some embodiments, the exon of the AR gene is exon 2 corresponding to SEQ ID NO: 4 or a variant thereof. In some embodiments, the splice site is 3′ of the splice donor site of the exon of the AR gene. In some embodiments, the exon of the AR gene is exon 1 corresponding to SEQ ID NO: 4 or a variant thereof. In some embodiments, after administration, the expression of the AR gene in the subject is reduced by at least 40%.

[0017] 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 an AR gene. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from Table 41A or Table 41B. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of 5′-ACUUACCGCAUGUCCCCGUAAGG-3′, 5′-AGUGCAGUUAGGGCUGGGAAGGG-3′, 5′-AAGUGCAGUUAGGGCUGGGAAGG-3′, and their complements.

[0018] In some embodiments, the subject is a mammal or a human. In some embodiments, administration is performed by delivery to a subject's central nervous system (CNS) cells. In some embodiments, the cells are motor neurons.

[0019] In some aspects, provided herein is a method for modifying a target gene associated with a neurological disorder or a regulatory element thereof, the method comprising: contacting the target gene or its regulatory element with: (i) an adenosine base editor or a nucleic acid sequence encoding an adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding a guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the guide polynucleotide guides the adenosine base editor to effect a single nucleobase change at a splice site of the target gene.

[0020] In some embodiments, the adenosine deaminase comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2. In some embodiments, the single nucleobase change results in alternative splicing of the 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 some embodiments, the target gene is the superoxide dismutase 1 (SOD1) gene, and wherein the neurological disease is amyotrophic lateral sclerosis (ALS). In some embodiments, the target gene is the androgen receptor (AR) gene, and wherein the neurological disease is spinal bulbar muscular atrophy (SBMA).

[0021] In some aspects, provided herein is a method for regulating superoxide dismutase (SOD1) gene expression, the method comprising: contacting the SOD1 gene or a regulatory element thereof with: (i) a base editor or a nucleic acid sequence encoding a base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding a guide polynucleotide, wherein the base editor comprises a programmable DNA binding domain and a deaminase domain, and wherein the guide polynucleotide guides the adenosine base editor to effect a single nucleobase change at a splice site of the superoxide dismutase 1 (SOD1) gene.

[0022] In some aspects, provided herein is a method for 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 a base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding a guide polynucleotide, wherein the guide polynucleotide guides the adenosine base editor to effect a single nucleobase change at a splice site of a superoxide dismutase 1 (SOD1) gene in a subject, and wherein the single nucleobase change results in a premature stop codon in the SOD1 gene.

[0023] In some embodiments, the single nucleobase change is an A to G change. In some embodiments, the nucleobase change is located at a splice acceptor site of the SOD1 gene. In some embodiments, the splice site is 5' to a splice acceptor site of an exon of the SOD1 gene. In some embodiments, the exon of the SOD1 gene is exon 3 corresponding to SEQ ID NO: 3, or a variant thereof. In some embodiments, exon 3 of the SOD1 gene is adjacent to a splice acceptor located at nucleotide position 6828 in the SOD1 polynucleotide sequence as numbered in SEQ ID NO: 3, or is a variant thereof. In some embodiments, the exon of the SOD1 gene is exon 3 corresponding to SEQ ID NO: 4, or a variant thereof. In some embodiments, the single nucleobase change produces a transcription product that lacks exons 3-5 of the SOD1 gene corresponding to SEQ ID NO: 3, or a variant thereof.

[0024] 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 SOD1 gene. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from Table 19 or Table 23. In some embodiments, 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′.

[0025] In some aspects, provided herein is a method for regulating expression of an androgen receptor (AR) gene, the method comprising: contacting the AR gene or a regulatory element thereof with: (i) a base editor or a nucleic acid sequence encoding a base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding a guide polynucleotide, wherein the base editor comprises a programmable DNA binding domain and a deaminase domain, and wherein the guide polynucleotide guides the adenosine base editor to effect a single nucleobase change at a splice site of the androgen receptor (AR) gene.

[0026] In some aspects, provided herein is a method for modifying an androgen receptor (AR) gene, the method comprising: contacting the AR gene or a regulatory element thereof with: (i) a base editor or a nucleic acid sequence encoding a base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding a guide polynucleotide, wherein the base editor comprises a programmable DNA binding domain and a deaminase domain, and wherein the guide polynucleotide guides the adenosine base editor to effect a single nucleobase change at a splice site of an androgen receptor (AR) gene in a subject, and wherein the single nucleobase change results in a premature stop codon in the AR gene.

[0027] In some embodiments, the deaminase is an adenosine deaminase comprising an amino acid replacement at amino acid position 82 or 166 as numbered in SEQ ID NO: 2. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the single nucleobase change is a C to T change. In some embodiments, the change in nucleobase results in a CAG-TAG codon change in the AR gene. In some embodiments, the codon change is in exon 1 or exon 2 of the AR gene. In some embodiments, the single nucleobase change is an A to G change. In some embodiments, the nucleobase change of A to G is located at the splice acceptor site of the AR gene.

[0028] In some embodiments, the splice site is a splice acceptor site 5′ of an exon of an AR gene. In some embodiments, the exon of an AR gene is exon 2 corresponding to SEQ ID NO: 4 or a variant thereof. In some embodiments, the splice site is a splice donor site 3′ of an exon of an AR gene. In some embodiments, the exon of an AR gene is exon 1 corresponding to SEQ ID NO: 4 or a variant thereof.

[0029] In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from Table 41A or Table 41B. 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 an AR gene. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from Table 41A or Table 41B. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of 5′-ACUUACCGCAUGUCCCCGUAAGG-3′, 5′-AGUGCAGUUAGGGCUGGGAAGGG-3′, 5′-AAGUGCAGUUAGGGCUGGGAAGG-3′, and their complements.

[0030] In some embodiments, the contacting is performed in a cell. In some embodiments, the single nucleobase modification results in less than 15% indels in the cell's genome. In some embodiments, the single nucleobase modification results in less than 5% indels in the cell's genome. In some embodiments, the single nucleobase modification results in less than 2% indels in the cell's genome. 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.

[0031] In some embodiments, contacting is performed in a population of cells. In some embodiments, at least 40% of the population of cells comprises the single nucleobase modification after contacting.

[0032] In some embodiments, at least 50% of the cell population comprises a single nucleobase modification after contacting. In some embodiments, at least 60% of the cell population comprises a single nucleobase modification after contacting. In some embodiments, at least 85% of the cell population survives after contacting. In some embodiments, the cell population is mammalian cells or human cells. In some embodiments, the cell population is central nervous system cells. In some embodiments, the cell population is motor neurons.

[0033] In some embodiments, the adenosine deaminase comprises a TadA deaminase. In some embodiments, the adenosine deaminase is TadA7.10. In some embodiments, the adenosine deaminase is TadA comprising a V28S mutation or a T166R mutation as numbered in SEQ ID NO: 2, or a corresponding mutation thereof.

[0034] In various aspects and embodiments provided herein, the adenosine deaminase comprises one or more of the following mutations: Y147T, Y147R, Q154S, Y123H, and Q154R as numbered in SEQ ID NO: 2, or their corresponding mutations. In various aspects and embodiments provided herein, the adenosine deaminase comprises a combination of mutations 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+Y123 3H+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.

[0035] In some embodiments, the adenosine deaminase comprises a C-terminal deletion starting at a residue selected from the group consisting of: 149, 150, 151, 152, 153, 154, 155, 156, and 157. In some embodiments, the adenosine deaminase comprises a TadA dimer. In some embodiments, the adenosine deaminase comprises an adenosine deaminase monomer.

[0036] In the various aspects and embodiments provided herein, 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 comprises a D10A and / or H840A amino acid substitution as numbered in SEQ ID NO: 1, or its corresponding amino acid substitution. In some embodiments, the Cas9 domain comprises a SaCas9 domain. In some embodiments, the Cas9 domain has specificity for an altered 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, wherein N is A, G, C, or T, and wherein R is A or G.

[0037] In some aspects, provided herein are cell populations produced by the methods described herein.

[0038] In some aspects, provided herein is a base editor system comprising: (i) a base editor or a nucleic acid sequence encoding a base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding a guide polynucleotide, wherein the base editor comprises a programmable DNA binding domain and a deaminase domain, and wherein the guide polynucleotide guides the adenosine base editor to achieve a single nucleobase modification located at a superoxide dismutase 1 (SOD1) splice site.

[0039] In some aspects, provided herein is a base editor system comprising: (i) a base editor or a nucleic acid sequence encoding a base editor, and (ii) a guide polynucleotide or a nucleic acid sequence encoding a guide polynucleotide, wherein the base editor comprises a programmable DNA binding domain and a deaminase domain, wherein the guide polynucleotide guides the adenosine base editor to effect a single nucleobase modification located in a superoxide dismutase 1 (SOD1) gene, and wherein the single nucleobase modification results in a premature stop codon in the SOD1 gene.

[0040] In some embodiments, the deaminase is an adenosine deaminase comprising an amino acid substitution at amino acid position 82 or 166, as numbered in SEQ ID NO: 2. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the single nucleobase modification is an A-to-G substitution. In some embodiments, the A-to-G substitution is located at a splice acceptor site of the SOD1 gene. In some embodiments, the splice site is 5′ of a splice acceptor site of an exon of the SOD1 gene. In some embodiments, the exon of the SOD1 gene is exon 3 corresponding to SEQ ID NO: 3, or a variant thereof. In some embodiments, exon 3 of the SOD1 gene is adjacent to a splice acceptor AG located at nucleotide position 6828 in the SOD1 polynucleotide sequence, as numbered in SEQ ID NO: 3, or is a variant thereof. In some embodiments, alternative splicing of the SOD1 transcript produces a transcript lacking exons 3-5 of the SOD1 gene corresponding to SEQ ID NO: 3, or a variant thereof. In some embodiments, the exon of the SOD1 gene is exon 3 corresponding to SEQ ID NO: 4 or a variant thereof.

[0041] 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 SOD1 gene. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from Table 19 or Table 23. In some embodiments, 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.

[0042] In some aspects, provided herein is a base editor system comprising: (i) a base editor or a nucleic acid sequence encoding a base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding a guide polynucleotide, wherein the base editor comprises a programmable DNA binding domain and a deaminase domain, and wherein the guide polynucleotide guides the adenosine base editor to achieve a single nucleobase modification located at an androgen receptor (AR) splice site.

[0043] In some aspects, provided herein is a base editor system comprising: (i) a base editor or a nucleic acid sequence encoding a base editor, and (ii) a guide polynucleotide or a nucleic acid sequence encoding a guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and a deaminase domain, and wherein the guide polynucleotide guides the adenosine base editor to effect a single nucleobase modification located in an androgen receptor (AR) gene of a subject, and wherein the single nucleobase modification results in a premature stop codon in the AR gene.

[0044] In some embodiments, the deaminase is an adenosine deaminase comprising an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the single nucleobase modification is a C-to-T substitution. In some embodiments, the single nucleobase modification causes a CAG-TAG codon change in the AR gene. In some embodiments, 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 some embodiments, the single nucleobase modification is an A-to-G substitution. In some embodiments, the nucleobase modification of A-to-G substitution is located at a splice acceptor site of the AR gene. In some embodiments, the splice site is 5′ of a splice acceptor site of an exon of the AR gene. In some embodiments, the exon of the AR gene is exon 2 corresponding to SEQ ID NO: 4, or a variant thereof. In some embodiments, the splice site is 3′ of a splice donor site of an exon of the AR gene. In some embodiments, the exon of the AR gene is exon 1 corresponding to SEQ ID NO: 4 or a variant thereof.

[0045] 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 an AR gene. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from Table 41A or Table 41B. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of 5′-ACUUACCGCAUGUCCCCGUAAGG-3′, 5′-AGUGCAGUUAGGGCUGGGAAGGG-3′, 5′-AAGUGCAGUUAGGGCUGGGAAGG-3′, and their complements.

[0046] In some embodiments, the adenosine deaminase comprises a TadA deaminase. In some embodiments, the adenosine deaminase is TadA7.10. In some embodiments, the adenosine deaminase is TadA comprising a V28S mutation or a T166R mutation as numbered in SEQ ID NO: 2, or a corresponding mutation thereof. In some embodiments, the adenosine deaminase comprises one or more of the following mutations: Y147T, Y147R, Q154S, Y123H, and Q154R as numbered in SEQ ID NO: 2, or a corresponding mutation thereof. In some embodiments, the adenosine deaminase comprises a combination of mutations selected from the group consisting of: Y147T, Y147R, Q154S, Y123H, and Q154R as numbered in SEQ ID NO: 2, or a corresponding mutation thereof. NO: 2 numbered Y147T+Q154K, Y147T+Q154S, Y147R+Q154S, V82S+Q154S, V82S+Y147R, V82S+Q154R, V82S+Y123H, I76Y+V82S, V82S+Y123H+Y147T, V82S+Y123H+Y147R, V82S+Y123 H+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.

[0047] In some embodiments, the adenosine deaminase comprises a C-terminal deletion starting at a residue selected from the group consisting of: 149, 150, 151, 152, 153, 154, 155, 156, and 157. In some embodiments, the adenosine deaminase comprises a TadA dimer. In some embodiments, the adenosine deaminase comprises an adenosine deaminase monomer.

[0048] 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 comprises a D10A and / or H840A amino acid substitution as numbered in SEQ ID NO: 1, or its corresponding amino acid substitution. In some embodiments, the Cas9 domain comprises a SaCas9 domain. In some embodiments, the Cas9 domain has specificity for an altered 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, wherein N is A, G, C, or T, and wherein R is A or G.

[0049] In some aspects, a vector is provided herein comprising a nucleic acid sequence encoding a polynucleotide programmable DNA binding domain in a base editor system described herein and a nucleic acid sequence encoding an adenosine deaminase domain. In some embodiments, the vector further comprises a nucleic acid sequence encoding a guide polynucleotide. In some embodiments, the vector is a viral vector.

[0050] In some aspects, a cell is provided herein comprising a base editor system or vector as 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.

[0051] In some aspects, the present invention provides a cell population comprising a base editor system or vector described herein. In some embodiments, the cell population is a mammalian cell, a human cell, or a motor neuron. In some embodiments, the cell population is in vivo, ex vivo, or in vitro. In some embodiments, the cell is an autologous cell isolated from a subject.

[0052] In some aspects, a pharmaceutical composition is provided herein comprising a base editor, a vector or a cell as 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.

[0053] In some aspects, provided herein is a kit comprising a base editor system or vector described herein.

[0054] In various embodiments of the methods described herein, at least one nucleotide of the guide polynucleotide comprises a non-naturally occurring modification. In various embodiments of the methods described herein, at least one nucleotide of the nucleic acid sequence comprises a non-naturally occurring 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-naturally occurring modification. In some embodiments, the non-naturally occurring modification is a chemical modification. In some embodiments, the chemical modification is 2'-O-methylation. In some embodiments, the nucleic acid sequence comprises a phosphorothioate.

[0055] The description and examples herein illustrate embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and is therefore variable. It will be appreciated by those skilled in the art that there are numerous changes and modifications to the present disclosure, and such changes and modifications are within the scope of the present disclosure.

[0056] The practice of certain embodiments disclosed herein employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); FM Ausubel et al., the series Current Protocols in Molecular Biology; the series Methods In Enzymology (Academic Press, Inc.); MJ MacPherson, BD Hames and GR Taylor, PCR 2: A Practical Approach (1995); Harlow and Lane, Antibodies, A Laboratory Manual (1988); and RI Freshney, Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. 6th Edition (2010).

[0057] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0058] Although 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, although the present disclosure may be described herein in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0059] 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 may 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 reference to the accompanying drawings as described below.

[0060] definition

[0061] The following definitions supplement those in the art and are specific to this application and are not attributed to any related or unrelated circumstances, such as any commonly owned patents or applications. Although methods and materials similar or equivalent to those disclosed herein can be used to test the practice of the present disclosure, preferred methods and materials are described herein. Accordingly, the technology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs. The following references provide general definitions of various terms used in the present invention to those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer-Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0063] In this application, unless specifically specified otherwise, the use of the singular includes the plural. It must be noted that, as used in this specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly excludes them. In this application, the use of "or" means "and / or" and is understood to be inclusive, unless otherwise specified. In addition, the term "including" as well as other forms (such as "include," "includes," and "included") are not restrictive.

[0064] As used in this specification and claims, the words "comprising" (and other forms of comprising, such as "comprise" and "comprises"), "having" (and other forms of having, such as "have" and "has"), "including" (and other forms of including, such as "includes" and "include"), or "containing" (and other forms of containing, 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 in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0065] The term "about" or "approximately" means within an acceptable error range for a particular value, as determined by one skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of at most 20%, at most 10%, at most 5%, or at most 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, such as within 5-fold or within 2-fold of a value. If a specific value is described in the application and claims, it should be assumed that the term "about" means within an acceptable error range for that specific value unless otherwise specified.

[0066] 47, 48, 49, or 50.

[0067] Reference in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some, but not necessarily all, embodiments of the present disclosure.

[0068] “Abasic base editor” means a reagent that can excise a nucleobase and insert a DNA nucleobase (A, T, C or G). The abasic base editor comprises a nucleic acid glycosidase polypeptide or a fragment thereof. In one embodiment, the nucleic acid glycosidase is a mutant human uracil DNA glycosidase or an active fragment thereof, which comprises Asp at amino acid 204 or at the corresponding position in uracil DNA glycosidase in the following sequence (e.g., replacing Asn at amino acid 204) and has cytosine-DNA glycosidase activity. In one embodiment, the nucleic acid glycosidase is a mutant human uracil DNA glycosidase or an active fragment thereof, which comprises Ala, Gly, Cys or Ser at amino acid 147 or at the corresponding position in uracil DNA glycosidase in the following sequence (e.g., replacing Tyr at amino acid 147) and has thymine-DNA glycosidase activity. The sequence of exemplary human uracil-DNA glycosidase subtype 1 is as follows:

[0069]

[0070] The sequence of human uracil-DNA glycosylase isoform 2 is as follows:

[0071]

[0072] In other embodiments, the abase editor is any of the abase editors described in PCT / JP2015 / 080958 and US20170321210, which are incorporated herein by reference. In a specific embodiment, the abase editor comprises a mutation at a position shown in bold and with an underline in the above sequence, or a mutation at the corresponding amino acid in any other abase editor or uracil deglycosylase known in the art. In one embodiment, the abase editor comprises a mutation at Y147, N204, L272 and / or R276 or a corresponding position. In another embodiment, the abase editor comprises a Y147A or Y147G mutation or a corresponding mutation. In another embodiment, the abase editor comprises an N204D mutation or a corresponding mutation. In another embodiment, the abase editor comprises an L272A mutation or a corresponding mutation. In another embodiment, the abase editor comprises an R276E or R276C mutation or a corresponding mutation.

[0073] "Adenosine deaminase" means a polypeptide or fragment thereof that can catalyze the hydrolytic deamination reaction of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination reaction of adenosine to inosine or the deoxygenation reaction of adenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination reaction of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) can be from any organism such as bacteria.

[0074] In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is a TadA variant. In some embodiments, the TadA variant is TadA*8. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to a naturally occurring deaminase. For example, deaminase domains are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is herein incorporated by reference in its entirety.See also, Komor, AC et al., "Programmable base editing of a target base in genomicDNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, NM et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage" Nature 551, 464-471 (2017); Komor, AC et al., "Improved base excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T: A 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: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0075] Wild-type TadA (wt) adenosine deaminase has the following sequence (also known as the TadA reference sequence):

[0076]

[0077] In some embodiments, the adenosine deaminase comprises an alteration in the following sequence:

[0078]

[0079] (also known as TadA*7.10).

[0080] In some embodiments, TadA*7.10 comprises at least one alteration. In some embodiments, TadA*7.10 comprises alterations at amino acids 82 and / or 166. In specific embodiments, variants of the above-referenced sequences comprise one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. Alteration Y123H is also referred to herein as H123H (alteration H123Y in TadA*7.10 is repaired to Y123H (wt)). In other embodiments, variants of the TadA*7.10 sequence comprise a combination of alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+ Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I 76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.

[0081] In other embodiments, the present invention provides adenosine deaminases comprising deletions, e.g., TadA*8, comprising a C-terminal deletion starting 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 comprising one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, the adenosine deaminase variant is a TadA8 (e.g., TadA*8) monomer comprising a combination of alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V 82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T 166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R;

[0082] In yet other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each adenosine deaminase domain having one or more of the following mutations: Y147T, Y147R, Q154S, Y123H, V82S, T166R and / or Q154R. In other embodiments, the adenosine deaminase variant is a polyadenylated homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each adenosine deaminase domain having a combination of alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H 3H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q1 54R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R;

[0083] In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type TadA adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8), wherein the adenosine deaminase variant domain comprises one or more of the following mutations: Y147T, Y147R, Q154S, Y123H, V82S, T166R and / or Q154R. 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 alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S ;V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147 R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R;

[0084] In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8), wherein the adenosine deaminase variant domain comprises one or more of the following mutations: Y147T, Y147R, Q154S, Y123H, V82S, T166R and / or Q154R. 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 alterations: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y1 47R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; I76Y+V82S+Y123H+Y147R+Q154R.

[0085] In one embodiment, the adenosine deaminase is TadA*8, which comprises or consists essentially of the following sequence, or a fragment thereof having adenosine deaminase activity:

[0086] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0087] In some embodiments, TadA*8 is truncated. In some embodiments, the truncated TadA*8 has lost 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 has lost 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.

[0088] In certain embodiments, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from:

[0089] In certain embodiments, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from:

[0090] Escherichia coli TadA:

[0091]

[0092] Escherichia coli TadA (N-terminal truncated):

[0093]

[0094]

[0095] Staphylococcus aureus (S. aureus) TadA:

[0096]

[0097] Bacillus subtilis (B. subtilis) TadA:

[0098]

[0099] Salmonella typhimurium (S. typhimurium) TadA:

[0100]

[0101] Shewanella putrefaciens (S. putrefaciens) TadA:

[0102]

[0103] Haemophilus influenzae F3031 (H. influenzae) TadA:

[0104]

[0105] Caulobacter crescentus (C. crescentus) TadA:

[0106]

[0107] Geobacter sulfurreducens (G.sulfurreducens) TadA:

[0108]

[0109] TadA*7.10

[0110]

[0111] Other TadA7.10 or TadA7.10 variants contemplated as components of heterodimers with TadA*8 include:

[0112]

[0113] TadA7.10 CP65

[0114]

[0115] TadA7.10 CP83

[0116]

[0117] TadA7.10 CP136

[0118]

[0119]

[0120] TadA7.10 C-terminal truncation

[0121]

[0122] TadA7.10 C-terminal truncation 2

[0123]

[0124] TadA7.10δ59-66+C-terminal truncation

[0125]

[0126] TadA7.10δ59-66

[0127]

[0128] In some embodiments, the adenosine deaminase variant comprises an alteration in TadA7.10. In some embodiments, TadA7.10 comprises an alteration at amino acid 82 or 166. In specific embodiments, the variant in the above-referenced sequence comprises one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R. In other embodiments, the adenosine deaminase variant comprises a combination of alterations selected from the group consisting of: Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y147T+Q154R: Y147T+Q154S; and Y123H+Y147R+Q154R+I76Y.

[0129] In other embodiments, the present invention provides adenosine deaminases comprising deletions, for example, TadA*7.10 comprising a C-terminal deletion starting at residue 149, 150, 151, 152, 153, 154, 155, 156, or 157. In other embodiments, the adenosine deaminase variant is a TadA monomer comprising one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, Q154R. In other embodiments, the adenosine deaminase variant is a monomer comprising the following changes: Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y147T+Q154R; Y147T+Q154S; and Y123H+Y147 R+Q154R+I76Y. In yet other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains, each adenosine deaminase domain having one or more of the following mutations: 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 TadA7.10 and an adenosine deaminase variant domain comprising one or more of the following mutations: 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, wherein the adenosine deaminase variant domain comprises the following changes: Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y147T+Q154R; Y147T+Q154S; and Y123H+Y147R+Q154R+I76Y.

[0130] Herein, "administering" refers to providing one or more compositions described herein to a patient or subject. For example and without limitation, composition administration (e.g., injection) can be performed by intravenous (iv) injection, subcutaneous (sc) injection, intradermal (id) injection, intraperitoneal (ip) injection, or intramuscular (im) injection. One or more such routes can be used. For example, parenteral administration can be performed by a single rapid injection or by progressive perfusion over time. In some embodiments, parenteral administration includes intravascular, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, and intrasternal infusion or injection. Alternatively, or simultaneously, administration can be by oral route.

[0131] "Agent" means any small molecule chemical compound, antibody, nucleic acid molecule, or peptide, or fragment thereof.

[0132] "Alteration" means a change (increase or decrease) in the structure, expression level, or activity of a gene or polypeptide, as detected by methods known in the art, such as those described herein. As used herein, an alteration includes a change in a polynucleotide or polypeptide sequence or a change in expression level, such as a 10% change, a 25% change, a 40% change, a 50% change, or more.

[0133] "Relieve" means to reduce, inhibit, attenuate, diminish, retard or stabilize the development or progression of a disease.

[0134] "Analog" means 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 native polynucleotide or polypeptide but has certain modifications that enhance the analog's function relative to the native polynucleotide or polypeptide. Such modifications may increase the analog's affinity for DNA, efficiency, specificity, protease or nuclease resistance, membrane permeability, and / or half-life without altering, for example, ligand binding. Analogs may include non-natural nucleotides or amino acids.

[0135] "Base editor (BE)" or "nucleobase editor (NBE)" means an agent that binds to a polynucleotide and has nucleobase modification activity. In various embodiments, the base editor comprises a nucleobase modifying polypeptide (e.g., a deaminase) and a nucleic acid programmable nucleotide binding domain, which cooperates with a guide polynucleotide (e.g., a guide RNA). In various embodiments, the agent is a biomolecular complex comprising a protein domain with base editing activity, i.e., a domain capable of modifying a base (e.g., A, T, C, G, or U) in a nucleic acid molecule (e.g., DNA). In some embodiments, the polynucleotide programmable DNA binding domain is fused or linked to a deaminase domain. In one embodiment In one embodiment, the reagent is a fusion protein comprising a domain having base editing activity. In another embodiment, the protein domain having base editing activity is connected to a guide RNA (e.g., via an RNA binding motif on the guide RNA and fusion to an RNA binding domain of a deaminase). In some embodiments, the domain having base editing activity is capable of deaminating bases within a nucleic acid molecule. In some embodiments, the base editor is capable of deaminating one or more bases within a DNA molecule. In some embodiments, the base editor is capable of deaminating adenosine (A) within DNA. In some embodiments, the base editor is an adenosine base editor (ABE).

[0136] "Cytidine deaminase" means a polypeptide or fragment thereof that can catalyze 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 APOBEC or AID. In one embodiment, the cytidine deaminase converts cytosine to uracil or converts 5-methylcytosine to thymine. PmCDA1 (sea lamprey cytosine deaminase 1, "PmCDA1") from sea lamprey (Petromyzon marinus), AID (initiation-induced cytidine deaminase; AICDA) from mammals (e.g., humans, pigs, cattle, horses, monkeys, etc.), and APOBEC are exemplary cytidine deaminases.

[0137] 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 a Cas9 fused to a deaminase (e.g., adenosine deaminase or cytidine deaminase). In some embodiments, the base editor is a nuclease-inactivated Cas9 (dCas9) fused to a deaminase (e.g., adenosine deaminase or cytidine deaminase). In some embodiments, Cas9 is a cyclic hypermutated Cas9 (e.g., spCas9 or saCas9). Circular hypermutated Cas9 is known in the art and is described in, for example, Oakes et al., Cell 176, 254-267, 2019. In some embodiments, the base editor is fused to an inhibitor of base excision repair (BER), for example, 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.

[0138] In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, 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, napDNAbp is Cas12a (Cpf1) with an internally fused deaminase domain. In some embodiments, napDNAbp is Cas12b (c2c1) with an internally fused deaminase domain. In some embodiments, napDNAbp is Cas12c (c2c3) with an internally fused deaminase domain. In some embodiments, napDNAbp is Cas12d (CasX) with an internally fused deaminase domain. In some embodiments, napDNAbp is Cas12e (CasY) with an internally fused deaminase domain. In some embodiments, napDNAbp is Cas12g with an internally fused deaminase domain. In some embodiments, napDNAbp is Cas12h with an internally fused deaminase domain. In some embodiments, the napDNAbp is a 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.

[0139] In some embodiments, a base editor (e.g., ABE8) is produced by cloning an adenosine deaminase variant (e.g., TadA*8) into a scaffold comprising a cyclic hypermutated Cas9 (e.g., spCAS9 or saCAS9) and a bipartite nuclear localization sequence. Circular hypermutated Cas9 is known in the art and is described, for example, in Oakes et al., Cell 176, 254-267, 2019. Exemplary cyclic hypermutants are as follows, where bold sequences indicate sequences derived from Cas9, italic sequences represent linker sequences, and bottom line sequences represent bipartite nuclear localization sequences.

[0140] CP5 (with MSP "NGC = Pam variant with canonical Cas9 mutations such as NGG", PID = protein interaction domain and "D10A" nickase):

[0141]

[0142]

[0143] In some embodiments, ABE8 is selected from a base editor from Tables 6-9, 13, or 14 above. In some embodiments, ABE8 contains an adenosine deaminase variant evolved from TadA. In some embodiments, the adenosine deaminase variant of ABE8 is a TadA*8 variant as described in Tables 7, 9, 13, or 14 above. In some embodiments, the adenosine deaminase variant is a TadA*7.10 variant (e.g., TadA*8) comprising one or more changes selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In various embodiments, ABE8 comprises a TadA*7.10 variant (e.g., TadA*8) having a combination of alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+

[0144] Y147R; V82S + Q154R; V82S + Y123H, I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y147R + Q154R + Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76Y; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R. In some embodiments, ABE8 is a monomeric construct. In some embodiments, ABE8 is a heterodimeric construct. In some embodiments, the ABE8 base editor comprises the sequence:

[0145]

[0146] In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-related (e.g., Cas or Cpf1) enzyme. In some embodiments, the base editor is a catalytically dead Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. In some embodiments, the base editor is fused to an inhibitor of base excision repair (BER). In some embodiments, the inhibitor of base excision repair is a uracil DNA glycosidase inhibitor (UGI). In some embodiments, the inhibitor of base excision repair is an inosine base excision repair inhibitor.

[0147] Details of base editors are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is incorporated herein by reference in its entirety. See also, Komor, AC et al., "Programmable base editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, NM et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage" Nature 551.464-471 (2017); Komor, AC et al., "Improved base excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T: A 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: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0148] For example, the cytidine base editor used in the base editing compositions, systems, and methods described herein has the following nucleic acid sequence (8877 base pairs) as provided below:

[0149] (Addgene, Watertown, MA.; Komor AC et al., 2017, Sci Adv., 30; 3(8):eaao4774. doi: 10.1126 / sciadv.aao4774.) Also included are polynucleotide sequences that are at least 95% identical or higher to the BE4 nucleic acid sequence.

[0150]

[0151]

[0152]

[0153]

[0154] BE4 amino acid sequence:

[0155]

[0156]

[0157] For example, the adenosine base editor (ABE) used in the base editing compositions, systems, and methods described herein has a nucleic acid sequence (8877 base pairs) as provided below (Addgene, Watertown, MA.; Gaudelli NM, et al., Nature. 2017 Nov 23; 551(7681): 464-471. doi: 10.1038 / nature24644; Koblan LW et al., Nat Biotechnol. 2018 Oct; 36(9): 843-846. doi: 10.1038 / nbt.4172.). Polynucleotide sequences having at least 95% or higher identity to the ABE nucleic acid sequence are also included.

[0158]

[0159]

[0160]

[0161] "Base editing activity" means acting to chemically change a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, for example, converting the target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, for example, converting A·T to G·C. In another embodiment, the base editing activity is cytidine deaminase activity, for example, converting the target C·G to T·A, and adenosine or adenine deaminase activity, for example, converting A·T to G·C. In some embodiments, the base editing activity is assessed by the efficiency of editing. The base editing efficiency can be measured by any suitable means, for example, by Sanger sequencing or next-generation sequencing. In some embodiments, the base editing efficiency is measured by the total percentage of sequencing reads with nuclear base conversions caused by the base editor, for example, the total percentage of sequencing reads with target AT base pairs converted to GC base pairs. In some embodiments, when base editing is performed in a population of cells, the base editing efficiency is measured by the total percentage of cells with a nucleobase conversion resulting from the base editor.

[0162] The term "base editor system" refers to a system for editing the nucleobases of a target nucleotide sequence. In various embodiments, the base editor system comprises: (1) a polynucleotide programmable nucleotide binding domain (e.g., Cas9); (2) a deaminase domain (e.g., adenosine deaminase or cytidine deaminase) for deaminating the nucleobase; and (3) one or more guide polynucleotides (e.g., guide RNA). In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is ABE8.

[0163] In some embodiments, the base editor system comprises more than one base editing component. For example, the base editor system can include more than one deaminase. In some embodiments, the base editor system can include one or more adenosine deaminases. In some embodiments, a single guide polynucleotide can be used to target different deaminases to a target nucleic acid sequence. In some embodiments, a pair of guide polynucleotides can be used to target different deaminases to a target nucleic acid sequence.

[0164] The deaminase domain and the polynucleotide programmable nucleotide binding component of the base editor system can be covalently or non-covalently associated with each other, or any combination of their association and interaction. For example, in some embodiments, the deaminase domain can be targeted to the target nucleotide sequence by the polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or connected to the deaminase domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the deaminase domain to the nucleotide sequence by non-covalently interacting or associating with the deaminase domain. For example, in some embodiments, the deaminase domain can include an additional heterologous portion or domain that is capable of interacting, associating or forming a complex with an additional heterologous portion or domain that is part of the polynucleotide programmable nucleotide binding domain. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide, interacting, associating or forming a complex. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide, interacting, associating or forming a complex. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous moiety can be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous moiety can be capable of binding to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a Kappa homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu coat protein domain, a sterile alpha motif, the telomerase Ku binding motif and Ku protein, the telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0165] The base editor system may further include a guide polynucleotide component. It should be understood that the components of the base editor system can be associated with each other via covalent bonds, non-covalent interactions, or any combination of their association and interaction. In some embodiments, the deaminase domain can be targeted to the target nucleotide sequence by the guide polynucleotide. For example, in some embodiments, the deaminase domain may include an additional heterologous portion or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein), which can interact, associate or form a complex with a portion or segment (e.g., a polynucleotide motif) of the guide polynucleotide. In some embodiments, an additional heterologous portion or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or connected to the deaminase domain. In some embodiments, an additional heterologous portion may be able to bind to a polypeptide, interact, associate or form a complex. In some embodiments, an additional heterologous portion may be able to bind to a polynucleotide, interact, associate or form a complex. In some embodiments, an additional heterologous portion may be able to bind to a guide polynucleotide. In some embodiments, the additional heterologous moiety can be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous moiety can be capable of binding to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a Kappa homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu coat protein domain, a sterile alpha motif, the telomerase Ku binding motif and Ku protein, the telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0166] In some embodiments, the base editor system may further include an inhibitor of base excision repair (BER) components. It should be understood that the components of the base editor system can be associated with each other via covalent bonds, non-covalent interactions, or any combination of their association and interaction. The inhibitor of the BER component may include a BER inhibitor. In some embodiments, the inhibitor of BER is a uracil DNA glycosidase inhibitor (UGI). In some embodiments, the inhibitor of BER is an inosine BER inhibitor. In some embodiments, the inhibitor of BER can be targeted to the target nucleotide sequence by a polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or connected to an inhibitor of BER. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or connected to a deaminase domain and an inhibitor of BER. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the inhibitor of BER to a nucleotide sequence by non-covalently interacting or associating with an inhibitor of BER. For example, in some embodiments, an inhibitor of a BER component may comprise an additional heterologous moiety or domain that is capable of interacting, associating, or forming a complex with an additional heterologous moiety or domain that is part of a programmable nucleotide binding domain of a polynucleotide.

[0167] In some embodiments, inhibitors of BER can be targeted to a target nucleotide sequence via a guide polynucleotide. For example, in some embodiments, an inhibitor of BER can comprise an additional heterologous portion or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) that is capable of interacting, associating, or forming a complex with a portion or segment of a guide polynucleotide (e.g., a polynucleotide motif). In some embodiments, an additional heterologous portion or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) of a guide polynucleotide can be fused or linked to an inhibitor of BER. In some embodiments, an additional heterologous portion can be capable of binding, interacting, associating, or forming a complex with a polynucleotide. In some embodiments, an additional heterologous portion can be capable of binding to a guide polynucleotide. In some embodiments, an additional heterologous portion can be capable of binding to a polypeptide linker. In some embodiments, an additional heterologous portion can be capable of binding to a polynucleotide linker. An additional heterologous portion 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, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0168] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active, inactivated, or partially active DNA cleavage domain of Cas9 and / or a gRMA binding domain of Cas9). Cas9 nuclease is sometimes also referred to as Casn1 nuclease or CRISPR (clustered regularly interspaced short palindromic repeat)-related nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic factors (viruses, transposable elements, and conjugative plasmids). The CRISPR cluster contains spacer sequences, sequences complementary to preceding mobile elements, and nucleic acids that invade the target. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein are required for correct processing of pre-crRNA. tracrRNA acts as a guide in ribonuclease 3-assisted pre-crRNA processing. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer sequence. The target chain that is not complementary to the crRNA is first cut endonucleolytically and then trimmed by 3′-5′ exonucleolytically. In essence, DNA binding and cleavage usually require proteins and two RNAs. However, single guide RNA (“sgRNA” or “gNRAs” for short) can be engineered to merge various aspects of both crRNA and tracrRNA into a single RNA species. See, for example, Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes short motifs (PAM or pre-spacer adjacent motifs) in CRISPR repeats to help distinguish self from non-self.The Cas9 nuclease sequence and structure are known to those skilled in the art (see, for example, "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E. et al., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M. et al., Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in a variety of species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus. Other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0169] An exemplary Cas9 is Streptococcus pyogenes Cas9 (spCas9), the amino acid sequence of which is provided below:

[0170]

[0171] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0172] Cas9 proteins inactivated by nucleases can be interchangeably referred to as "dCas9" proteins (Cas9 "dead" for nucleases) or catalytically inactive Cas9. Methods for generating Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains are known (see, for example, Jinek et al., Science. 337: 816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152(5): 1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations in these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337: 816-821 (2012); Qi et al., Cell. 28; 152(5): 1173-83 (2013)). In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, in other words, Cas9 is a nickase, referred to as a "nCas9" protein (for "nickase" Cas9). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, the protein comprises one or two Cas9 domains: (1) a gRNA binding domain of Cas9; or (2) a DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or a fragment thereof are referred to as "Cas9 variants". Cas9 variants share homology with Cas9 or a fragment thereof. For example, the Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9.In some embodiments, the Cas9 variant can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% the amino acid length of the corresponding wild-type Cas9.

[0173] In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0174] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, nucleotide and amino acid sequences below).

[0175]

[0176]

[0177]

[0178]

[0179] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0180] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence:

[0181]

[0182]

[0183]

[0184]

[0185] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0186] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2 (nucleotide sequence below); and Uniprot Reference Sequence: Q99ZW2 (amino acid sequence below)).

[0187]

[0188]

[0189]

[0190]

[0191] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0192] In some embodiments, Cas9 refers to Cas9 from the following microorganisms: 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 taiwanensis (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_021284.1); Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1) or Neisseria meningitidis (NCBI Ref: Yp_002342100.1), or refers to Cas9 from any other organism.

[0193] In some embodiments, dCas9 partially or completely corresponds to or comprises a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises D10A and H840A mutations or corresponding mutations in another Cas9. In some embodiments, dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A):

[0194]

[0195]

[0196] (Single underline: HNH domain; double underline: RuvC domain).

[0197] In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequences provided above, or at the corresponding position in any of the amino acid sequences provided herein.

[0198] In other embodiments, dCas9 variants are provided having mutations other than D10A and H840A, which, for example, result in nuclease-inactivated Cas9 (dCas9). For example, such mutations include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs of dCas9 are provided 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, wherein the amino acid sequence of the variant is 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.

[0199] In some embodiments, the Cas9 fusion protein as provided herein comprises the full-length amino acid sequence of the Cas9 protein, for example, one of the Cas9 sequences provided herein. However, in other embodiments, the fusion protein as provided herein does not comprise the full-length Cas9 sequence, but only comprises one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and other suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0200] It will be appreciated that other Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including variants and homologs thereof, are within the scope of the present disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is a nuclease-dead Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease-active Cas9.

[0201] Exemplary catalytically inactive Cas9 (dCas9):

[0202]

[0203] Exemplary catalytic Cas9 nickase (nCas9):

[0204]

[0205] Exemplary catalytically active Cas9:

[0206]

[0207]

[0208] In some embodiments, Cas9 refers to Cas9 from Archaea (e.g., Nanoarchaeum), which builds the domains and kingdoms of unicellular prokaryotic microorganisms. In some embodiments, Cas9 refers to CasX or CasY, which have been described in, for example, Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genome-resolved metagenomics, a large number of CRISPR-Cas systems have been identified, including Cas9, which was first reported in the Archaea domain of life. This divergent Cas9 protein was discovered in the rarely studied Nanoarchaeum as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, were found, which are among the most compact systems discovered so far. In some embodiments, Cas9 refers to CasX or a variant of CasX. In some embodiments, Cas9 refers to CasY or a variant of CasY. It will be appreciated that other RNA-guided DNA binding proteins can be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0209] In certain embodiments, napDNAbp useful in the methods of the invention include circular hypermutators, which are known in the art and described, for example, by Oakes et al., Cell 176, 254-267, 2019. Exemplary circular hypermutators are as follows, where bold sequences indicate sequences derived from Cas9, italic sequences indicate linker sequences, and underlined sequences indicate bipartite nuclear localization sequences.

[0210] CP5 (with MSP "NGC = Pam variant with canonical Cas9 mutations such as NGG", PID = protein interaction domain and "D10A" nickase):

[0211]

[0212]

[0213] 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).

[0214] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to a 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 that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any CasX or CasY protein described herein. It will be appreciated that Cas12b / C2c1, CasX, and CasY from other bacterial species can also be used according to the present disclosure.

[0215] Cas12b / C2c1(uniprot.org / uniprot / TOD7A2#2)

[0216] sp|TOD7A2 C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acido-terrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB13137 / GD3B) GN=c2c1 PE=1 SV=1

[0217]

[0218]

[0219] CasX(uniprot.org / uniprot / FONN87;uniprot.org / uniprot / FONH53)

[0220] >tr|FONN87|FONN87_SULIH CRISPR-associated Casx protein OS=Sulfolobus islandicus (strain HVE10 / 4) GN=SiH_0402 PE=4 SV=1

[0221]

[0222] >tr FONH53 FONH53_SULIR CRISPR-associated protein, Casx OS=Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4 SV=1

[0223]

[0224] Deltaproteobacterial CasX

[0225]

[0226] CasY(ncbi.nlm.nih.gov / protein / APG80656.1)

[0227] >APG80656.1 CRISPR-associated protein CasY [uncultured bacteria from the Thresholdia spp. community]

[0228]

[0229] 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, inactivated, or partially active DNA cleavage domain of Cas12 and / or a gRMA binding domain of Cas12). Cas12 belongs to a Class 2, Type V CRISPR / Cas system. Cas12 nucleases are sometimes also referred to as CRISPR (clustered regularly interspaced short palindromic repeats)-associated nucleases. The sequence of an exemplary Bacillus hisashii Cas 12b (BhCas12b) Cas 12 domain is provided below:

[0230]

[0231] Amino acid sequences having at least 85% or greater identity to the BhCas12b amino acid sequence can also be used in the methods of the invention.

[0232] "Cytidine deaminase" means a polypeptide or fragment thereof that can catalyze a deamination reaction that converts an amino group to a carbonyl group. In one embodiment, the cytidine deaminase converts cytosine to uracil or converts 5-methylcytosine to thymine. PmCDA1 (sea lamprey cytosine deaminase 1, "PmCDA1") from the sea lamprey (Petromyzon marinus), AID (initiation-induced cytidine deaminase; AICDA) from mammals (e.g., humans, pigs, cattle, horses, monkeys, etc.), and APOBEC are exemplary cytidine deaminases.

[0233] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid by another amino acid with common properties. A functional approach to defining common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Based on such analysis, groups of amino acids can be defined in which the amino acids within a group are preferentially exchanged with each other and are therefore most similar to each other in their effect on the overall protein structure (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include amino acid substitutions of the following amino acids: for example, amino acid substitutions of arginine and vice versa such that a positive charge can be maintained; substitutions of glutamic acid for aspartic acid and vice versa such that a negative charge can be maintained; substitutions of serine for threonine such that a free -OH can be maintained; and substitutions of glutamine for asparagine such that a free -NH2 can be maintained.

[0234] As used interchangeably herein, the terms "coding sequence" or "protein coding sequence" refer to a polynucleotide segment that encodes a protein. This region or sequence has a start codon near the 5' end and a stop codon near the 3' end. A coding sequence may also be referred to as an open reading frame.

[0235] As used herein, the term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination reaction of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination reaction of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the deoxygenation reaction of adenosine or deoxyinosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination reaction of adenosine in deoxyribonucleic acid (DNA). Provided herein are adenosine deaminases (e.g., engineered adenosine deaminases, evolved adenosine deaminases) that can be from any organism such as bacteria. In some embodiments, the adenosine deaminase is from a bacterium, such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Caulobacter crescentus.

[0236] In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is a TadA variant. In some embodiments, the TadA variant is TadA*8. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to a naturally occurring deaminase. For example, deaminase domains are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is herein incorporated by reference in its entirety.See also, Komor, AC et al., "Programmable base editing of a target base in genomicDNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, NM et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage" Nature 551.464-471 (2017); Komor, AC et al., "Improved base excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T: A 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: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0237] "Detection" refers to confirming the presence, absence, or amount of an analyte to be detected. In one embodiment, sequence changes in a polynucleotide or polypeptide are detected. In another embodiment, the presence of insertions or deletions (indels) is detected.

[0238] "Detectable label" means a composition that, when attached to a molecule of interest, renders the latter detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic microbeads, metal microbeads, colloidal ions, fluorescent dyes, electron-dense reagents, enzymes (e.g., as typically used in enzyme-linked immunosorbent assays (ELISAs)), biotin, digoxigenin, or haptens.

[0239] "Disease" means any condition or disorder that damages or interferes with the normal function of cells, tissues, or organs.

[0240] As used herein, the term "effective amount" refers to an amount of a bioactive agent sufficient to cause a desired biological response. The effective amount of the active compound used to practice the present invention for therapeutic treatment of diseases is variable and depends on the mode of administration and the age, weight and general health of the subject. Basically, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. This amount is referred to as an "effective" amount. In one embodiment, an effective amount is an amount of a base editor of the present invention (e.g., a fusion protein comprising a programmable DNA binding protein, a nuclear base editor and a gRNA) sufficient to introduce a change in a gene of interest into a cell (e.g., a cell in vitro or in vivo). In some embodiments, an effective amount of a fusion protein provided herein (e.g., a nuclear base editor comprising an nCas9 domain and a deaminase domain (e.g., adenosine deaminase or cytidine deaminase)) may refer to an amount of a fusion protein sufficient to induce editing of a target site that is specifically bound and edited by the nuclear base editor. In one embodiment, an effective amount is the amount of a base editor required to achieve a therapeutic effect (e.g., to weaken or control a disease or symptom or condition). Such therapeutic effects need not be sufficient to alter the gene of interest in all cells of a subject, tissue or organ, but rather only in about 1%, 5%, 10%, 25%, 50%, 75% or more of the cells present in the subject, tissue or organ.

[0241] In some embodiments, an effective amount of a fusion protein (e.g., a nucleobase editor comprising an nCas9 domain and a deaminase domain (e.g., an adenosine deaminase or cytidine deaminase)) provided herein refers to an amount of the fusion protein sufficient to induce editing of a target site that is specifically bound and edited by a nucleobase editor as described herein. As will be appreciated by those skilled in the art, the effective amount of an agent, such as a fusion protein, a nucleobase, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide can vary based on a variety of factors, for example, based on the desired biological response, for example, based on the specific allele, genome, or target to be edited, based on the cell or assembly being targeted, and / or based on the agent used.

[0242] "Fragment" means a portion of a polypeptide or nucleic acid molecule. This portion contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of a reference nucleic acid molecule or polypeptide. A fragment can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0243] "Guide RNA" or "gRNA" means a polynucleotide that can be specific for a target sequence and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpf1). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs, or as a single RNA molecule. A gRNA that exists as a single RNA molecule can be referred to as a single guide RNA (sgRNA), but "gRNA" is used 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., and guides the Cas9 complex to bind to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and includes a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. gRNAs (e.g., those including domain (2)) can be found in U.S. Provisional Patent Application No. 61 / 874,682, entitled "Switchable Cas9 Nucleases and Uses Thereof," and U.S. Provisional Patent Application No. 61 / 874,746, entitled "Delivery System For Functional Nucleases," filed on September 6, 2013, the entire contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the gRNA comprises two or more domains (1) and (2) and can be referred to as an "extended gRNA." The extended gRNA will bind two or more Cas9 proteins at two or more different regions and bind to the target nucleic acid, as described herein. The gRNA comprises a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site and provides sequence specificity to the nuclease:RNA complex.

[0244] "Hybridization" means hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen, and reversed Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds.

[0245] The term "inhibitor of base repair" or "IBR" refers to a protein that can inhibit the activity of a nucleic acid repair enzyme (e.g., base excision repair (BER) enzyme). In some embodiments, IBR is an inhibitor of inosine base excision repair. Exemplary base repair inhibitors include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGG1, hNEIL1, T7Endol, T4PDG, UDG, hSMUG1, and hAAG. In some embodiments, IBR is an inhibitor of Endo V or hAAG. In some embodiments, IBR is catalytically inactive EndoV or catalytically inactive hAAG. In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, the base repair inhibitor is catalytically inactive EndoV or catalytically inactive hAAG.

[0246] In some embodiments, the base repair inhibitor is a uracil glycosidase inhibitor (UGI). UGI refers to a protein that can inhibit the uracil-DNA glycosidase base excision repair enzyme. In some embodiments, the UGI domain comprises a wild-type UGI or a fragment of wild-type UGI. In some embodiments, the UGI protein provided herein includes a fragment of UGI and a protein homologous to UGI or a UGI fragment. In some embodiments, the base repair inhibitor is an inhibitor of inosine base excision repair. In some embodiments, the base repair inhibitor is a "catalytically inactive inosine-specific nuclease" or a "dead inosine-specific nuclease." Without being bound by any particular theory, a catalytically inactive inosine glycosidase (e.g., alkyladenine glycosidase (AAG)) can bind to inosine but cannot create an abasic site or remove the inosine, thereby spatially blocking the effects of the DNA damage / repair mechanism on the newly formed inosine portion. In some embodiments, a catalytically inactive inosine-specific nuclease can bind to inosine within a nucleic acid but does not cleave the nucleic acid. Non-limiting exemplary catalytically inactive inosine-specific nucleases include, for example, catalytically inactive alkyladenosine glycosidase (AAG nuclease) from humans, and, for example, catalytically inactive endonuclease V (EndoV nuclease) from Escherichia coli. In some embodiments, the catalytically inactive AAG nuclease comprises an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0247] By "increase" is meant a positive change of at least 10%, 25%, 50%, 75% or 100%.

[0248] "Intein" is a fragment of a protein that can excise itself and join the remaining fragment (extein) through a peptide bond in a process called protein splicing. Intein is also called "protein intron". The process in which an intein excises itself and joins the remaining part of a protein is referred to herein as "protein splicing" or "intein-mediated protein splicing". In some embodiments, the intein of a precursor protein (a protein containing an intein before intein-mediated protein splicing) comes from two genes. Herein, such inteins are referred to as split inteins (e.g., split intein-N and split intein-C). For example, in cyanobacteria, DnaE (the catalytic subunit of DNA polymerase III) is encoded by two independent genes (dnaE-n and dnaE-c). The intein encoded by the dnaE-n gene can be referred to herein as "intein-N". The intein encoded by the dnaE-c gene can be referred to herein as "intein-C".

[0249] Other intein systems can also be used. For example, synthetic inteins based on the dnaE intein, the Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C) intein pairs, have been described (e.g., in Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7): 2162-5, which is incorporated herein by reference). Non-limiting examples of intein pairs that 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. Pat. No. 8,394,604, which is incorporated herein by reference).

[0250] Exemplary nucleotide and amino acid sequences of inteins are provided.

[0251] DnaE intein-N DNA:

[0252]

[0253] DnaE intein-N protein:

[0254]

[0255] DnaE intein-C DNA:

[0256]

[0257] Intein-C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN

[0258] Cfa-N DNA:

[0259]

[0260] Cfa-N protein:

[0261]

[0262] Cfa-C DNA:

[0263]

[0264] Cfa-C protein:

[0265]

[0266] Intein-N and intein-C can be fused to the N-terminal portion of split-Cas9 and the C-terminal portion of split-Cas9, respectively, to join the N-terminal portion of split-Cas9 and the C-terminal portion of split-Cas9. For example, in some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of split-Cas9, i.e., to form a structure of N-[N-terminal portion of split-Cas9]-[intein-N]--C. In some embodiments, intein-C is fused to the N-terminus of the C-terminal portion of split-Cas9, i.e., to form a structure of N-[intein-C]--[C-terminal portion of split-Cas9]-C. The mechanism of intein-mediated protein splicing for joining proteins to which intein is fused (e.g., split-Cas9) is known in the art, for example, as described in Shah et al., Chem Sci. 2014; 5(1): 446-461, which is incorporated herein by reference. Methods for designing and using inteins are known in the art and are described in, for example, WO2014004336, WO2017132580, US20150344549, and US20180127780, each of which is herein incorporated by reference in its entirety.

[0267] The terms "isolated," "purified," or "biologically pure" refer to a material that is free, to varying degrees, from the normal accompanying components found in its native state. "Isolated" refers to a degree of separation from the original source or surrounding material. "Purified" refers to a degree of separation greater than "isolated." A "purified" or "biologically pure" protein is sufficiently free of other substances such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. In other words, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or 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 a nucleic acid or protein gives rise to substantially a single band in an electrophoretic gel. For proteins that can be modified, such as phosphorylation or glycosylation, different modifications can give rise to different isolated proteins that can be purified independently.

[0268] "Isolated polynucleotide" means a nucleic acid (e.g., DNA) that is free of the genes that flank it in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. The term thus includes, for example, recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryotic or eukaryotic organism, or that exists as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments prepared by PCR or restriction endonuclease digestion). In addition, the term includes RNA molecules transcribed from a DNA molecule, as well as recombinant DNA that is part of a hybrid gene encoding an accessory polypeptide sequence.

[0269] "Isolated polypeptide" means a polypeptide of the present invention that has been separated from its naturally associated components. Typically, a polypeptide is isolated when it is at least 60% free, by weight, of the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation contains at least 75%, more preferably at least 90%, and most preferably 99%, by weight of the polypeptide of the present invention. An isolated polypeptide of the present invention can be obtained, for example, by extraction from a natural source, expression of a recombinant nucleic acid encoding the polypeptide, or chemical synthesis of the protein. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0270] As used herein, the term "linker" may refer to a covalent linker

[0271] (e.g., covalent bond), non-covalent linker, chemical group or molecule that connects two molecules or parts, for example, two components of a protein complex or a ribonucleic acid complex, or two domains of a fusion protein, for example, a polynucleotide programmable DNA binding domain (e.g., dCas9) and a deaminase domain (e.g., adenosine deaminase, 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 into the Cas protein or its fragment. The linker can engage different components of the base editor system or different parts of multiple components. For example, in some embodiments, the linker can engage the guide polynucleotide binding domain of the polynucleotide programmable nucleotide binding domain with the catalytic domain of the deaminase. In some embodiments, the linker can engage the CRISPR polypeptide with the deaminase. In some embodiments, the linker can engage Cas9 with the deaminase. In some embodiments, the linker can connect dCas9 to a deaminase. In some embodiments, the linker can connect nCas9 to a deaminase. For example, in some embodiments, the linker can connect Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, or Cas12i to a deaminase. In some embodiments, the linker can connect a guide polynucleotide to a deaminase. In some embodiments, the linker can connect the deamination component of a base editor system to a polynucleotide programmable nucleotide binding component. In some embodiments, the linker can bind the RNA binding portion of the deamination component of the base editor to the napDNAbp component. In some embodiments, the linker can bind the RNA binding portion of the deamination component of the base editor to the polynucleotide programmable nucleotide binding component. In some embodiments, the linker can bind the RNA binding portion of the deamination component of the base editor to the RNA binding portion of the polynucleotide programmable nucleotide binding component. A linker can be positioned between or flanked by two groups, molecules, or other moieties and linked to each group, molecule, or other moiety via a covalent bond or non-covalent interaction, thereby linking the two. In some embodiments, the linker can be an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, the linker can 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, the linker can comprise an aptamer that can be derived from a riboswitch. The riboswitch from which the aptamer is derived can be selected from the group consisting of theophylline riboswitch, thiamine pyrophosphate (TPP) riboswitch, adenosylcobalamin (AdoCbl) riboswitch, S-adenosylmethionine (SAM) riboswitch, SAH riboswitch, flavin mononucleotide (FMN) riboswitch, tetrahydrofolate riboswitch, lysine riboswitch, glycine riboswitch, purine riboswitch, GlmS riboswitch, or pre-deazaguanosine 1 (PreQ1) riboswitch. In some embodiments, the linker can comprise an aptamer that binds to a polypeptide or protein domain, such as a polypeptide ligand. In some embodiments, the polypeptide ligand can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif. In some embodiments, the polypeptide ligand can be part of a base editor system component. For example, a nucleobase editing component can include a deaminase domain and an RNA recognition motif.

[0272] In some embodiments, the connexon can be an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the connexon can be a length of about 5-100 amino acids, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids in length. In some embodiments, the connexon can be a length of about 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 amino acids in length. Longer or shorter connexons can also be envisioned.

[0273] In some embodiments, the linker engages the gRNA engagement domain of an RNA programmable nuclease (including a Cas9 nuclease domain) and the catalytic domain of a nucleic acid editing protein (e.g., a cytidine or adenosine deaminase). In some embodiments, the linker engages dCas9 and a nucleic acid editing protein. For example, the linker can be positioned between two groups, molecules, or other moieties or flanked by two groups, molecules, or other moieties and linked to each group, molecule, or other moiety via a covalent bond, thereby linking the two. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-200 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 65, 70, 75, 80, 85, 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.

[0274] In some embodiments, multiple domains of a nucleobase editor are fused via a linker comprising or In some embodiments, the multiple domains of the base editor are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES, which may also be referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker comprises (SGGS) n 、(GGGS) n 、(GGGGS) n 、(G) n 、(EAAAK) n 、(GGS) n , SGSETPGTSESATPES or (XP) n motif, or a combination of any of these motifs, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0275] In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence

[0276]

[0277] "Marker" means any protein or polynucleotide whose expression level or activity is altered in a manner associated with a disease or disorder.

[0278] As used herein, the term "mutation" refers to a residue within a sequence (e.g., a nucleic acid or amino acid sequence) being replaced with another residue, or a deletion or insertion of one or more residues within the sequence. Typically, mutations are described herein by identifying the original residue, then identifying the position of the residue within the sequence and the identity of the newly replaced residue. The methods for performing amino acid substitutions (mutations) provided herein are well known in the art and are provided in, for example, "Molecular Cloning: A Laboratory Manual (Fourth Edition)" (Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). In some embodiments, the base editors disclosed herein can effectively produce "expected mutations" such as point mutations in nucleic acids (e.g., nucleic acids within the subject's genome) without producing a significant number of unexpected mutations such as unexpected point mutations. In some embodiments, the expected mutation is a mutation generated by a specific base editor (e.g., a cytidine base editor or an adenosine base editor) bound to a guide polynucleotide (e.g., gRNA), which is specifically directed to produce the expected mutation.

[0279] Typically, mutations made or identified in a sequence (e.g., an amino acid sequence as described herein) are numbered relative to a reference (or wild-type) sequence, i.e., a sequence that does not contain the mutation. Those skilled in the art will readily understand how to determine the position of mutations in amino acid and nucleic acid sequences relative to a reference sequence.

[0280] The term "non-conservative mutation" includes amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the wild-type protein.

[0281] The term "nuclear localization element sequence," "nuclear localization element signal," or "NLS" refers to an amino acid sequence that facilitates the entry of a protein into the nucleus of a cell. Nuclear localization sequences are known in the art and are described, for example, in International PCT Application PCT / EP2000 / 011690, filed November 23, 2000, by Plank et al., and published as WO / 2001 / 038547 on May 31, 2001, which is incorporated herein by reference for its disclosure of exemplary nuclear localization element sequences. In other embodiments, the NLS is an optimized NLS, for example, as described in Koblan et al., Nature Biotech. 2018 doi: 10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence or

[0282] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a compound comprising a core base and an acidic moiety, for example, a polymer of nucleosides, nucleotides or nucleotides. Typically, a polymeric nucleic acid (for example, a nucleic acid molecule) comprising three or more nucleotides is a linear molecule in which adjacent nucleotides are linked to each other via phosphodiester bonds. In some embodiments, "nucleic acid" refers to an individual nucleic acid residue (for example, a nucleotide and / or nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably and refer to a polymer of nucleotides (for example, a string of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA and single-stranded and / or double-stranded DNA. Nucleic acid can naturally occur in the context of, for example, genomes, transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plasmids, cosmids, chromosomes, chromatids or other naturally occurring nucleic acid molecules. On the other hand, nucleic acid molecules can be the molecule that non-natural occurs, for example, recombinant DNA or RNA, artificial chromosome, genome or its fragment through through engineering approaches, or synthetic DNA, RNA, DNA / RNA hybrid, or comprise nucleotide or nucleoside that non-natural occurs.In addition, term " nucleic acid ", " DNA ", " RNA " and / or similar term comprise nucleic acid analogs, for example, have the analog of the main chain except phosphodiester.Nucleic acid can be purified from natural source, uses recombinant expression system to produce and optionally purifies, chemically synthesizes etc.If suitable, for example, in the case of chemically synthesized molecule, nucleic acid can comprise nucleoside analogs such as have chemically modified base or sugar and the analog of main chain modification.Unless otherwise noted, otherwise nucleotide sequence presents with 5 ' to 3 ' direction. In some embodiments, the nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyuridine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, guanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (2' modifications, e.g., fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0283] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" can be used interchangeably with "polynucleotide programmable nucleotide binding domain" to refer to a protein (e.g., DNA or RNA) that associates with a nucleic acid, such as a guide nucleic acid or a 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 associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, e.g., a nuclease-active Cas9, a Cas9 nickase (nCas9), or a Cas9 without nuclease activity (dCas9). Non-limiting examples of nucleic acid-programmable DNA-binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2c1, 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, CasSh, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2 , Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a type II Cas effector protein, a type V Cas effector protein, a type VI Cas effector protein, CARF, DinG, a homolog thereof, or a modified or engineered version thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, but they may not be specifically listed in the present disclosure. See, for example, Makarova et al. "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR J. 2018 Oct; 1: 325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., "Functionally diverse type V CRISPR-Cas systems" Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, the entire contents of each of which are incorporated herein by reference.

[0284] As used herein, the terms "nucleobase," "nitrogenous base," or "base" are used interchangeably to refer to biological compounds containing nitrogen that form nucleosides, which in turn become components of nucleotides. The ability of nucleobases to form base pairs and stack one upon another directly leads to long helical structures, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Five nucleobases, i.e., adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), are referred to as basic bases or standard bases. Adenine and guanine are derived from purines, while cytosine, uracil, and thymine are derived from pyrimidines. DNA and RNA may also contain other (non-basic) bases that are modified. Non-limiting exemplary modified nucleobases may include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydroxymethylcytosine. Hypoxanthine and xanthine can be produced by the presence of mutagens, and both are produced by deamination (replacing the amino group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be obtained from the deamination reaction of cytosine. "Nucleoside" consists of a nucleobase and a five-carbon sugar (or ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. Examples of nucleosides with modified nucleobases include inosine (I), xanthine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C) and pseudouridine (Ψ). "Nucleotide" consists of a nucleobase, a five-carbon sugar (or ribose or deoxyribose) and at least one phosphate group.

[0285] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" refers to a protein (e.g., DNA or RNA) that associates with a nucleic acid, such as a guide nucleic acid that guides napDNAbp to a specific nucleic acid sequence. For example, the Cas12 protein can associate with a guide RNA that guides the Cas12 protein to a specific DNA sequence, the DNA sequence being complementary to the guide RNA. In some embodiments, napDNAbp is a Cas12 domain, for example, a nuclease-active Cas12 domain. Examples of napDNAbp include Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Other napDNAbps are also within the scope of the present disclosure, but they may not be specifically listed in the present disclosure. See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct; 1: 325-336. doi: 10.1089 / crispr.2018.0033; Yan et al. “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, the entire contents of each of which are incorporated herein by reference.

[0286] As used herein, the term "nucleobase editing domain" or "nucleobase editing protein" refers to a protein or enzyme that can catalyze nucleobase modifications in RNA or DNA, such as cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine) and adenine (or adenosine) to hypoxanthine (or inosine), as well as non-templated nucleotide additions and insertions. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., an adenine deaminase or adenosine deaminase; or a cytidine deaminase or a cytosine deaminase). In some embodiments, the nucleobase editing domain is more than one deaminase domain (e.g., an adenine deaminase or adenosine deaminase; and a 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 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 excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A baseeditors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017)), the entire contents of which are incorporated herein by reference.

[0287] As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing or otherwise obtaining the agent.

[0288] As used herein, "patient" or "subject" refers to a mammalian subject or individual who has been diagnosed with, is at risk of, or is susceptible to or develops a disease or disorder. In some embodiments, the term "patient" refers to a mammalian subject who has a higher than average likelihood of developing a disease or disorder. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cows, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), and other mammals that may benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female.

[0289] As used herein, a "patient in need thereof" or "subject in need thereof" refers to a patient who has been diagnosed with, is at risk of, is previously identified as having, or is susceptible to a disease or disorder.

[0290] The terms "pathogenic mutation," "pathogenic variant," "disease casing mutation," "disease causing variant," "deleterious mutation," or "predisposing mutation" refer to a genetic mutation or alteration that increases an individual's susceptibility to or predisposition to a disease or condition. In some embodiments, the pathogenic mutation comprises a substitution of at least one wild-type amino acid in a protein encoded by a gene with at least one pathogenic amino acid.

[0291] The term "pharmaceutically-acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate, zinc stearate, or stearic acid), or solvent 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 in the body (e.g., an organ, tissue, or part of the body). A pharmaceutically acceptable carrier is "acceptable" in terms of compatibility with the other ingredients of the formulation and is not harmful to the tissues of the subject (e.g., physiologically compatible, sterile, physiological pH, etc.). Terms such as "excipient," "carrier," "pharmaceutically acceptable carrier," "vehicle," etc. are used interchangeably herein.

[0292] The term "pharmaceutical composition" may refer to a composition formulated for pharmaceutical use.

[0293] As used herein, the terms "protein," "peptide," "polypeptide," and their grammatical equivalents are used interchangeably and refer to polymers of amino acid residues linked together by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids in length. A protein, peptide, or polypeptide may refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modifications, or other modifications. A protein, peptide, or polypeptide may also be a single molecule or may be a multimolecular complex. A protein, peptide, or polypeptide may be merely a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. A protein can be positioned at the amino terminal (N-terminal) part of the fusion protein or positioned at the carboxyl terminal (C-terminal) protein, thereby forming an amino terminal fusion protein or a carboxyl terminal fusion protein respectively. Protein can include different domains, for example, a nucleic acid binding domain (for example, a gRNA binding domain that guides the protein to bind to the target site of Cas9) and a nucleic acid cleavage domain or a catalytic domain of a nucleic acid editing protein. In some embodiments, the protein includes a protein portion (for example, an amino acid sequence constituting a nucleic acid binding domain) and an organic compound (for example, a compound that can serve as a nucleic acid cleavage agent). In some embodiments, the protein is compounded or associated with nucleic acids such as RNA or DNA. Any protein provided herein can be produced by any method known in the art. For example, the protein provided herein can be produced via recombinant protein expression and purification, and this method is particularly suitable for fusion proteins comprising peptide linkers. Methods for recombinant protein expression and purification are well known and include those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), the entire contents of which are incorporated herein by reference.

[0294] The polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) may comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-yl ...

[00145] The polypeptides and proteins may be associated with post-translational modifications of one or more amino acids of the polypeptide construct. Non-limiting examples of post-transcriptional modifications include phosphorylation, acylation (including acetylation and formylation), glycosylation (including N-linked glycosylation and O-linked glycosylation), amidation, hydroxylation, alkylation (including methylation and ethylation), ubiquitination, addition of pyrrolidonecarboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, prenylation, farnesylation, geranylation, glycosylphosphatidylinositolization, lipidation, and iodination.

[0295] The term "polynucleotide programmable nucleotide binding domain" or "nucleic acid programmable DNA binding domain (napDNAbp)" refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide polynucleotide (e.g., guide RNA) that guides 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.

[0296] As used herein, the term "recombinant" in the context of a protein or nucleic acid refers to a protein or nucleic acid that does not occur in nature but is instead a product of artificial engineering. For example, in some embodiments, a recombinant protein or nucleic acid comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any naturally occurring sequence.

[0297] "Decrease" means a negative change of at least 10%, 25%, 50%, 75% or 100%.

[0298] "Reference" means a standard or control condition. In one embodiment, the reference is a wild-type or healthy cell. In other embodiments and without limitation, the reference is an untreated cell that has not been subjected to the test condition, or has been subjected to a placebo or saline, culture medium, buffer, and / or control vector that does not carry the polynucleotide of interest.

[0299] A "reference sequence" is a defined sequence used as a basis for sequence alignment. A reference sequence can be a portion or the entirety of a particular sequence; for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will typically be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will typically be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides, or any integer close thereto or therebetween. In some embodiments, the reference sequence is the wild-type sequence of a protein of interest. In other embodiments, the reference sequence is a polynucleotide sequence encoding a wild-type protein.

[0300] The terms "RNA programmable nuclease" and "RNA-guided nuclease" are used in conjunction with (e.g., bound to or associated with) one or more RNAs that are not cleavage targets. In some embodiments, when complexed with RNA, the RNA programmable nuclease can be referred to as a nuclease:RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs, or as a single RNA molecule. A gRNA that exists as a single RNA molecule can be referred to as a single guide RNA (sgRNA), but "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or as a complex of two or more molecules. Typically, a gRNA that exists as a single RNA species contains two domains: (1) a domain that shares homology with the target nucleic acid (e.g., and guides the Cas9 complex to the target); and (2) a domain that binds the Cas9 protein. In some embodiments, domain (2) corresponds to the sequence known as tracrRNA and includes a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. gRNAs (e.g., those including domain (2)) can be found in U.S. Provisional Patent Application Serial No. 61 / 874,682, entitled "Switchable Cas9 Nucleases and Uses Thereof," filed on September 6, 2013, and U.S. Provisional Patent Application Serial No. 61 / 874,746, entitled "Delivery System For Functional Nucleases," filed on September 6, 2013, the entire contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the gRNA comprises two or more domains (1) and (2) and can be referred to as an "extended gRNA." For example, an extended gRNA will bind to two or more Cas9 proteins and bind to a target nucleic acid, for example, at two or more different regions, as described herein. The gRNA comprises a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site and provides sequence specificity to the nuclease:RNA complex.

[0301] In some embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 from Streptococcus pyogenes (Casn1) (see, e.g., Complete genome sequence of an Mlstrain 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)).

[0302] Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins can in principle be targeted to any sequence that is specific to a guide RNA. Methods for site-specific cleavage (e.g., to modify the genome) using RNA-programmable nucleases such as Cas9 are known in the art (see, e.g., Cong, L. et al., Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, WY et al., Efficient genome editing 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).

[0303] The term "single nucleotide polymorphism (SNP)" is a single nucleotide variation that occurs at a specific location in the genome, where each variation exists to some appreciable degree (e.g., >1%) within a population. For example, at a specific base position in the human genome, a C nucleotide may appear in most individuals, but in a minority of individuals, this position is occupied by an A. This means that a SNP exists at this specific position, and the two possible nucleotide variations, C or A, are called alleles at this position. SNPs form the basis for differences in susceptibility to disease. The severity of a disease and the way our bodies respond to treatment are also manifestations of genetic variation. SNPs can fall within the coding region of a gene, the non-coding region of a gene, or within the intergenic region (the region between genes). In some embodiments, due to the degeneracy of the genetic code, SNPs within the coding sequence do not necessarily change the amino acid sequence of the protein produced. There are two types of SNPs in the coding region: synonymous SNPs and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, while non-synonymous SNPs change the amino acid sequence of the protein. There are two types of non-synonymous SNPs: missense and nonsense. SNPs that are not in protein coding regions may still affect gene splicing, transcription factor binding, messenger RNA degradation or the sequence of non-coding RNA. The gene expression affected by this type of SNP is called eSNP (expression SNP) and can be located upstream or downstream of the gene. Single nucleotide variants (SNVs) are variations in single nucleotides, without any frequency restrictions, and can occur in somatic cells. Somatic single nucleotide variations can also be referred to as single nucleotide changes.

[0304] "Specifically binds" means that 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 recognizes and binds to a polypeptide and / or nucleic acid molecule of the invention, but they do not substantially recognize and bind to other molecules in a sample, such as a biological sample.

[0305] 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 will exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing to 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 will exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" means the pairing of complementary polynucleotide sequences (e.g., a gene described herein) or portions thereof under various stringency conditions to form a double-stranded molecule. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

[0306] 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 and more preferably at least about 50% formamide. Stringent temperature conditions will generally include temperatures of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Additional factors that can be varied, such as hybridization time, the concentration of detergents such as sodium dodecyl sulfate (SDS), and the presence or absence of saturated carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as desired. In one embodiment, hybridization will occur in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30°C. In another embodiment, hybridization will occur in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C. In another embodiment, hybridization will occur in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations on these conditions will be apparent to those skilled in the art.

[0307] For most applications, the stringency of the post-hybridization wash step will also vary. Wash stringency conditions can be defined by salt concentration and temperature. As described above, wash stringency can be increased by reducing the salt concentration or increasing the temperature. For example, the stringent salt concentration for the wash step is preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash step will generally include a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In one embodiment, the wash step will occur in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS at 25°C. In a more preferred embodiment, the wash step will occur in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 42°C. In an even more preferred embodiment, the wash step will occur in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 68°C. Additional variations on these conditions will be readily apparent to those skilled 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.

[0308] "Split" means to divide into two or more fragments.

[0309] "Split Cas9 protein" or "split Cas9" refers to a Cas9 protein that is provided as an N-terminal fragment and a C-terminal fragment encoded by two independent nucleotide sequences. The polypeptides corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein can be split to form a "reconstructed" Cas9 protein. In a particular embodiment, the Cas9 protein is divided into two fragments that are within the disordered region of the protein, for example, as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp.935-949, 2014 or as described in Jiang et al. (2016) Science 351: 867-871.PDB file: 5F9R, each of which is incorporated herein by reference. In some embodiments, the protein is split into two fragments at any C, T, A, or S located in the region between approximately amino acids A292-G364, F445-K483, or E565-T637 of SpCas9, or at corresponding positions of any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other napDNAbp. In some embodiments, the protein is split into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of splitting a protein into two fragments is referred to as "splicing" the protein.

[0310] In other embodiments, the N-terminal portion of the Cas9 protein comprises amino acids 1-573 or 1-637 of Streptococcus pyogenes Cas9 wild type (SpCas9) (NCBI reference sequence: NC_002737.2, Uniprot reference sequence: Q99ZW2), and the C-terminal portion of the Cas9 protein comprises amino acids 574-1368 or 638-1368 of SpCas9 wild type.

[0311] The C-terminal portion of the split Cas9 can be joined to the N-terminal portion of the split Cas9 to form a complete Cas9 protein. In some embodiments, the C-terminal portion of the Cas9 protein begins where the N-terminal portion of the Cas9 protein ends. Therefore, in some embodiments, the C-terminal portion of the split Cas9 comprises the amino acid (551-651)-1368 portion of spCas9. "(551-651)-1368" means starting at the amino acids between amino acids 551-651 (inclusive) and ending at amino acid 1368.For example, the C-terminal portion of the split-Cas9 can comprise amino acids 551-1368, 552-1368, 553-1368, 554-1368, 555-1368, 556-1368, 557-1368, 558-1368, 559-1368, 560-1368, 561-1368, 562-1368, 563-1368, 564-1368, 565-1368, 566-1368, 567-1368, 568-1368, 569-1368, 570-1368, 571-1368, 572-1368, 573-1368, 574-1368 74-1368、575-1368、576-1368、577-1368、578-1368、579-1368、580-1368、581-1368、582-1368、583-1368、584-1368、585-1368、586-1368、587-1368、588-1368、589-1368、590-1368、591-1368、592-1368、593-1368、594-1368、595-1368、596-1368、597-1368、598-1368、599-1368、600 -1368, 601-1368, 602-1368, 603-1368, 604-1368, 605-1368, 606-1368, 607-1368, 608-1368, 609-1368, 610-1368, 611-1368, 612-1368, 613-1368, 614-1368, 615-1368, 616-1368, 617-1368, 618-1368, 619-1368, 620-1368, 621-1368, 622-1368, 623-1368, 624-1368, 625-1368, 626-1368 or a portion of any of: 368, 627-1368, 628-1368, 629-1368, 630-1368, 631-1368, 632-1368, 633-1368, 634-1368, 635-1368, 636-1368, 637-1368, 638-1368, 639-1368, 640-1368, 641-1368, 642-1368, 643-1368, 644-1368, 645-1368, 646-1368, 647-1368, 648-1368, 649-1368, 650-1368, or 651-1368. In some embodiments, the C-terminal portion of the split-Cas9 protein comprises amino acids 574-1368 or 638-1368 of spCas9.

[0312] "Subject" means a mammal, including but not limited to humans and non-human mammals, such as cattle, horses, dogs, sheep or cats. Subjects include livestock and domesticated animals used to produce labor and provide commodities such as food, including but not limited to cattle, goats, chickens, horses, pigs, rabbits and sheep.

[0313] "Substantially identical" means that the polypeptide or nucleic acid molecule exhibits at least 50% identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). In one embodiment, the sequence is at least 60%, 80%, 85%, 90%, 95%, or even 99% identical at the amino acid level or nucleic acid level to the sequence used for comparison.

[0314] Sequence identity is typically measured using sequence analysis software (e.g., BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs from the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). The software matches identical or similar sequences by setting the degree of homology for a variety of 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; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program can be used, where between e -3 With e -100 Likelihood scores between indicate closely related sequences.

[0315] For example, COBALT with the following parameters was used:

[0316] a) Alignment parameters: gap penalties of -11, -1, and end gap penalties of -5, -1,

[0317] b) CDD parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved columns and Recompute on, and

[0318] c) Query clustering parameters: Use query clustering enabled; word length 4; maximum inter-cluster distance 0.8; autotype style is Regular.

[0319] For example, the EMBOSS Needle with the following parameters was used:

[0320] a) Matrix: BLOSUM62;

[0321] b) GAP OPEN: 10;

[0322] c) GAP EXTEND: 0.5;

[0323] d) Output format: Yes;

[0324] e) End gap penalty: False;

[0325] f) Start of terminal gap: 10; and

[0326] g) Terminal gap extension: 0.5.

[0327] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a base editor. In one embodiment, the target site is deaminated by a deaminase or a fusion protein comprising a deaminase (e.g., cytidine or adenine deaminase).

[0328] As used herein, "treat," "treating," "treatment," and the like refer to alleviating or relieving the pathology and / or symptoms associated therewith or obtaining a desired pharmacological and / or physiological effect. It should be understood that, although not excluded, treating a condition or symptom does not require the complete elimination of the condition, symptom, or symptoms associated with the condition or symptom. In some embodiments, the effect is therapeutic, that is, without limitation, partially or completely weakening, reducing, abolishing, attenuating, alleviating, reducing the intensity of the disease and / or adverse symptoms attributable to the disease, or curing the disease and / or adverse symptoms attributable to the disease. In some embodiments, the effect is prophylactic, that is, an effect that protects or prevents the onset or recurrence of a disease or condition. In this regard, the methods disclosed herein comprise administering a therapeutically effective amount of a composition as described herein.

[0329] "Uracil glycosylase inhibitor" or "UGI" means an agent that inhibits the uracil excision repair system. In one embodiment, the agent is a protein or a fragment thereof that binds to the host uracil-DNA glycosidase and prevents uracil residues from being removed from DNA. In a delivery method, UGI refers to a protein, a fragment or a domain thereof that is capable of inhibiting the uracil-DNA glycosidase base excision repair enzyme. In some embodiments, the UGI domain comprises a wild-type UGI or a modified version thereof. In some embodiments, the UGI domain comprises a fragment of the exemplary amino acid sequence described in detail below. In some embodiments, the UGI fragment comprises an amino acid sequence comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the exemplary UGI sequence provided below. In some embodiments, UGI comprises an amino acid sequence homologous to an exemplary GI amino acid sequence or a fragment thereof, as described in detail below. In some embodiments, UGI or a portion thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to wild-type UGI or a UGI sequence or a portion thereof, as described in detail below. An exemplary UGI comprises the following amino acid sequence:

[0330] >splP14739IUNGI_BPPB2 uracil-DNA glycosidase inhibitor

[0331]

[0332] The term "vector" refers to the process by which a nucleic acid sequence is introduced into a cell to transform the cell. Vectors include plasmids, transposons, phages, viruses, liposomes, and episomes. An "expression vector" is a nucleic acid sequence that contains a nucleotide sequence to be expressed in a recipient cell. An expression vector may include additional nucleic acid sequences to facilitate and / or promote expression of the introduced sequence, such as initiators, terminators, promoters, and secretion sequences.

[0333] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0334] DNA editing has emerged as a viable approach to modify disease states by correcting pathogenic mutations at the genetic level. Until recently, all DNA editing platforms functioned by introducing DNA double-strand breaks (DSBs) at specific genomic loci and relying on endogenous DNA repair pathways to determine product outcomes in a semi-stochastic manner, resulting in a complex population of gene products. Although precise, user-defined repair outcomes can be achieved through the homology-directed repair (HDR) pathway, numerous challenges have prevented the use of HDR to achieve high-efficiency repair in therapeutically relevant cell types. In practice, this pathway is inefficient relative to the competing, error-prone non-homologous end joining pathway. Furthermore, HDR is strictly confined to the G1 and S phases of the cell cycle, preventing precise repair in post-mitotic cells. As a result, it has proven difficult or impossible to efficiently alter genomic sequences in these populations in a user-defined, programmable manner.

[0335] Superoxide dismutase 1 (SOD1) is an enzymatic protein encoded by the SOD1 gene. The SOD1 enzyme is present in large quantities within cells throughout the body. The SOD1 enzyme combines copper (Cu) and zinc (Zn) to degrade toxic, charged oxygen molecules known as superoxide radicals or reactive oxygen species (ROS). These oxygen molecules are byproducts of normal cellular processes and must be regularly degraded to avoid cell damage, transformation, or death. It has been found that at least 200 mutations in the SOD1 gene cause amyotrophic lateral sclerosis (ALS), a disease characterized by progressive muscle weakness, loss of muscle mass, and inability to control movement. Most of these mutations change an amino acid in the superoxide dismutase. Worldwide, SOD1 gene mutations cause 15% to 20% of familial ALS. Of all Americans with ALS caused by SOD1 gene mutations, approximately half have a specific mutation in which the amino acid residue alanine (A) at position 5 of the enzyme is replaced by the amino acid residue valine (V), i.e., Ala5Val or A5V. ALS caused by the A5V mutation is generally associated with a shorter life expectancy compared to ALS caused by mutations in other genes.

[0336] ALS is caused by the death of nerve cells that control muscle movement (motor neurons). It is not clear why motor neurons are particularly sensitive to mutations in the SOD1 gene, but the large size of these types of neurons may make them more sensitive to disruptions in the normal function of the SOD1 enzyme. There are several ways in which the altered SOD1 enzyme may cause motor neuron death, including: (i) an increase in harmful superoxide radicals, particularly in the cell's lysosomes and / or proteasomes; (ii) an increase in the production of other types of toxic free radicals and increased cell death; and / or (iii) the accumulation of clumps (aggregates) of misfolded superoxide dismutase, which can be toxic to cells.

[0337] "SOD1 protein" means a polypeptide or fragment thereof having at least about 95% amino acid sequence identity to NCBI Accession No. NP_000445 and having demethylase activity. An exemplary human SOD1 amino acid sequence is provided below.

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[0339] "SOD1 polynucleotide" means a nucleic acid molecule encoding a SOD1 protein or a fragment thereof. The genomic sequence of an exemplary human SOD1 polynucleotide, available at NCBI Accession No. NC_000021.9:31659622-31668931 (Homo sapiens chromosome 21, GRCh38.p13 basic assembly), is provided below (SEQ ID NO: 3).

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[0345] In the above SOD1 genomic nucleic acid sequence, the "ag" splice acceptor site located at the 5' end of exon 3 of the SOD1 gene is indicated in bold. In the above SOD1 genomic sequence, the nucleic acid sequence of exon 3 of the SOD1 gene is indicated in italics and double underlined.

[0346] An exemplary mRNA / cDNA sequence of a human SOD1 polynucleotide (which can be obtained as NCBI reference sequence: NM_000454.4) is provided below:

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[0349] "Androgen receptor (AR) polynucleotide" means any polynucleotide encoding an androgen receptor polypeptide. An exemplary androgen receptor polynucleotide is provided below (SEQ ID NO: 4):

[0350] Homo sapiens androgen receptor (AR), transcript variant 1, mRNA (NM_000044.6)

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[0353] BRIEF DESCRIPTION OF THE DRAWINGS

[0354] Figures 1A to 1C Describe the plasmid. Figure 1A It is an expression vector encoding the TadA7.10-dCas9 base editor. Figure 1B The plasmid contains nucleic acid molecules encoding proteins that confer resistance to chloramphenicol (CamR) and spectinomycin (SpectR). The plasmid also contains the kanamycin resistance gene disabled by two point mutations. Figure 1C The plasmid contains a nucleic acid molecule encoding proteins that confer resistance to chloramphenicol (CamR) and spectinomycin (SpectR). The plasmid also contains the kanamycin resistance gene, which has been disabled by three point mutations.

[0355] Figure 2 This is an image of a bacterial colony transduced with the expression vector shown in Figure 1, which contains a defective kanamycin resistance gene. Vectors containing ABE7.10 variants were generated using error-prone PCR. Bacterial cells expressing these "evolved" ABE7.10 variants were collected and tested 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, thereby restoring kanamycin resistance. Kanamycin-resistant cells were collected for further analysis.

[0356] Figure 3A and 3B Shown is editing of the regulatory region of the hemoglobin subunit gamma (HGB1) locus, a therapeutically relevant site for upregulating fetal hemoglobin. Figure 3A A diagram showing a portion of the regulatory region of the HGB1 gene. Figure 3B The efficiency and specificity of the adenosine deaminase variants were quantified. Editing was measured in HEK293T cells at the hemoglobin subunit gamma 1 (HGB1) locus, a therapeutically relevant site for upregulating hemoglobin. The figure above depicts the nucleotide residues in the target region within the regulatory sequence of the HGB1 gene. A5, A8, A9, and A11 represent the edited adenosine residues in HGB1.

[0357] Figure 4 Figure 2 shows the relative effectiveness of adenosine base editors containing dCas9 that recognize non-canonical PAM sequences. The top panel shows the coding sequence of hemoglobin subunits. The bottom panel is a graph illustrating the efficiency of adenosine deaminase variant base editors with guide RNAs of varying lengths.

[0358] Figure 5 Figure 2 is a graph showing the efficiency and specificity of the ABE8 base editor. The percentage of editing at the intended target nucleotide and the unintended target nucleotide (bystander) was quantified.

[0359] Figure 6 Figure 2 is a graph showing the efficiency and specificity of the ABE8 base editor. The percentage of editing at the intended target nucleotide and the unintended target nucleotide (bystander) was quantified.

[0360] Figures 7A to 7D The eighth-generation adenine base editor is shown to mediate outstanding A·T to G·C conversion in human cells. Figure 7A An overview of adenine base editing is shown: i) ABE8 generates an R-loop at the site targeted by the sgRNA in the genome; ii) TadA* deaminase chemically converts adenine on the ss-DNA portion of the R-loop to inosine via hydrolytic deamination; iii) Cas9's D10A nickase nicks the strand opposite the inosine-containing strand; iv) the inosine-containing strand can serve as a template during DNA replication; v) in the context of DNA polymerase, inosine preferentially base pairs with cytidine; and vi) after replication, inosine can be replaced with guanosine. Figure 7B The architecture of ABE8.xm and ABE8.xd is shown. Figure 7C Three perspective views of the alignment of E. coli TadA deaminase (PDB 1Z3A) and S. aureus TadA (not shown) in complex with tRNAArg2 (PDB 2B3J) are shown. Mutations identified in the eighth round of evolution are highlighted. Figure 7D Figure 2 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 loci in Hek293T cells. Values ​​and error bars reflect the means and standard deviations of three independent biological replicates performed on different days.

[0361] Figures 8A to 8C Figure 3. Cas9 PAM variant ABE8 and catalytically dead 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 of three independent biological replicates performed on different days. Figure 8A A diagram showing A·T to G·C conversion using NG-Cas9 ABE8 (-NG PAM) in Hek293T cells. Figure 8B Graph showing A·T to G·C conversion using Sa-Cas9ABE8 (-NNGRRT PAM) in Hek293T. Figure 8CGraph showing A·T to G·C conversion using catalytically inactive dCas9-ABE8 (D10A, H840A in Streptococcus pyogenes Cas9) in Hek293T cells.

[0362] Figures 9A to 9E Shown is a comparison of on-target and off-target editing frequencies between ABE7.10, ABEmax, and ABEmax with a BPNLS in Hek293T cells. Individual data points are shown, and error bars represent the standard deviation of n=3 independent biological replicates performed on different days. Figure 9A and 9B is a graph showing the frequency of on-target DNA editing. Figure 9B and 9C is a graph showing the frequency of sgRNA-guided off-target DNA editing. Figure 9E is a graph showing the frequency of RNA off-target editing.

[0363] Figures 10A to 10B Shown are the median A·T to G·C conversions and corresponding indel formation of TadA (C-terminal α-helical truncation ABE construct) in HEK293T cells. Figure 10A is a heat map showing the median editing conversion of A·T to G·C across 8 genomic sites. Figure 10B is a heat map showing indel formation. The delta residue values ​​correspond to the position of the deletion in TadA. The median values ​​were generated from n=3 biological replicates.

[0364] Figure 11 Figure 2 is a heatmap showing the median A·T to G·C conversion of 40 ABE8 constructs across 8 genomic sites in HEK293T cells. Median values ​​were determined from two or more biological replicates.

[0365] Figure 12 Figure 2 is a heatmap showing the median indel % for 40 ABE8 constructs across 8 genomic loci in HEK293T cells. Median values ​​were determined from two or more biological replicates.

[0366] Figure 13 Figure 2 is a graph showing the fold change in editing, ABE8:ABE7. It represents the average of A·T to G·C editing by ABE8:ABE7 across all A positions within the target across eight different genomic loci in HEK293T cells. Positions 2-12 represent the positions of the target adenine within the 20-nt pre-spacer sequence, with position 20 immediately 5' of the -NGG PAM.

[0367] Figure 14 A dendrogram of ABE8 is shown. Core ABE8 constructs selected for further study are highlighted in bold.

[0368] Figure 15 Figure 2 is a heatmap showing the median A·T to G·C conversion of eight core ABE8 constructs across eight genomic sites in HEK293T cells. Median values ​​were determined from three or more biological replicates.

[0369] Figure 16 is a heatmap showing the median indel frequencies of eight core ABE8s tested at eight genomic sites in HEK293T cells.

[0370] Figure 17 is a heat map 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 ​​were generated from n=3 biological replicates.

[0371] Figure 18 is a heatmap showing the median indel frequencies of core NG-ABE8 at six genomic loci tested in HEK293T cells. Median values ​​were generated from n=3 biological replicates.

[0372] Figure 19 Figure 2 is a heatmap showing the median A·T to G·C conversions of the core Sa-ABE8 construct (-NNGRRT PAM) at six genomic sites in HEK293T cells. Site positions are numbered from -2 to 20 (5' to 3') within the 22-nt protospacer sequence. Position 20 is 5' to the NNGRRT PAM. Median values ​​are generated from n = 3 biological replicates.

[0373] Figure 20 is a heatmap showing the median indel frequencies of core Sa-ABE8 at 8 genomic sites tested in HEK293T cells. Median values ​​were generated from n=3 biological replicates.

[0374] Figure 21 Figure 2 is a heatmap showing the median A·T to G·C conversion of the core dC9-ABE8-m construct at eight genomic sites in HEK293T cells. Dead Cas9 (dC9) is defined as the D10A and H840A mutations in Streptococcus pyogenes Cas9. Median values ​​are generated from n ≥ 3 biological replicates.

[0375] Figure 22Figure 2 is a heatmap showing the median A·T to G·C conversion 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 Streptococcus pyogenes Cas9. Median values ​​are generated from n ≥ 3 biological replicates.

[0376] Figure 23A and 23B Shown are the median indel frequencies of core dC9-ABE8 at eight genomic loci tested in HEK293T cells. Median values ​​were generated from n≥3 biological replicates. Figure 23A is a heat map showing the indel frequencies displayed for the dC9-ABE8-m variant relative to ABE7.10. Figure 23B is a heat map showing the indel frequencies displayed for the dC9-ABE8-d variant relative to ABE7.10.

[0377] Figure 24 Figure 2 is a graph showing C·G to T·A editing using HEK293T cells treated with ABE8 and ABE7.10. The editing frequency at each site is averaged across all C positions within the target. Cytosines within the protospacer are shaded.

[0378] Figures 25A to 25H Improved DNA specificity on-target and sgRNA-dependent off-target DNA editing by ABE8 constructs and ABE8 constructs with TadA mutations is shown. Individual data points are shown, and error bars represent the standard deviation of n=3 independent biological replicates performed on different days. Figure 25A and 25B is a graph showing the on-target DNA editing frequency of the core ABE8 construct compared to ABE7. Figure 25C and 25D is a graph showing the on-target DNA editing frequency of ABE8 with mutations that improve off-target RNA editing. Figure 25E and 25F is a graph showing the frequency of sgRNA-guided DNA off-target editing for the core ABE8 construct compared to ABE7. Figure 25G and 25H is a graph showing the frequency of DNA off-target editing guided by sgRNA of ABE8 with mutations that improve RNA off-target editing.

[0379] Figure 26is a graph showing the 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 standard deviation of n=3 independent biological replicates performed on different days.

[0380] Figure 27A and 27B Shown are A·T to G·C conversion in primary cells and phenotypic consequences. Figure 27A This graph shows the A·T to G·C conversion at the -198HBG1 / 2 locus in CD34+ cells from two independent donors treated with ABE. NGS analysis was performed at 48 and 144 hours after treatment. The -198HBG1 / 2 target region is highlighted as A7. The percentage of A·T to G·C conversion at A7 is plotted. Figure 27B is a graph showing the formation of gamma-globin as a fragment of alpha-globin. The values ​​shown are from two donors after ABE treatment and erythroid differentiation.

[0381] Figure 28A and 28B Describes A·T to G·C conversion at the -198 promoter site upstream of HBG1 / 2 in ABE8-treated CD34+ cells. Figure 28A is a heat map showing A to G editing by ABE8 in CD34+ cells from two donors, where donor 2 is heterozygous for sickle cell disease, 48 hours and 144 hours after editor treatment. Figure 28B is a graphical representation of the distribution of total sequencing reads containing either only A7 edits or combined (A7+A8) edits.

[0382] Figure 29 Figure 2 is a heat map showing the frequency of indels at position -198 of the γ-globin promoter in CD34+ cells treated with ABE8. Frequencies shown are from two donors at the 48 and 144 hour time points.

[0383] Figure 30 Shown are UHPLC UV-Vis traces (220 nm) and integration of globin chain levels of untreated differentiated CD34+ cells (Donor 1).

[0384] Figure 31 Shown are UHPLC UV-Vis traces (220 nm) and integration of globin chain levels in differentiated CD34+ cells (Donor 1) treated with ABE7.10-m.

[0385] Figure 32Shown are UHPLC UV-Vis traces (220 nm) and integration of globin chain levels in differentiated CD34+ cells (Donor 1) treated with ABE7.10-d.

[0386] Figure 33 Shown are UHPLC UV-Vis traces (220 nm) and integration of globin chain levels in differentiated CD34+ cells (Donor 1) treated with ABE8.8-m.

[0387] Figure 34 Shown are UHPLC UV-Vis traces (220 nm) and integration of globin chain levels in differentiated CD34+ cells (Donor 1) treated with ABE8.8-d.

[0388] Figure 35 Shown are UHPLC UV-Vis traces (220 nm) and integration of globin chain levels in differentiated CD34+ cells (Donor 1) treated with ABE8.13-m.

[0389] Figure 36 Shown are UHPLC UV-Vis traces (220 nm) and integration of globin chain levels in differentiated CD34+ cells (Donor 1) treated with ABE8.13-d.

[0390] Figure 37 Shown are UHPLC UV-Vis traces (220 nm) and integration of globin chain levels in differentiated CD34+ cells (Donor 1) treated with ABE8.17-m.

[0391] Figure 38 Shown are UHPLC UV-Vis traces (220 nm) and integration of globin chain levels in differentiated CD34+ cells (Donor 1) treated with ABE8.17-d.

[0392] Figure 39 Shown are UHPLC UV-Vis traces (220 nm) and integration of globin chain levels in differentiated CD34+ cells (Donor 1) treated with ABE8.20-m.

[0393] Figure 40 Shown are UHPLC UV-Vis traces (220 nm) and integration of globin chain levels in differentiated CD34+ cells (Donor 1) treated with ABE8.20-d.

[0394] Figure 41 Shown are UHPLC UV-Vis traces (220 nm) and integration of globin chain levels of untreated differentiated CD34+ cells (Donor 2). Note: Donor 2 is heterozygous for sickle cell disease.

[0395] Figure 42 Shown are UHPLC UV-Vis traces (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.

[0396] Figure 43 Shown are UHPLC UV-Vis traces (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.

[0397] Figure 44 Shown are UHPLC UV-Vis traces (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.

[0398] Figure 45 Shown are UHPLC UV-Vis traces (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.

[0399] Figure 46 Shown are UHPLC UV-Vis traces (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.

[0400] Figure 47 Shown are UHPLC UV-Vis traces (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.

[0401] Figure 48 Shown are UHPLC UV-Vis traces (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.

[0402] Figure 49 Shown are UHPLC UV-Vis traces (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.

[0403] Figure 50Shown are UHPLC UV-Vis traces (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.

[0404] Figures 51A to 51E We describe editing at two independent sites using ABE8.8, achieving greater than 90% editing by day 11 after erythroid differentiation before enucleation and approximately 60% more γ-globin than α-globin or total β-family globin by day 18 after erythroid differentiation. Figure 51A is a graph showing the average ABE8.8 editing in 2 healthy donors in 2 independent experiments. Editing efficiency was measured using primers that distinguish HBG1 from HBG2. Figure 51B is a graph showing the average of 2 independent experiments in 1 healthy donor. Editing efficiency was measured using primers recognizing both HBG1 and HBG2. Figure 51C is a graph showing editing of ABE8.8 in a donor with a heterozygous E6V mutation. Figure 51D and 51E is a graph showing the increase of gamma globin in ABE8.8-edited cells.

[0405] Figure 52A and 52B The percentage of editing using the ABE variant to correct the sickle cell mutation is shown. Figure 52A is a graph showing a screen for different editor variants with approximately 70% editing in SCD patient fibroblasts. Figure 52B Figure 2 is a graph showing CD34 cells from a healthy donor edited using a lead ABE variant targeting a synonymous mutation A13 in an adjacent proline, which is within the editing window and serves as an indicator for editing SCD mutations. The ABE8 variant showed an average editing efficiency of 40% at indicator A13.

[0406] Figure 53A and 53B RNA amplicon sequencing is shown to detect intracellular A to I editing of RNA associated with ABE treatment. Individual data points are shown, and error bars represent the standard deviation of n=3 independent biological replicates performed on different days. Figure 53A is a graph showing the frequency of A to I editing in targeted RNA amplicons for the core ABE8 construct versus ABE7 and Cas9(D10A) nickase controls. Figure 53B is a graph showing the A to I editing frequency in targeted RNA amplicons by ABE8, which has mutations that have been reported to improve RNA off-target editing.

[0407] Figure 54 Figure 2 is a bar graph showing the total percentage of A to G base editing at the splice acceptor target site (AG nucleic acid site) achieved by the tested adenosine base editors (ABE8 variants) versus controls. The protospacer PAM sequence for the ABEs evaluated was NGG ABE.

[0408] Figure 55 A diagram of the exon 3 splice acceptor of the SOD1 genomic nucleic acid sequence is shown, which serves as a target for an A to G nucleotide change to cause splicing disruption of the transcription of SOD1 exon 3. The figure shows a large number of genomic DNA locations in the exon 3 region of the SOD1 nucleic acid sequence (top); the SOD1 genomic nucleic acid sequence in the exon 3 region, the intron nucleic acid sequence 5' of exon 3; and the target splice acceptor nucleic acid sequence 5' of exon 3; and the nucleic acid sequence of the corresponding guide RNA (gRNA), wherein the splice acceptor

[0409] The ("AG") nucleic acid is immediately 5' of SOD1 exon 3, indicated by the upward-pointing arrow. Also of note in this figure is the bystander adenosine (A) nucleobase (boxed), which is located within the intronic sequence 5' of SOD1 exon 3 proximal to the AG splice acceptor.

[0410] Figure 56 : is a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained after PCR and deep sequencing of DNA using the base editor systems and methods described herein. The efficiency percentage of the precise A to G nucleobase change (change) in the target splicing acceptor (AG) nucleic acid sequence at 5' of exon 3 of the SOD1 genomic nucleic acid sequence is shown, as detected by deep sequencing (MySeq) after PCR of the genomic DNA in cells in which base editing has occurred. 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 (expressed in base pairs (bp)) is shown along the bottom of the table. The table shows that the A to G conversion efficiency at the splicing acceptor nucleic acid site (position 6 along the target site) is approximately 81% (80.77%) using the ABE8 base editor and systems and methods described herein. Compared to the editing of the target A in the splice acceptor nucleic acid sequence, the changes of the bystander A nucleobase to the G nucleobase (at positions 2, 3, and 4 shown in the box to the left of the A nucleobase of the targeted splice acceptor) are minimal.

[0411] Figures 57A to 57Lis a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained 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 (expressed in base pairs (bp)) is shown along the bottom of the table. See also the previous text Figure 56 .

[0412] Figure 58 Figure 5 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 the tested adenosine base editors (ABE8 variant) with guide 20 versus controls. The protospacer PAM sequence for the ABE evaluated was NGG ABE.

[0413] Figure 59 42 (ABE8 variant) and the control (left panel) show the percentage of A to G base editing at target position 5 of the splice acceptor target site (AG nucleic acid site) and the total percentage of A to G base editing at positions 2, 5, and 9 of the splice acceptor target site (AG nucleic acid site) (right panel). The protospacer PAM sequence for the ABE evaluated was NGG ABE.

[0414] Figure 60A is a bar graph showing the percentage of A to G base editing at target position 5 of the splice acceptor target site (AG nucleic acid site) achieved by the tested adenosine base editors with guide 41 (PV variant) versus controls. The protospacer PAM sequence for the ABE evaluated was an NGT ABE. Figure 60B is a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained 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 (expressed in base pairs (bp)) is shown along the bottom of the table. See also the previous text Figure 56 .

[0415] Figure 61 Figure 2 is a bar graph showing the total percentage of A to G base editing at the splice acceptor target site (AG nucleic acid site) achieved by the tested adenosine base editors (PV variant) with guide 24 and controls. The protospacer PAM sequence for the ABE evaluated was NGA ABE.

[0416] Figure 62Shown are SOD1 protein levels in HEK297T cells edited with ABE8.8 or ABE7.10 with guide 20 (left panel: Western blot; right panel: quantification). β-actin was used as a control.

[0417] Figure 63 Figure 2 is a bar graph showing the percentage of C to T base editing at target position 8 of the splice acceptor target site (AG nucleic acid site) achieved by the tested cytidine base editors with guide 19 (PV variant) versus controls. The protospacer PAM sequence for the ABE evaluated was NGC CBE.

[0418] Figure 64A is a bar graph (left) showing the percentage of A to G base editing at target position 5 of the splice acceptor target site (AG nucleic acid site) achieved by the tested adenosine base editor (PV variant) with guide 41 and the control. The pre-spacer PAM sequence for the ABE evaluated was NGT ABE. The right figure is a diagram depicting a large number of genomic DNA positions in the exon 3 region of the SOD1 nucleic acid sequence to which guides 41, 20, 40 and 21 are designed to bind. See also above Figure 55 . Figure 64B is a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained 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 (expressed in base pairs (bp)) is shown along the bottom of the table. See also the previous text Figure 56 .

[0419] Figure 65 is a bar graph (left) showing the total percentage of A to G base editing at the splice acceptor target site (AG nucleic acid site) achieved by the tested adenosine base editors (ABE variants) with guide 42 and controls. The pre-spacer PAM sequence for the ABE evaluated was NGG ABE. The right figure is a diagram depicting a large number of genomic DNA positions in the exon 3 region of the SOD1 nucleic acid sequence to which guides 42, 24, and 25 were designed to bind. See also above Figure 55 .

[0420] Figure 66A is a bar graph showing the total percentage of C to T base editing achieved by the assayed cytidine base editor with guide 40 (BE4 VRQR) versus a control. The protospacer PAM sequence for the evaluated CBE was NGACBE. Figure 66Bis a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained 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 (expressed in base pairs (bp)) is shown along the bottom of the table. See also the previous text Figure 56 .

[0421] Figure 67 Figure 5 is a bar graph showing the percentage of A to G base editing at target position 5 of the splice acceptor target site (AG nucleic acid site) achieved by the tested adenosine base editors (PV variant) with guide 18 versus controls. The protospacer PAM sequence for the ABE evaluated was an NGT ABE.

[0422] Figure 68 is a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained 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 (expressed in base pairs (bp)) is shown along the bottom of the table. See also the previous text Figure 56 .

[0423] Figure 69 is a bar graph showing the total percentage of C to T base editing achieved by the assayed cytidine base editor (BE4 VRQR) with guide 16 versus a control. The protospacer PAM sequence for the evaluated CBE was NGACBE.

[0424] Figure 70 is a bar graph showing the percentage of C to T base editing at target position 4 achieved by the assayed cytidine base editor with guide 17 (BE4 VRQR) versus a control. The protospacer PAM sequence for the evaluated CBE was NGA CBE.

[0425] Figure 71 is a bar graph showing the percentage of C to T base editing at target position 5 achieved by the assayed cytidine base editor with guide 21 (BE4 VRQR) versus a control. The protospacer PAM sequence for the evaluated CBE was NGA CBE.

[0426] Figures 72A to 72Lis a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained 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 (expressed in base pairs (bp)) is shown along the bottom of the table. See also the previous text Figure 56 .

[0427] Figures 73A to 73C is a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained 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 (expressed in base pairs (bp)) is shown along the bottom of the table. See also the previous text Figure 56 .

[0428] Figures 74A to 74C is a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained 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 (expressed in base pairs (bp)) is shown along the bottom of the table. See also the previous text Figure 56 .

[0429] Figures 75A to 75D is a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained 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 (expressed in base pairs (bp)) is shown along the bottom of the table. See also the previous text Figure 56 .

[0430] Figures 76A to 76L is a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained 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 (expressed in base pairs (bp)) is shown along the bottom of the table. See also the previous text Figure 56 .

[0431] Figures 77A to 77Lis a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained 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 (expressed in base pairs (bp)) is shown along the bottom of the table. See also the previous text Figure 56 .

[0432] Figures 78A to 78D is a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained 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 (expressed in base pairs (bp)) is shown along the bottom of the table. See also the previous text Figure 56 .

[0433] Figures 79A to 79L is a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained 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 (expressed in base pairs (bp)) is shown along the bottom of the table. See also the previous text Figure 56 .

[0434] Figure 80 showed that when a cytidine base editor (CBE) was used to target "CAG", a stop codon was introduced into exon 1 of the androgen receptor.

[0435] Figure 81 Two graphs are provided. The left graph shows the percentage of C to T editing by a specific cytidine base editor. The right graph shows the percentage of indels by each cytidine base editor.

[0436] Figures 82A to 82I is a table showing the percent efficiency of C to T editing in AR nucleic acid sequences using CBE base editor variants, as detected by deep sequencing (MySeq) following PCR of genomic DNA in cells in which base editing has occurred. Figures 82A-82I Shown are the percentages of C to T base editing at position 6 in the AR nucleic acid target site. A control reaction is shown in which water was used instead of CBE.

[0437] Figure 83ATwo graphs are provided. The left graph shows that the introduction of a premature stop codon into exon 1 results in functional knockout of the androgen receptor in most cells. The middle graph shows the percentage of indels generated by each cytidine base editor. The right graph is a diagram depicting a number of genomic DNA locations in the exon 1 region of the AR nucleic acid sequence to which Guide 8 is designed to bind. Figure 83B is a table showing the percent efficiency of C to T editing in AR nucleic acid sequences using CBE base editor variants, as detected by deep sequencing (MySeq) following PCR of genomic DNA in cells in which base editing has occurred. Figure 83B Shown are the percentages of C to T base editing at the target position in the AR nucleic acid target site. A control reaction is shown in which water was used instead of CBE.

[0438] Figure 84A is a bar graph (left) showing the percentage of C to T base editing at target position 6 achieved by the cytidine base editors (BGX5, BGX27, BGX 29, and BTX448) with guide 10 and the control; and the indel rate expressed as a percentage (middle). The pre-spacer PAM sequence for the CBE evaluated is NGGCBE. The right figure is a diagram depicting a large number of genomic DNA positions in the exon 1 region of the AR nucleic acid sequence to which guides 9 and 10 are designed to bind. See also above Figure 80 . Figure 84B is a table showing the percent efficiency of C to T editing in AR nucleic acid sequences using CBE base editor variants, as detected by deep sequencing (MySeq) following PCR of genomic DNA in cells in which base editing has occurred. Figure 84B Shown are the percentages of C to T base editing at position 6 in the AR nucleic acid target site. A control reaction is shown in which water was used instead of CBE.

[0439] Figure 85 Figure 2 is a bar graph showing the total percentage of A to G base editing at the splice acceptor target site (AG nucleic acid site) achieved by the tested adenosine base editors with guide 8 (ABE8 variant) (left panel) or guide 14 (right panel) versus controls. The protospacer PAM sequence for the ABEs evaluated was NGG ABE.

[0440] Figure 86A Figure 2 is a bar graph (top) and summary table (bottom) showing the percentage of C to T base editing at target position 8 achieved by the assayed cytidine base editor with guide 11 (BE4 VRQR) versus a control. The protospacer PAM sequence for the evaluated CBE was NGA CBE. Figure 86B is a table showing the percent efficiency of C to T editing in AR nucleic acid sequences using CBE base editor variants, as detected by deep sequencing (MySeq) following PCR of genomic DNA in cells in which base editing has occurred. Figure 86B Shown are the percentages of 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.

[0441] Figure 87A Figure 2 is a bar graph (top) and summary table (bottom) showing the percentage of C to T base editing at target position 5 achieved by the assayed cytidine base editor with guide 12 (BE4 VRQR) versus a control. The protospacer PAM sequence for the evaluated CBE was NGA CBE. Figure 87B is a table showing the percent efficiency of C to T editing in AR nucleic acid sequences using CBE base editor variants, as detected by deep sequencing (MySeq) following PCR of genomic DNA in cells in which base editing has occurred. Figure 87B Shown are the percentages of C to T base editing at position 5 in the AR nucleic acid target site. A control reaction is shown in which water was used instead of CBE.

[0442] Figure 88 is a bar graph showing the total percentage of C to T base editing achieved by the assayed cytidine base editor (BE4 VRQR) with guide 15 versus a control. The protospacer PAM sequence for the evaluated CBE was NGACBE.

[0443] 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 (expressed in base pairs (bp)) is shown along the bottom of the table.

[0444] Figures 89A to 89D Is a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained after PCR and deep sequencing of DNA using the base editor systems and methods described herein. The efficiency percentages of accurate C to T or A to G nucleobase changes (changes) in the target nucleic acid sequence of exon 1 of the AR genomic nucleic acid sequence are shown, as detected by deep sequencing (MySeq) after PCR of genomic DNA in cells in which base editing has occurred. 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 (expressed in base pairs (bp)) is shown along the bottom of the table.

[0445] Figures 90A to 90CIs a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained after PCR and deep sequencing of DNA using the base editor systems and methods described herein. The efficiency percentages of accurate C to T or A to G nucleobase changes (changes) in the target nucleic acid sequence of exon 1 of the AR genomic nucleic acid sequence are shown, as detected by deep sequencing (MySeq) after PCR of genomic DNA in cells in which base editing has occurred. 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 (expressed in base pairs (bp)) is shown along the bottom of the table.

[0446] Figures 91A to 91C Is a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained after PCR and deep sequencing of DNA using the base editor systems and methods described herein. The efficiency percentages of accurate C to T or A to G nucleobase changes (changes) in the target nucleic acid sequence of exon 1 of the AR genomic nucleic acid sequence are shown, as detected by deep sequencing (MySeq) after PCR of genomic DNA in cells in which base editing has occurred. 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 (expressed in base pairs (bp)) is shown along the bottom of the table.

[0447] Figure 92A and 92B Is a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained after PCR and deep sequencing of DNA using the base editor systems and methods described herein. The efficiency percentages of accurate C to T or A to G nucleobase changes (changes) in the target nucleic acid sequence of exon 1 of the AR genomic nucleic acid sequence are shown, as detected by deep sequencing (MySeq) after PCR of genomic DNA in cells in which base editing has occurred. 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 (expressed in base pairs (bp)) is shown along the bottom of the table.

[0448] Figures 93A to 93XIs a table showing the efficiency of target site nucleobase changes in target nucleic acid sequences obtained after PCR and deep sequencing of DNA using the base editor systems and methods described herein. The efficiency percentages of accurate C to T or A to G nucleobase changes (changes) in the target nucleic acid sequence of exon 1 of the AR genomic nucleic acid sequence are shown, as detected by deep sequencing (MySeq) after PCR of genomic DNA in cells in which base editing has occurred. 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 (expressed in base pairs (bp)) is shown along the bottom of the table. DETAILED DESCRIPTION

[0449] The present invention provides compositions with increased efficiency comprising novel adenosine base editors (e.g., ABE8) and methods of using them to generate modifications in target nucleobase sequences.

[0450] Nucleobase editors

[0451] Disclosed herein is a base editor or a core base editor for editing, modifying or changing a target nucleotide sequence of a polynucleotide. In specific embodiments, the base editor of the present invention modifies SOD1 polynucleotides. In specific embodiments, the base editor of the present invention introduces a stop codon into an AR polynucleotide or interrupts the splicing site of an AR polynucleotide. A core base editor or a base editor is described herein, comprising a polynucleotide programmable nucleotide binding domain (e.g., Cas9) and a core base editing domain (e.g., adenosine deaminase). When bound to a guide polynucleotide (e.g., gRNA), the polynucleotide programmable nucleotide binding domain (e.g., Cas9) can specifically bind to a target polynucleotide sequence (i.e., via complementary base pairing between the base of the bound guide nucleic acid and the base of the target polynucleotide sequence), and thus position the base editor at the target nucleic acid sequence where editing is desired. 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.

[0452] Polynucleotide programmable nucleotide binding domain

[0453] It should be understood that a polynucleotide programmable nucleotide binding domain can also include a nucleic acid programmable protein that binds RNA. For example, a polynucleotide programmable nucleotide binding domain can be associated with a nucleic acid that guides the polynucleotide programmable nucleotide binding domain to RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, but they are not specifically listed in this disclosure.

[0454] The polynucleotide programmable nucleotide binding domain of the base editor itself may include one or more domains. For example, the polynucleotide programmable nucleotide binding domain may include one or more nuclease domains. In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain may include an endonuclease or an exonuclease. Herein, the term "exonuclease" refers to a protein or polypeptide capable of digesting a nucleic acid (e.g., RNA or DNA) from a free end, and the term "endonuclease" refers to a protein or polypeptide capable of catalyzing (e.g., cleavage) an internal region in a nucleic acid (e.g., DNA or RNA). In some embodiments, an endonuclease can cleave one chain of a double-stranded nucleic acid. In some embodiments, an endonuclease can cleave two chains of a double-stranded nucleic acid molecule. In some embodiments, the polynucleotide programmable nucleotide binding domain may be a deoxyribonuclease. In some embodiments, the polynucleotide programmable nucleotide binding domain may be a ribonuclease.

[0455] In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain can cut zero, one or two chains of the target polynucleotide.In some embodiments, the polynucleotide programmable nucleotide binding domain can include a nickase domain.Herein, the term "nickase" refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain, which can only crack one of the two chains in a double-stranded nucleic acid molecule (e.g., DNA). In some embodiments, a nickase can be derived from a polynucleotide programmable nucleotide binding domain of complete catalytic activity (e.g., natural) form by introducing one or more mutations into an active polynucleotide programmable nucleotide binding domain. For example, if the polynucleotide programmable nucleotide binding domain includes a nickase domain derived from Cas9, the nickase domain derived from Cas9 can include a D10A mutation and a histidine at position 840. In such embodiments, residue H840 maintains catalytic activity and can therefore crack a chain of a nucleic acid double helix. In another embodiment, the nickase domain derived from Cas9 can include an H840A mutation, and the amino acid at position 10 remains D. In some embodiments, the nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide programmable nucleotide binding domain by removing all or part of the nuclease domain not required for nickase activity. For example, if the polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the nickase domain derived from Cas9 can comprise all or part of a deletion of a RuvC domain or a HNH domain.

[0456] The amino acid sequence of an exemplary catalytically active Cas9 is as follows:

[0457]

[0458]

[0459] A base editor comprising a polynucleotide programmable nucleotide binding domain with a nickase domain is capable of producing single-stranded DNA breaks (nicks) at a specific polynucleotide target sequence (e.g., determined by the complementary sequence of the bound guide nucleic acid). In some embodiments, the chain of the nucleic acid double helix target polynucleotide sequence cleaved by a base editor comprising a nickase domain (e.g., a nickase domain derived from Cas9) is not edited by the base editor (i.e., the chain cleaved by the base editor is relative to the chain comprising a methyl group to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., a nickase domain derived from Cas9) can cleave the chain of a DNA molecule targeted for editing. In such embodiments, the non-targeted chain is not cleaved.

[0460] Also provided herein are base editors comprising a polynucleotide programmable nucleotide binding domain that is catalytically dead (i.e., not capable of cleaving a target polynucleotide sequence).Herein, the terms “catalytically dead” and “nuclease dead” are used interchangeably and refer to a polynucleotide programmable nucleotide binding domain having one or more mutations and / or deletions, resulting in the loss of the ability to cleave nucleic acid chains. In some embodiments, as a result of specific point mutations in one or more nuclease domains, a catalytically dead polynucleotide programmable nucleotide binding domain methyl editor may lack nuclease activity. For example, in the case where a base editor comprises a Cas9 domain, the Cas9 may comprise both a D10A mutation and an H840A mutation. Such mutations render the two nuclease domains inactive, resulting in the loss of nuclease activity. In other embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain may comprise one or more deletions of all or part of a catalytic domain (e.g., a RuvC1 domain and / or an HNH domain). In further embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (eg, D10A or H840A) and a deletion of all or part of the nuclease domain.

[0461] It is also contemplated herein that mutations of catalytically dead polynucleotide programmable nucleotide binding domains can be generated from a previous functional version of the polynucleotide programmable nucleotide binding domain. For example, in the case of catalytically dead Cas9 ("dCas9"), variants with mutations other than D10A and H840A are provided that result in nuclease-free Cas9. For example, such mutations include other amino acid replacements at D10 and H840, or other replacements within the nuclease domain of Cas9 (e.g., replacements in the HNH nuclease subdomain and / or the RuvC1 subdomain). Based on the present disclosure and knowledge in the art, other suitable nuclease-free dCas9 domains will be apparent to those skilled in the art and are within the scope of the present disclosure. Such other exemplary suitable nuclease-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).

[0462] Non-limiting examples of polynucleotide programmable nucleotide binding domains that can be incorporated into base editors include domains derived from CRISPR proteins, restriction nucleases, large-range nucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some embodiments, the base editor comprises a polynucleotide programmable nucleotide binding domain having a natural or modified protein or portion thereof, which can bind to a nucleic acid sequence via a bound guide nucleic acid during CRISPR (i.e., regularly clustered interspaced short palindromic repeats)-mediated nucleic acid modification. Herein, such proteins are referred to as “CRISPR proteins.” Accordingly, disclosed herein are base editors comprising polynucleotide programmable nucleotide binding domains having all or part of a CRISPR protein (i.e., base editors comprising all or part of a CRISPR protein as a domain, which domain is also referred to as a “domain derived from a CRISPR protein” of a base editor). The domain derived from a CRISPR protein incorporated into the base editor can be modified compared to the wild-type or natural version of the CRISPR protein. For example, as described below, a domain derived from a CRISPR protein can comprise one or more mutations, insertions, deletions, rearrangements, and / or recombinations relative to a wild-type or naturally occurring version of the CRISPR protein.

[0463] CRISPR is an adaptive immune system that provides protection against mobile genetic factors (viruses, transposable elements, and conjugative plasmids). The CRISPR cluster contains a spacer sequence, a sequence complementary to the preceding mobile element, and a nucleic acid that invades the target. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In the type II CRISPR system, correct processing of pre-crRNA requires trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. TracrRNA acts as a guide in the ribonuclease 3-assisted pre-crRNA processing. Subsequently, Cas9 / crRNA / tracrRNA endonuclease solubilizes and cleaves linear or circular dsDNA targets complementary to the spacer sequence. The target chain that is not complementary to crRNA is first cut endonuclease solubilized and then trimmed by 3'-5' exonuclease solubilization. In essence, DNA binding and cleavage generally require protein and two types of RNA. However, single guide RNA ("sgRNA", or simply "gRNA") can be engineered to combine aspects of both 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 (PAM or protospacer adjacent motif) in the CRISPR repeat sequence to help distinguish self from non-self.

[0464] In some embodiments, the methods described herein can utilize engineered Cas proteins. Guide RNA (gRNA) is a short synthetic RNA composed of a scaffold sequence necessary for Cas binding and a user-defined spacer sequence of approximately 20 nucleotides that defines the genomic target to be modified. Thus, a skilled artisan can alter the genomic target of a Cas protein, with specificity determined in part by how specific the gRNA targeting sequence is for the genomic target versus the rest of the genome.

[0465] In some embodiments, the gRNA scaffold sequence is as follows:

[0466] In some embodiments, the domain derived from the CRISPR protein incorporated into the base editor is an endonuclease (e.g., a deoxyribonuclease or a ribonuclease) that can bind to a target polynucleotide when acting in concert with the bound guide nucleic acid. In some embodiments, the domain derived from the CRISPR protein incorporated into the base editor is a nickase that can bind to a target polynucleotide when acting in concert with the bound guide nucleic acid. In some embodiments, the domain derived from the CRISPR protein incorporated into the base editor is a catalytically dead domain that can bind to a target polynucleotide when acting in concert with the bound guide nucleic acid. In some embodiments, the target polynucleotide bound by the CRISPR protein derived from the base editor is DNA. In some embodiments, the target polynucleotide bound by the CRISPR protein derived from the base editor is RNA.

[0467] Cas proteins that can be used herein include Class 1 and Class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, C Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h and Cas12i, CARF, DinG, homologs thereof, or modified versions thereof. Unmodified CRISPR enzymes may have DNA cleavage activity, such as Cas9, which has two functional endonuclease domains: RuvC and HNH. The CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within the target sequence and / or within the complement of the target sequence. For example, the CRISPR enzyme can direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence.

[0468] A vector can be used that encodes a CRISPR enzyme that is mutated with respect to the wild-type enzyme so that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. Cas9 can refer to a polypeptide having at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from Streptococcus pyogenes). Cas9 can refer to a polypeptide having at most or at most about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., from Streptococcus pyogenes). Cas9 can refer to a wild-type or modified form of a Cas9 protein that can include amino acid changes such as deletions, insertions, substitutions, variations, mutations, fusions, chimeras, or any combination thereof.

[0469] In some embodiments, the CRISPR protein-derived domain of the base editor can include all or part of Cas9 from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanensis (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella bartica (NCBI Ref: NC_021315.1); 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.

[0470] Cas9 domains of nucleobase editors

[0471] The Cas9 nuclease sequence and structure are known to those skilled in the art (see, for example, "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E. et al., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M. et al., Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in a variety of species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus. Other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0472] In some embodiments, nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-restrictive exemplary Cas9 domains are provided herein. The Cas9 domain can be a nuclease activity Cas9 domain, a Cas9 domain without nuclease activity (dCas9) or a Cas9 nickase (nCas9). In some embodiments, the Cas9 domain is a nuclease activity domain. For example, the Cas9 domain can be a Cas9 domain that cuts two chains of a double-helical nucleic acid (e.g., two chains of a double-helical DNA molecule). In some embodiments, the Cas9 domain includes any one of the amino acid sequences described in detail herein. In some embodiments, the Cas9 domain includes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 99.5% identical to any one of the amino acid sequences described in detail herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences detailed herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any one of the amino acid sequences detailed herein.

[0473] In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, the protein comprises one or two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or a fragment thereof are referred to as "Cas9 variants." Cas9 variants share homology with Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70% identical to wild-type Cas9, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the Cas9 variant can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of the corresponding wild-type Cas9. In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0474] In some embodiments, the Cas9 fusion protein as provided herein comprises the full-length amino acid sequence of the Cas9 protein, for example, one of the Cas9 sequences provided herein. However, in other embodiments, the fusion protein as provided herein does not comprise the full-length Cas9 sequence, but only comprises one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and other suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0475] The Cas9 protein can be associated with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 domain, for example, a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Examples of nucleic acid programmable DNA binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b / C2C1, and Cas12c / C2C3.

[0476] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, nucleotide and amino acid sequences below).

[0477]

[0478]

[0479]

[0480]

[0481] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0482] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence:

[0483]

[0484]

[0485]

[0486]

[0487]

[0488] (Single underline: HNH domain; double underline: RuvC domain).

[0489] In some embodiments, the wild-type Cas9 corresponds to a protein from Streptococcus pyogenes.

[0490] pyogenes) (NCBI reference sequence: NC_002737.2 (nucleotide sequence as follows); and Uniprot reference sequence: Q99ZW2 (amino acid sequence as follows)):

[0491]

[0492]

[0493]

[0494] (Single bottom line: HNH domain; double bottom line: RuvC domain)

[0495] In some embodiments, Cas9 refers to Cas9 from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma yrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanensis (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Bacteroides spp. baltica)(NCBI Ref: NC_018010.1); Psychroflexus torquisI(NCBI Ref: NC_018721.1); Streptococcus thermophilus(NCBI Ref: YP_820832.1); Listeria innocua(NCBI Ref: NP_472073.1); Campylobacter jejuni(NCBI Ref: YP_002344900.1); Neisseria meningitidis(NCBI Ref: YP_002342100.1) or refers to Cas9 from any other organism.

[0496] It will be appreciated that other Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including variants and homologs thereof, are within the scope of the present disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is a nuclease-dead Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease-active Cas9.

[0497] In some embodiments, the Cas9 domain is a Cas9 domain (dCas9) without nuclease activity. For example, the dCas9 domain can be bound to a double-helix nucleic acid molecule (for example, via a gRNA molecule) without cleaving any chain of the double-helix nucleic acid molecule. In some embodiments, the dCas9 domain without nuclease activity comprises a D10X mutation and an H840X mutation of the amino acid sequence described in detail herein, or a corresponding mutation of any amino acid sequence provided herein, wherein X is any amino acid change. In some embodiments, the dCas9 domain without nuclease activity comprises a D10A mutation and an H840A mutation of the amino acid sequence described in detail herein, or a corresponding mutation of any amino acid sequence provided herein. As an example, the Cas9 domain without nuclease activity comprises the amino acid sequence described in detail in the cloning vector pPlatTET-gRNA2 (accession number BAV54124).

[0498] The amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows:

[0499]

[0500]

[0501] (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).

[0502] Based on the present disclosure and the knowledge in the art, other suitable nuclease-free dCas9 domains will be apparent to those skilled in the art and are within the scope of the present disclosure. Such other exemplary suitable nuclease-free Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference).

[0503] In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, in other words, Cas9 is a nickase, referred to as a "nCas9" protein (for "nickase" Cas9). Cas9 proteins inactivated by nucleases can be interchangeably referred to as "dCas9" proteins (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 entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations in these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337: 816-821 (2012); Qi et al., Cell. 28; 152(5): 1173-83 (2013)).

[0504] In some embodiments, the dCas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 99.5% identical to any one of the dCas9 domains provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences detailed herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any one of the amino acid sequences detailed herein.

[0505] In some embodiments, the dCas9 partially or completely corresponds to or comprises a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises D10A and H840A mutations or corresponding mutations in another Cas9.

[0506] In some embodiments, the dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A):

[0507] (Single underline: HNH domain; double underline: RuvC domain).

[0508] In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequences provided above, or at the corresponding position in any of the amino acid sequences provided herein.

[0509] In other embodiments, dCas9 variants are provided having mutations other than D10A and H840A, which, for example, result in nuclease-inactivated Cas9 (dCas9). For example, such mutations include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs of dCas9 are provided 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, wherein the amino acid sequence of the variant is 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.

[0510] In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase can be a Cas9 protein that can only cleave a chain of a double-helical nucleic acid molecule (e.g., a double-helical DNA molecule). In some embodiments, the target chain of the Cas9 nickase cleavage double-helical nucleic acid molecule means that the Cas9 nickase cleaves the chain that is base-paired (complementary) with the gRNA (e.g., sgRNA) bound to Cas9. In some embodiments, the Cas9 nickase comprises a D10A mutation and has a histidine at position 840. In some embodiments, the non-target, unbase-edited chain of the Cas9 nickase cleavage double-helical nucleic acid molecule means that the Cas9 nickase cleaves the chain that is not base-paired (complementary) with the gRNA (e.g., sgRNA) bound to Cas9. In some embodiments, the Cas9 nickase comprises an H840A mutation and has an aspartic acid residue at position 10, or a corresponding mutation. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 99.5% identical to any one of the Cas9 nickases provided herein. Other suitable Cas9 nickases 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.

[0511] The amino acid sequence of an exemplary catalytic Cas9 nickase (nCas9) is as follows:

[0512]

[0513] In some embodiments, Cas9 refers to Cas9 from an archaeon (e.g., Nanoarchaeum), which builds the domains and kingdoms of unicellular prokaryotic microorganisms. In some embodiments, the programmable nucleotide binding protein can be a CasX or CasY protein, which has been described in, for example, Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genome-resolved metagenomics, a large number of CRISPR-Cas systems have been identified, including the first Cas9 reported in the Archaea domain of life. This divergent Cas9 protein was discovered in the little-studied Nanoarchaeum as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, were discovered that are among the most compact systems discovered to date. In some embodiments, in the base editor systems described herein, Cas9 is replaced with CasX or a variant of CasX. In some embodiments, in the base editor system described herein, Cas9 is replaced with CasY or a variant of CasY. It should be understood that other RNA-guided DNA binding proteins can be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0514] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to a 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 that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any CasX or CasY protein described herein. It should be understood that CasX and CasY from other bacterial species can also be used in accordance with the present disclosure.

[0515] An exemplary CasX ((uniprot.org / uniprot / FONN87; uniprot.org / uniprot / FONH53)tr|FONN87|FONN87_SULIHCRISPR-associated Casx protein OS=Sulfolobus icelandica (strain HVE10 / 4) GN=SiH_0402 PE=4 SV=1) amino acid sequence is as follows:

[0516]

[0517]

[0518] An exemplary CasX (>tr|FONH53|FONH53_SULIR CRISPR-associated protein, Casx OS=Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4 SV=1) amino acid sequence is as follows:

[0519]

[0520] Deltaproteobacterial CasX

[0521]

[0522]

[0523] An exemplary CasY ((ncbi.nlm.nih.gov / protein / APG80656.1)>APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacteria]) amino acid sequence is as follows:

[0524]

[0525] In some embodiments, Cas9 is Neisseria meningitidis Cas9 (NmeCas9) or a variant thereof. The NmeCas9 features and PAM sequences described in Edraki et al. Mol. Cell. (2019) 73(4): 714-726 are incorporated herein by reference in their entirety.

[0526] An exemplary Nme1Cas9 amino acid sequence is provided below:

[0527] type II CRISPR RNA-guided endonuclease Cas9[Neisseria meningitidis]WP_002235162.1

[0528]

[0529] An exemplary Nme2Cas9 amino acid sequence is provided below:

[0530] Type II CRISPR RNA-guided endonuclease Cas9 [Meningococcus]

[0531] WP_002230835.1

[0532]

[0533]

[0534] The Cas9 nuclease has two functional nuclease domains: RuvC and HNH. When targeting those positions of the nuclease domain to cleave the opposite strand of the target DNA, Cas9 undergoes a conformational change. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (approximately 3-4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired by one of two conventional repair pathways: (1) the efficient but error-prone non-homologous end joining (NHEK) pathway; or (2) the inefficient but high-fidelity homology-directed repair (HDR) pathway.

[0535] The "efficiency" of non-homologous end joining (NHEJ) and / or homology-guided repair (HDR) can be calculated by any conventional method. For example, in some embodiments, efficiency can be expressed as a percentage of successful HDR. For example, an inspector nuclease assay can be used to generate a cleavage result, and the ratio of product to substrate can be used to calculate the percentage. For example, an inspector nuclease can be used that directly cleaves DNA containing newly incorporated restriction sequences as a result of successful HDR. More cleaved substrates indicate a higher percentage of HDR (higher HDR efficiency). As an illustrative example, the score (percentage) of HDR can be calculated using the following equation: [(cleavage product) / (substrate plus cleavage product)] (e.g., (b+c) / (a+b+c), where "a" is the band intensity of the DNA substrate, and "b" and "c" are cleavage products).

[0536] In some embodiments, the efficiency can be expressed as a percentage of successful NHEJ. For example, a T7 endonuclease I assay can be used to generate a cleavage result, and the ratio of product to substrate can be used to calculate the percentage NHEJ. T7 endonuclease I cleaves mismatched heteroduplex DNA resulting from hybridization of wild-type and mutant DNA strands (NHEJ produces small random insertions or deletions (indels) at the site of the original strand). More cleavage indicates a higher percentage of NHEJ (higher NHEJ efficiency). As an illustrative example, the fraction (percentage) of NHEJ can be calculated using the following equation: (1-(1-(b+c) / (a+b+c)) 1 / 2 )×100, where “a” is the band intensity of the DNA substrate, and “b” and “c” are the cleavage products (Ran et al., Cell. 2013 Sep. 12; 154(6): 1380-9; and Ran et al., Nat Protoc. 2013 Nov.; 8(11): 2281-2308).

[0537] The NHEJ repair pathway is the most active repair mechanism, and it often causes small nucleotide insertions or deletions (indels) at DSB sites. The randomness of NHEJ-mediated DSB repair has important practical significance because cell populations or guide polynucleotides expressing Cas9 and gRNA can lead to diverse mutations. In most embodiments, NHEJ gives small insertions and deletions in the target DNA, which result in amino acid deletions, insertions, or frameshift mutations that result in the extraction of stop codons within the open reading frame (ORF) of the targeted gene. The ideal end result is a disabling mutation within the targeted gene.

[0538] Although NHEJ-mediated DSB repair often disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to generate specific nucleotide changes ranging from single nucleotide changes to large insertions such as the addition of fluorophores or tags.

[0539] To perform gene editing using HDR, a DNA repair template containing the desired sequence can be delivered to the cell type of interest using gRNA and Cas9 or Cas9 nickase. The repair template can contain the desired edit and additional homologous sequences located immediately upstream or downstream of the target (called the left homology arm and the right homology arm). The length of each homology arm may depend on the size of the variation introduced, with larger insertions requiring longer homology arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. Even in cells expressing Cas9, gRNA, and exogenous gRNAs, the efficiency of HDR is generally low (<10% of modified alleles). The efficiency of HDR can be improved by synchronizing the cells because HDR occurs in the S and G2 phases of the cell cycle. Chemically or genetically inhibiting genes involved in NHEJ may also increase the frequency of HDR.

[0540] In some embodiments, Cas9 is a modified Cas9. A given gRNA targeting sequence may have additional sites throughout the genome where there is partial homology. These sites are called off-targets, and these sites need to be considered when designing gRNA. In addition to optimizing gRNA design, CRISPR specificity can also be increased by modifying Cas9. Cas9 produces double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase, the D10A mutant of SpCas9, retains one nuclease domain and produces DNA nicks instead of DSBs. The nickase system can also be combined with HDR-mediated gene editing for specific gene editing.

[0541] In some embodiments, Cas9 is a variant Cas9 protein. The variant Cas9 polypeptide has an amino acid sequence that differs by one amino acid (e.g., with a deletion, insertion, substitution, fusion) when compared to the amino acid sequence of the wild-type Cas9 protein. In some 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 has no substantial nuclease activity. When the test Cas9 protein is a variant Cas9 protein without substantial nuclease activity, it can be referred to as "dCas9".

[0542] In some embodiments, the variant Cas9 protein has reduced nuclease activity. For example, the variant Cas9 protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1% or less than about 0.1% of the endonuclease activity of the wild-type Cas9 protein (e.g., wild-type Cas9 protein).

[0543] In some embodiments, the variant Cas9 protein can cleave the complementary chain of the guide target sequence, but the ability to cleave the non-complementary chain of the double-stranded guide target sequence is reduced. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, the variant Cas9 protein has D10A (aspartic acid at amino acid position 10 is changed to alanine), and therefore the complementary chain of the double-stranded guide target sequence can be cleaved, but the ability to cleave the non-complementary chain of the double-stranded guide target sequence is reduced (therefore, when the variant Cas9 protein cleaves double-stranded target nucleic acid, single-strand breaks (SSBs) rather than double-strand breaks (DSBs) are caused) (see, for example, Jinek et al., Science. 2012 Aug. 17; 337 (6096): 816-21).

[0544] In some embodiments, the variant Cas9 protein can cleave the non-complementary chain of the double-stranded guide target sequence, but the ability to cleave the complementary chain of the guide target sequence is reduced. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif). As a non-limiting example, in some embodiments, the variant Cas9 protein has an H840A (histidine at amino acid position 840 becomes alanine) mutation, and therefore can cleave the non-complementary chain of the guide target sequence but the ability to cleave the complementary chain of the guide target sequence is reduced (thus, when the variant Cas9 protein cleaves the target sequence, it causes SSB rather than DSB). The ability of such Cas9 proteins to cleave guide target sequences (e.g., single-stranded guide target sequences) is reduced, but the ability to bind to guide target sequences (e.g., single-stranded guide target sequences) is retained.

[0545] In some embodiments, the variant Cas9 protein has a reduced ability to cleave both the complementary and non-complementary strands of a double-stranded target DNA. As a non-limiting example, in some embodiments, the variant Cas9 protein has two mutations, D10A and H840A, which reduce the ability of the polypeptide to cleave the complementary and non-complementary strands of a double-stranded target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0546] As another non-limiting example, in some embodiments, the variant Cas9 protein carries W476A and W1126A mutations, which reduce the ability of the polypeptide 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).

[0547] As another non-limiting example, in some embodiments, the variant Cas9 protein carries P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, such that the ability of the polypeptide to cleave target DNA is reduced. 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).

[0548] As another non-limiting example, in some embodiments, the variant Cas9 protein has H840A, W476A and W1126A mutations, so that the ability of the polypeptide to cleave target DNA is reduced. The ability of such Cas9 protein to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind target DNA (e.g., single-stranded target DNA) is retained. As another non-limiting example, in some embodiments, the variant Cas9 protein has H840A, D10A, W476A and W1126A mutations, so that the ability of the polypeptide to cleave target DNA is reduced. The ability of such Cas9 protein to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind target DNA (e.g., single-stranded target DNA) is retained. In some embodiments, the variant Cas9 has a repaired His residue (A840H) located at position 840 in the Cas9 HNH domain.

[0549] As another non-limiting example, in some embodiments, the variant Cas9 protein has H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, so that the ability of the polypeptide to cleave target DNA is reduced. The ability of such Cas9 protein to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to target DNA (e.g., single-stranded target DNA) is retained. As another non-limiting example, in some embodiments, the variant Cas9 protein has D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, so that the ability of the polypeptide to cleave target DNA is reduced. The ability of such Cas9 protein to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to target DNA (e.g., single-stranded target DNA) is retained. In some embodiments, when the variant Cas9 protein carries W476A and W1126A mutations or when the variant Cas9 protein carries P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, the variant Cas9 protein does not effectively bind to the PAM sequence. Thus, in some such embodiments, when such variant Cas9 proteins are used in a binding method, the method does not require a PAM sequence. In other words, in some embodiments, when such variant Cas9 proteins are used in a grafting method, the method can include a guide RNA, but the method can be performed in the absence of a PAM sequence (thus, the specificity of binding is provided by the targeting segment of the guide RNA). Other residues can be mutated to achieve the above effects (i.e., partially inactivating one or the other strand of the nuclease). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 may be altered (i.e., substituted). Furthermore, mutations other than alanine substitutions are suitable.

[0550] In some embodiments, a variant Cas9 protein with reduced catalytic activity (e.g., when the Cas9 protein has D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986 and / or A987 mutations such as D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A and / or D986A), the variant Cas9 protein can still bind to the target DNA in a site-specific manner (because it is still guided to the target DNA sequence by the guide RNA), as long as it retains the ability to interact with the guide RNA.

[0551] 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.

[0552] In some embodiments, a modified SpCas9 comprising amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and having specificity for an altered PAM 5'-NGC-3' is used.

[0553] As an alternative to S. pyogenes Cas9, RNA-guided endonucleases from the Cpf1 family can be included, which exhibit cleavage activity in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA editing technology homolog of the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of the class II CRISPR / Cas system. This acquired immunity mechanism is found in Prevotella and Francisella bacteria. The Cpf1 gene is associated with the CRISPR locus 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 the Cas9 nuclease, the DNA cleavage mediated by Cpf1 results in a double-strand break with a short 3' nucleotide. The staggered cleavage pattern of Cpf1 can open up the possibility of targeted gene transfer, similar to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Similar to the aforementioned Cas9 variants and orthologs, Cpf1 can also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM sites favored by SpCas9. The Cpf1 locus contains a mixed α / β domain, followed by RuvC-I, RuvC-II, and zinc finger-like domains, followed by a helical region. The Cpf1 protein possesses a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, Cpf1 lacks the HNH endonuclease domain, and its N-terminus lacks the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain architecture reveals that Cpf1 is functionally unique and is classified as a type V CRISPR system. The Cpf1 locus, encoding the Cas1, Cas2, and Cas4 proteins, is more similar to type I and type III systems than to type II systems. Functional Cpf1 does not require a reverse-priming CRISPR RNA (tracrRNA); therefore, only CRISPR (crRNA) is required. This is beneficial for genome editing because Cpf1 is not only smaller than Cas9, but also has a smaller sgRNA molecule (about half the number of nucleotides as Cas9). Unlike the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex cleaves the target DNA or RNA by identifying the protospacer adjacent motif 5'-YTN-3'. After identifying the PAM, Cpf1 introduces a sticky-end-like DNA double-strand break with a 4 or 5 nucleotide overhang.

[0554] Cas12 domain of nucleobase editors

[0555] Typically, microbial CRISPR-Cas systems are divided into Class 1 systems and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while Class 2 systems have single-protein effectors. For example, Cas9 and Cpf1 are Class 2 effectors, but belong to different types (Type II and Type V, respectively). In addition to Cpf1, Class 2 Type V CRISPR-Cas systems also include Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. See, for example, Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems,” Mol. Cell, 2015 Nov. 5; 60(3): 385-397; Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR Journal, 2018, 1(5): 325-336; and Yan et al., “Functionally Diverse Type VCRISPR-Cas Systems,” Science, 2019 Jan. 4; 363: 88-91; the entire contents of each are incorporated herein by reference. Type V Cas proteins contain a RuvC (or RuvC-like) nuclease domain. Although the production of mature CRISPR RNA (crRNA) is generally independent of tracrRNA, for example, Cas12b / C2c1 requires tracrRNA to produce crRNA. Cas12b / C2c1 relies on both crRNA and tracrRNA for DNA cleavage.

[0556] The nucleic acid programmable DNA binding proteins contemplated by the present invention include Cas proteins (Cas12 proteins) classified as 2 types of V. Non-limiting examples of 2 types of V-type Cas proteins include Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h and Cas12i, their homologues or modified versions thereof. As used herein, Cas12 protein may also be referred to as Cas12 nuclease, Cas12 domain or Cas12 protein domain. In some embodiments, the Cas12 protein of the present invention comprises an amino acid sequence interrupted by an internally fused protein domain such as a deaminase domain.

[0557] In some embodiments, the Cas12 domain is a Cas12 domain or a Cas12 nickase without nuclease activity. In some embodiments, the Cas12 domain is a nuclease activity domain. For example, the Cas12 domain can be a Cas12 domain that cuts a chain of a double-helical nucleic acid (for example, a double-helical DNA molecule). In some embodiments, the Cas12 domain includes any one of the amino acid sequences described in detail herein. In some embodiments, the Cas12 domain includes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 99.5% identical to any one of the amino acid sequences described in detail herein. In some embodiments, the Cas12 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences detailed herein. In some embodiments, the Cas12 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any one of the amino acid sequences detailed herein.

[0558] In some embodiments, proteins comprising fragments of Cas12 are provided. For example, in some embodiments, the protein comprises one or two Cas12 domains: (1) a gRNA binding domain of Cas12; or (2) a DNA cleavage domain of Cas12. In some embodiments, 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 to wild-type Cas12, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the Cas12 variant can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas12. In some embodiments, the Cas12 variant comprises a fragment of Cas12 (e.g., a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical to the corresponding fragment of wild-type Cas12, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% of the amino acid length of the corresponding wild-type Cas12. In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0559] In some embodiments, Cas12 partially or completely corresponds to or comprises a Cas12 amino acid sequence having one or more mutations that alter the Cas12 nuclease activity. For example, such mutations include amino acid replacements within the RuvC nuclease domain of Cas2. In some embodiments, a variant or homolog of Cas12 is provided, which is at least about 70% identical to wild-type Cas12, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, a variant of Cas12 is provided, the amino acid sequence of the variant being shorter or longer, being 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.

[0560] In some embodiments, the Cas12 fusion protein as provided herein comprises the full-length amino acid sequence of the Cas12 protein, for example, one of the Cas12 sequences provided herein. However, in other embodiments, the fusion protein as provided herein does not comprise the full-length Cas12 sequence, but only comprises one or more fragments thereof. Exemplary amino acid sequences of suitable Cas12 domains are provided herein, and other suitable sequences of Cas12 domains and fragments will be apparent to those skilled in the art.

[0561] Typically, Class 2 V-type Cas proteins have a single functional RuvC endonuclease domain (see, e.g., Chen et al., “CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity,” Science 360: 436-439 (2018)). In some cases, the Cas12 protein is a variant Cas12 protein. (See, Strecker et al., Nature Communications, 2019, 10(1): Art. No.: 212). In one embodiment, the variant Cas12 polypeptide has an amino acid sequence that differs by 1, 2, 3, 4, 5 or more amino acids (e.g., having a deletion, insertion, substitution, fusion) when compared to the amino acid sequence of the wild-type Cas12 protein. In some examples, the variant Cas12 polypeptide has an amino acid change (e.g., a deletion, insertion, or substitution) that reduces the activity of the Cas12 polypeptide. For example, in some instances, the variant Cas12 polypeptide is a Cas2b polypeptide having less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 protein. In some cases, the variant Cas12b protein has no substantial nickase activity.

[0562] In some cases, the variant Cas12b protein has reduced nickase activity. For example, the variant Cas12 protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1% or less than about 0.1% of the nickase activity of the wild-type Cas12b protein.

[0563] In some embodiments, the Cas12 protein includes an RNA-guided nuclease from the Cas12a / Cpf1 family, which exhibits activity in mammalian cells. CRISPR (CRISPR / Cpf1) from Prevotella and Francisella 1 is a DNA editing technology homolog of the CRISPR / Cas9 system. Cpf1 is an RNA-guided nuclease of the Class II CRISPR / Cas system. This acquired immunity mechanism is found in Prevotella and Francisella bacteria. The Cpf1 gene is associated with the CRISPR locus 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 limitations of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, the DNA cleavage result mediated by Cpf1 is a double-strand break with a short 3' nucleotide. The staggered cleavage pattern of Cpf1 can open the possibility of directional gene transfer, similar to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Similar to the aforementioned Cas9 variants and orthologs, Cpf1 can also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM sites favored by SpCas9. The Cpf1 locus contains a mixed α / β domain, followed by RuvC-I, RuvC-II, and zinc finger-like domains, followed by a helical region. The Cpf1 protein possesses a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, unlike Cas9, Cpf1 lacks the HNH endonuclease domain, and its N-terminus lacks the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain architecture reveals that Cpf1 is functionally unique and is classified as a type V CRISPR system. The Cas1, Cas2, and Cas4 proteins encoded by the Cpf1 locus are more similar to type I and type III systems than to type II systems. Functional Cpf1 does not require reverse initiation of CRISPR RNA (tracrRNA); therefore, only CRISPR (crRNA) is required. This is beneficial for genome editing because Cpf1 is not only smaller than Cas9, but it also has a smaller sgRNA molecule (about half the number of nucleotides of Cas9). Unlike the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex cleaves the target DNA or RNA by identifying the protospacer adjacent motif 5'-YTN-3' or 5'-TTTN-3'. After identifying the PAM, Cpf1 introduces a sticky end-like DNA double-strand break with a 4 or 5 nucleotide overhang.

[0564] In some aspects of the invention, a vector can be used that encodes a CRISPR enzyme that is mutated with respect to the wild-type enzyme so that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. 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 sp.). Cas12 can refer to a polypeptide having at most or at most about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas12 polypeptide, e.g., from Bacillus houttuyniae (BhCas12b), from Bacillus sp. V3-13 (BvCas12b), and from Alicyclobacillus acidiphilus (AaCas12b). Cas12 can refer to a wild-type or modified form of a Cas12 protein that can include amino acid changes such as deletions, insertions, substitutions, variations, mutations, fusions, chimeras, or any combination thereof.

[0565] Nucleic acid programmable DNA binding protein

[0566] Some 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 guide proteins such as base editors to specific nucleic acid (e.g., DNA or RNA) sequences. In certain embodiments, the fusion protein comprises a nucleic acid programmable DNA binding protein domain or a deaminase domain. Non-limiting examples of nucleic acid programmable DNA binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2 , Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, Cs0, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, a type II Cas effector protein, a type V Cas effector protein, a type VI Cas effector protein, CARF, DinG, a homolog thereof, or a modified or engineered version thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, but they may not be specifically listed in this disclosure.See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” Crispr J. 2018 Oct; 1: 325-336. doi: 10.1089 / crispr.2018.0033; Yah et al. “Functionally diverse type VCRISPR-Cas systems” Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, the entire contents of each of which are incorporated herein by reference.

[0567] An example of a nucleic acid-programmable DNA-binding protein with a different PAM specificity than Cas9 is the clustered regularly interspaced short palindromic repeats from Prevotella and Francisella 1 (Cpf1). Similar to Cas9, Cpf1 is also a Class 2 CRISPR effector. Cpf1 has been shown to mediate robust DNA interference with characteristics distinct from those of Cas9. Cpf1 is a single RNA-guided endonuclease-deficient tracrRNA that utilizes a T-rich protospacer adjacent motif (TTN, TTTN, or YTN). In addition, Cpf1 cleaves DNA via staggered DNA double-strand breaks. Of the 16 Cpf1 family proteins, two enzymes from the genera Acidaminococcus and Lachnospiraceae have been shown to possess potent genome editing activity in human cells. Cpf1 protein is known in the art and has been previously described, for example, in Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962, the entire contents of which are incorporated herein by reference.

[0568] Can be used in the compositions and methods of the present invention are Cpf1 (dCpf1) variants without nuclease activity, which can be used as guide nucleotide sequence programmable DNA binding protein domains. The Cpf1 protein has a RuvC-like nuclease domain similar to the RuvC domain of Cas9 but does not have an HNH nuclease domain, and the N-terminus of Cpf1 does not have the α-helical recognition lobe of Cas9. Zetsche et al., Cell, 163, 759-771, 2015 (which is incorporated herein by reference) show that the main responsibility of the RuvC-like domain of Cpf1 is to crack the two DNA chains, and the inactivation of the RuvC-like domain inactivates the Cpf1 nuclease activity. For example, mutations corresponding to D917A, E1006A or D1255A in Francisella novicida Cpf1 inactivate the Cpf1 nuclease activity. In some embodiments, the dCpf1 of the present disclosure comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It should be understood that any mutation that inactivates the RuvC domain of Cpf1, e.g., a substitution mutation, deletion, or insertion, can be used in accordance with the present disclosure.

[0569] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In some embodiments, the Cpf1 protein is a Cpf1 without nuclease activity (dCpf1). In some embodiments, Cpf1, nCpf1, or dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a Cpf1 sequence disclosed herein. In some embodiments, dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a Cpf1 sequence disclosed herein and comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It will be appreciated that Cpf1 from other bacterial species can also be used in accordance with the present disclosure.

[0570] Wild-type Francisella novo Cpf1 (D917, E1006, and D1255 are bold and underlined)

[0571]

[0572]

[0573] Francisella novogenes Cpf1 D917A (A917, E1006, and D1255 are bold and underlined)

[0574]

[0575]

[0576] Francisella novo Cpf1 E1006A (D917, A1006, and D1255 are bold and underlined)

[0577]

[0578]

[0579] Francisella novo Cpf1 D1255A (D917, E1006, and A1255 are bold and underlined)

[0580]

[0581] Francisella novogenes Cpf1 D917A / E1006A (A917, A1006, and D1255 are bold and underlined)

[0582]

[0583] Francisella novogenes Cpf1 D917A / D1255A (A917, E1006, and A1255 are bold and underlined)

[0584]

[0585]

[0586] Francisella novo Cpf1 E1006A / D1255A (D917, A1006, and A1255 are bold and underlined)

[0587]

[0588]

[0589] Francisella novogenes Cpf1 D917A / E1006A / D1255A (A917, A1006, and A1255 are bold and underlined)

[0590]

[0591]

[0592] In some embodiments, one of the Cas9 domains present in the fusion protein can be replaced with a guide nucleotide sequence-programmable DNA-binding protein domain that does not require a PAM sequence.

[0593] In some embodiments, the Cas9 domain is a Cas9 domain from Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domain is a nuclease-active SaCas9, a SaCas9 without nuclease activity (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, SaCas9 comprises an N579A mutation, or a corresponding mutation in any amino acid sequence provided herein.

[0594] In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence having a non-classical PAM. In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence having a NNGRRT or NNGRRT PAM. In some embodiments, the SaCas9 domain comprises one or more of E781X, N967X, and R1014X mutations, or corresponding mutations of any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of E781K, N967K, and R1014H mutations, or corresponding mutations of any amino acid sequence provided herein. In some embodiments, the SaCas9 domain comprises E781K, N967K, or R1014H mutations, or corresponding mutations of any amino acid sequence provided herein.

[0595] Exemplary SaCas9 sequences

[0596]

[0597] Residue N579, which is underlined and shown in bold, can be mutated (e.g., to A579) to generate the SaCas9 nickase.

[0598] Exemplary SaCas9n sequences

[0599]

[0600]

[0601] Residue A579, which can be mutated from N579 to yield the SaCas9 nickase, is underlined and shown in bold.

[0602] Exemplary SaKKH Cas9

[0603]

[0604]

[0605] Residue A579, which can be mutated from N579 to obtain the SaCas9 nickase, is underlined and shown in bold. Residues K781, K967, and H1014, which can be mutated from E781, N967, and R1014 to obtain the SaKKH Cas9, are underlined and shown in italics.

[0606] In some embodiments, the napDNAbp is circular hypermutated. In the following sequences, plain text represents adenosine deaminase sequences, bold sequences indicate sequences derived from Cas9, italic sequences represent linker sequences, and underlined sequences represent bipartite nuclear localization sequences.

[0607] CP5 (with MSP "NGC" PID and "D10A" nickase):

[0608]

[0609]

[0610] In some embodiments, nucleic acid programmable DNA binding protein (napDNAbp) is a single effector of a microbial CRISPR-Cas system. Single effectors of a microbial CRISPR-Cas system include, but are not limited to, Cas9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 systems and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while Class 2 systems have single protein effectors. For example, Cas9 and Cpf1 are Class 2 effectors. In addition to Cas9 and Cpf1, three distinct Class 2 CRISPR-Cas systems (Cas12b / C2c1 and Cas12c / C2c3) have been described by Shmakov et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems," Mol. Cell, 2015 Nov. 5; 60(3): 385-397, the entire contents of which are incorporated herein by reference. The effectors of two systems, Cas12b / C2c1 and Cas12c / C2c3, contain a Ruv-like endonuclease domain associated with Cpf1. A third system contains an effector with two domains called HEPN RNases. Unlike CRISPR RNA production by Cas12b / C2c1, the retrieval of mature CRISPR RNA is independent of tracrRNA. Cas12b / C2c1 relies on both CRISPR RNA and tracrRNA for DNA cleavage.

[0611] The crystal structure of Alicyclobaccillus acidoterrastris Cas12b / C2c1 (AacC2c1) has been reported as a complex with a chimeric single-molecule guide RNA (sgRNA). See, for example, Liu et al., "C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism," Mol. Cell, 2017 Jan. 19; 65(2): 310-322, the entire contents of which are incorporated herein by reference. The crystal structure has been reported as a ternary complex of Alicyclobaccillus acidoteirastris C2c1 bound to target DNA. See, for example, Yang et al., "PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Casendonuclease," Cell, 2016 Dec. 15; 167(7): 1814-1828, the entire contents of which are incorporated herein by reference. Catalytically active conformations of AacC2c1 with target and non-target DNA strands have been captured and independently localized in a single RuvC catalytic zone, and Cas12b / C2c1-mediated cleavage results in staggered heptanucleotide breaks of the target DNA. Structural comparisons between the Cas12b / C2c1 ternary complex and previously identified Cas9 and Cpf1 partners demonstrate the diversity of mechanisms employed by the CRISPR-Cas9 system.

[0612] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can be Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, napDNAbp is Cas12b / C2c1 protein. In some embodiments, napDNAbp is Cas12c / C2c3 protein. In some embodiments, napDNAbp comprises an amino acid sequence, and the amino acid sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, napDNAbp is naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the napDNAbp sequences provided herein. It should be understood that Cas12b / C2c1 or Cas12c / C2c3 from other bacterial species can also be used according to the present disclosure.

[0613] The amino acid sequence of Cas12b / C2c1 ((uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2 C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acido-terrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) GN=c2c1 PE=1SV=1) is as follows:

[0614]

[0615]

[0616] AacCas12b (Alicyclobacillus acidiphilus)-WP_067623834

[0617]

[0618]

[0619] BhCas12b (Bacillus hisashii) NCBI reference sequence: WP_095142515

[0620]

[0621]

[0622] The variant named BvCas12b V4 (relative to the wild type, with S893R / K846R / E837G changes) is expressed as follows: 5'mRNA Cap---5'UTR---bhCas12b---STOP sequence---3'UTR---120polyA tail

[0623] 5'UTR:

[0624]

[0625] 3'UTR (TriLink standard UTR)

[0626]

[0627] Nucleic acid sequence of bhCas12b(V4)

[0628]

[0629]

[0630]

[0631] In some embodiments, Cas12b is BvCas12B. In some embodiments, BvCas12B comprises the following changes relative to the wild-type sequence: S893R, K846R, and E837G, as numbered in the BvCas12B exemplary sequence provided below.

[0632] BvCas12b (Bacillus sp. V3-13) NCBI reference sequence: WP_101661451.1

[0633]

[0634]

[0635] In some embodiments, Cas12b is BTCas12b.

[0636] BTCas12b (Bacillus thermoamylovorans) NCBI reference sequence: WP041902512

[0637]

[0638] In some embodiments, napDNAbp refers to Cas12c. In some embodiments, the Cas12c protein is a variant of Cas12c1 or Cas12c1. In some embodiments, the Cas12 protein is a variant of Cas12c2 or Cas12c2. In some embodiments, the Cas12 protein is a Cas12c protein (i.e., OspCas12c) or a variant of OspCas12c from Oleiphilus sp. HI0009. These Cas12c molecules have been described in Yan et al., “Functionally DiverseType V CRISPR-Cas Systems,” Science, 2019 Jan. 4; 363: 88-91, the entire contents of which are incorporated herein by reference. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring Cas12c1, Cas12c2, or OspCas12c protein. In some embodiments, the napDNAbp is a naturally occurring Cas12c1, Cas12c2, or OspCas12c protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any Cas12c1, Cas12c2, or OspCas12c protein described herein. It will be appreciated that Cas12c1, Cas12c2, or OspCas12c from other bacterial species may also be used according to the present disclosure.

[0639] Cas12c1

[0640]

[0641]

[0642] Cas12c2

[0643]

[0644] 0spCas12c

[0645]

[0646]

[0647] In some embodiments, napDNAbp refers to Cas12g, Cas12h or Cas12i, which have been described in, for example, Yan et al., “Functionally Diverse Type V CRISPR-Cas Systems,” Science, 2019 Jan. 4; 363: 88-91, the entire contents of each of which are incorporated herein by reference. By assembling more than 10TB of sequence data, a new classification of V-type Cas proteins was identified that showed weak similarity to previously characterized V-type proteins, including Cas12g, Cas12h, and Cas12i. In some embodiments, the Cas12 protein is a variant of Cas12g or Cas12g. In some embodiments, the Cas12 protein is a variant of Cas12h or Cas12h. In some embodiments, the Cas12 protein is a variant of Cas12i or Cas12i. It should be understood that other RNA-guided DNA binding proteins can be used as napDNAbp and are within the scope of the present disclosure. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring Cas12g, Cas12h, or Cas12i protein. In some embodiments, the napDNAbp is a naturally occurring Cas12g, Cas12h, or Cas12i protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any Cas12g, Cas12h, or Cas12i protein described herein. It will be appreciated that Cas12g, Cas12h or Cas12i from other bacterial species may also be used according to the present disclosure. In some embodiments, the Cas12i protein is Cas12il or Cas12i2.

[0648] Cas12g1

[0649]

[0650] Cas12h1

[0651]

[0652] Cas12i1

[0653]

[0654]

[0655] Cas12i2

[0656]

[0657] Representative nucleic acid and protein sequences of base editors are as follows:

[0658] BhCas12b GGSGGS-ABE8-Xten20 located at P153

[0659]

[0660]

[0661]

[0662]

[0663] BhCas12b GGSGGS-ABE8-Xten20 at K255

[0664]

[0665]

[0666]

[0667]

[0668] BhCas12b GGSGGS-ABE8- located in D306

[0669]

[0670]

[0671]

[0672]

[0673] BhCas12b GGSGGS-ABE8-Xten20 at D980

[0674]

[0675]

[0676]

[0677]

[0678] BhCas12b GGSGGS-ABE8-Xten20 at K1019

[0679]

[0680]

[0681]

[0682]

[0683] For the above sequences, the Kozak sequence is bold and underlined; the N-terminal nuclear localization signal (NLS) is marked; lowercase letters indicate the GGGSGGS linker; _________ marks the sequence encoding ABE8, and the unmodified sequence encodes BhCas12b; the double underline indicates the Xten20 linker; the single underline indicates the C-terminal NLS; GGATCC represents the GS linker; and italic letters represent the coding sequence for the 3× hemagglutinin (HA) tag.

[0684] Guide polynucleotides

[0685] In one embodiment, the guide polynucleotide is a guide RNA. As used herein, the term "guide polynucleotide" 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. As used herein, the term "guide RNA (gRNA)" and its grammatical equivalents can refer to RNA that can be specific for a target DNA and can form a complex with a Cas protein. The RNA / Cas complex can assist in "guiding" the Cas protein to the target DNA. Cas9 / crRNA / tracrRNA endonucleases and solubilizes linear or circular dsDNA targets that are complementary to the spacer sequence. The target chain that is not complementary to the crRNA is first cut endonucleases and solubilized, and then trimmed by 3'-5' exonucleases. In essence, DNA binding and cleavage typically require protein and two RNAs. However, single guide RNA ("sgRNA", or simply "gRNA") can be engineered to combine aspects of both 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 (PAM or protospacer adjacent motif) in the CRISPR repeat sequence to help distinguish self from non-self. The Cas9 nuclease sequence and structure are known to those skilled in the art (see, for example, "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti, JJ et al., Natl. Acad. Sci. USA 98: 4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E. et al., Nature 471: 602-607 (2011); and "Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M. et al., Science 337: 816-821 (2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in a variety of species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus.Based on this disclosure, other suitable Cas9 nucleases and sequences may be apparent to those skilled in the art, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, in other words, Cas9 is a nickase.

[0686] In some embodiments, the guide polynucleotide is at least one single guide RNA ("sgRNA" or "gRNA"). In some embodiments, the guide polynucleotide is at least one tracrRNA. In some embodiments, the guide polynucleotide does not require a PAM sequence to guide a polynucleotide programmable DNA binding domain (e.g., Cas9 or Cpf1) to a target nucleotide sequence.

[0687] The polynucleotide programmable nucleotide binding domains of the base editors disclosed herein (e.g., domains derived from CRISPR) can identify target polynucleotide sequences by associating with guide polynucleotides. Guide polynucleotides (e.g., gRNA) are typically single-stranded and can be programmed to site-specifically bind (i.e., via complementary base pairing) to the target sequence of the polynucleotide, thereby directing the base editor that cooperates with the guide nucleic acid to the target sequence. The guide polynucleotide can be DNA. The guide polynucleotide can be RNA. In some embodiments, the guide polynucleotide comprises natural nucleotides (e.g., adenosine). In some embodiments, the guide polynucleotide comprises non-natural (non-natural) nucleotides (e.g., peptide nucleic acids or nucleotide analogs). In some embodiments, the targeting region of the guide nucleic acid sequence can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. The targeting region of the guide nucleic acid can be between 10-30 nucleotides in length, or between 15-25 nucleotides in length, or between 15-20 nucleotides in length.

[0688] In some embodiments, a guide polynucleotide comprises two or more individual polynucleotides that can interact with each other via, for example, complementary base pairing (e.g., a dual guide polynucleotide). For example, a guide polynucleotide can comprise a CRISPR RNA (crRNA) and a reverse priming CRISPR RNA (tracrRNA). For example, a guide polynucleotide can comprise one or more reverse priming CRISPR RNAs (tracrRNAs).

[0689] In a type II CRISPR system, targeting nucleic acids by a CRISPR protein (e.g., Cas9) typically requires complementary base pairing between a first RNA molecule (crRNA) containing a sequence that recognizes the target sequence and a second RNA molecule (trRNA) containing a repeat sequence, which forms a scaffold region that stabilizes the guide RNA-CRISPR protein complex. Such dual-guide RNA systems can be used as guide polynucleotides to direct the base editors disclosed herein to the target polynucleotide sequence.

[0690] In some embodiments, the base editors provided herein utilize a single guide polynucleotide (e.g., gRNA). In some embodiments, the base editors provided herein utilize a dual guide polynucleotide (e.g., dual gRNA). In some embodiments, the base editors provided herein utilize one or more guide polynucleotides (e.g., multiple gRNAs). In some embodiments, a single guide polynucleotide is used in different base editors described herein. For example, a single guide polynucleotide can be used for a cytidine base editor and an adenosine base editor.

[0691] In other embodiments, the guide polynucleotide can comprise both a polynucleotide targeting portion of a nucleic acid and a scaffold portion of a nucleic acid in a single molecule (i.e., a single-molecule guide nucleic acid). For example, a single-molecule guide polynucleotide can be a single guide RNA (sgRNA or gRNA). As used herein, the term guide polynucleotide contemplates any single, double, or multi-molecule nucleic acid that is capable of interacting with a base editor and directing it to a target polynucleotide sequence.

[0692] Typically, a guide polynucleotide (e.g., a crRNA / trRNA complex or gRNA) includes a "polynucleotide targeting segment" that includes a sequence capable of recognizing and binding to a target polynucleotide sequence and a guide polynucleotide "protein binding segment" within the polynucleotide programmable nucleotide binding domain of the base editor. In some embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to a DNA polynucleotide, thereby facilitating the editing of bases in the DNA. In other embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to an RNA polynucleotide, thereby facilitating the editing of bases in the RNA. Herein, "segment" refers to a segment or region of a molecule, for example, a continuous extension of nucleotides in a guide polynucleotide. A segment may also refer to a region / segment of a complex, such that a segment may include a region of more than one molecule. For example, if the guide polynucleotide comprises a plurality of nucleic acid molecules, the protein binding segment may include all or part of a plurality of independent molecules that hybridize, for example, along complementary regions. In some embodiments, a protein-binding segment of a DNA-targeting RNA comprising two separate molecules can comprise: (i) base pairs 40-75 of a first RNA molecule that is 100 base pairs in length; and (ii) base pairs 10-25 of a first RNA molecule that is 50 base pairs in length. Unless specifically defined otherwise in a particular context, the definition of "segment" is not limited to a specific number of total base pairs, is not limited to any specific number of base pairs from a given RNA molecule, is not limited to a specific number of separate molecules within a complex, and can include regions of any total length of an RNA molecule and can include regions that have complementarity with other molecules.

[0693] The guide RNA or guide polynucleotide can comprise two or more RNAs, for example, CRISPR RNA (crRNA) and reverse initiation crRNA (tracrRNA). The guide RNA or guide polynucleotide can sometimes comprise single-stranded RNA, or a single guide RNA (sgRNA) formed by fusing a portion (e.g., a functional portion) of crRNA and tracrRNA. The guide RNA or guide polynucleotide can also be a double RNA comprising crRNA and tracrRNA. In addition, crRNA can hybridize with target DNA.

[0694] As described above, the guide RNA or guide polynucleotide can be an expression product. For example, the DNA encoding the guide RNA can be a vector comprising a sequence encoding the guide RNA. The guide RNA or guide polynucleotide can be transferred into the cell by transfecting the cell with an isolated guide RNA or a plasmid DNA comprising a sequence encoding the guide RNA and a promoter. The guide RNA or guide polynucleotide can also be transferred into the cell by other routes, such as using viral-mediated gene delivery.

[0695] The guide RNA or guide polynucleotide can be isolated. For example, the guide RNA can be transferred into a cell or organism in the form of isolated RNA. The guide RNA can be prepared by in vitro transcription using in vitro transcription systems known in the art. The guide RNA can be transferred into the cell in the form of isolated RNA rather than in the form of a plasmid containing the coding sequence of the guide RNA.

[0696] A guide RNA or guide polynucleotide can comprise three regions: a first region at the 5' end that can complement the target site in the chromosomal sequence, a second internal region that can form a stem-loop structure, and a third 3' region that can be single-stranded. The first region of each guide RNA can also be different, allowing each guide RNA to guide the fusion protein to a specific target site. Furthermore, the second and third regions of each guide RNA can be identical across all guide RNAs.

[0697] The first region of the guide RNA or guide polynucleotide can be complementary to the sequence at the target site in the chromosomal sequence, and the first region of the appropriate guide RNA can be base paired with the target site. In some embodiments, the first region of the guide RNA can include or include about 10 nucleotides to 25 nucleotides (i.e., 10 nucleotides to nucleotides; or about 10 nucleotides to about 25 nucleotides; or 10 nucleotides to about 25 nucleotides; or about 10 nucleotides to 25 nucleotides) or more. For example, the region that performs base pairing between the first region of the guide RNA and the target site of the chromosomal sequence can be or can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25 or more nucleotides in length. Sometimes, the first region of the guide RNA can be or can be a length of about 19, 20 or 21 nucleotides.

[0698] The guide RNA or guide polynucleotide can also include a second region that forms a secondary structure. For example, the secondary structure formed by the guide RNA can include a stem (or hairpin) and a loop. The length of the loop and the stem can be variable. For example, the length range of the loop can be or can be about 3 to 10 nucleotides, and the length range of the stem can be or can be about 6 to 20 base pairs. The stem can include one or more 1 to 10 or about 10 nucleotide protrusions. The overall length of the second region can be or can be within the length range of about 16 to 60 nucleotides. For example, the loop can be or can be a length of about 4 nucleotides, and the stem can be or can be about 12 base pairs.

[0699] The guide RNA or guide polynucleotide can also include a third region at the 3' end, which can be substantially single-stranded. For example, the third region is sometimes not complementary to any chromosomal sequence in the cell of interest, and sometimes not complementary to the remainder of the guide RNA. Furthermore, the length of the third region can vary. The third region can be greater than or greater than about 4 nucleotides in length. For example, the overall length of the third region can be or can be in the range of about 5 to 60 nucleotides in length.

[0700] A guide RNA or guide polynucleotide can target any exon or intron of a gene target. In some embodiments, a guide can target exon 1 or 2 of a gene; in other embodiments, a guide can target exon 3 or 4 of a gene. A composition can include multiple RNAs that all target the same exon, or in some embodiments, multiple RNAs that can target different exons. Exons and introns of a gene can be targeted.

[0701] The guide RNA or guide polynucleotide can target a nucleic acid sequence of about 20 nucleotides. The target nucleic acid can be less than about 20 nucleotides. The target nucleic acid can be at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 nucleotides, or anywhere between 1-100 nucleotides in length. The target nucleic acid can be up to about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50 nucleotides, or anywhere between 1-100 nucleotides in length. The target nucleic acid sequence can be about 20 bases immediately 5' of the first nucleotide of the PAM. The guide RNA can target a nucleic acid sequence. The target nucleic acid can be at least about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, or 1-100 nucleotides.

[0702] A guide polynucleotide, e.g., a guide RNA, can refer to a nucleic acid that can hybridize to another nucleic acid in the genome of a cell (e.g., a target nucleic acid or a pre-spacer sequence). A guide polynucleotide can be RNA. A guide polynucleotide can be DNA. A guide polynucleotide can be programmed or designed to site-specifically bind to a nucleic acid sequence. A guide polynucleotide can comprise one polynucleotide chain and can be referred to as a single guide polynucleotide. A guide polynucleotide can comprise two polynucleotide chains and can be referred to as a dual guide polynucleotide. A guide RNA can be introduced into a cell as an RNA molecule. For example, an RNA molecule can be transcribed and / or chemically synthesized in vitro. RNA can be derived from a synthetic DNA molecule (e.g., Gene fragment) is transcribed. The guide RNA can then be introduced into the cell as an RNA molecule. The guide RNA can also be introduced into the cell in the form of a non-RNA nucleic acid molecule (e.g., a DNA molecule). For example, the DNA encoding the guide RNA can be operably linked to a promoter control sequence to express the guide RNA in the cell of interest. The RNA coding sequence can be operably linked to a promoter sequence recognized by RNA polymerase III (Pol III). Plasmid vectors that can be used to express guide RNA include, but are not limited to, px330 vectors and px333 vectors. In some embodiments, the plasmid vector (e.g., px333 vector) may contain at least two DNA sequences encoding the guide RNA.

[0703] Methods for selecting, designing and verifying guide polynucleotides (e.g., guide RNAs and targeting sequences) are described herein and are known to those skilled in the art. For example, in order to minimize the impact of potential substrate promiscuity of a deaminase domain (e.g., an AID domain) in a nucleobase editor system, the number of residues that may be inadvertently targeted by a deamination reaction (e.g., off-target C residues that may potentially reside on ssDNA within the target nucleic acid locus) can be minimized. In addition, software tools can be used to optimize the gRNA corresponding to the target nucleic acid sequence, for example, to minimize the total off-target activity across the locus. For example, for each possible targeting domain selection using Streptococcus pyogenes Cas9, all off-target sequences (previously selected PAMs, e.g., NAG or NGG) can be identified across the genome, containing up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base pairs. The first region of the gRNA that is complementary to the target site can be identified, and all first regions (e.g., crRNAs) can be ranked according to the predicted total off-target score; the top-ranked targeting domains represent those that are likely to have the greatest on-target activity and the least off-target activity. Functional assessment of candidate gRNAs can be performed using methods known in the art and / or as described in detail herein.

[0704] As a non-limiting example, the target DNA hybridization sequence in the crRNA of the guide RNA used in ...

Claims

1. A use of (i) an adenosine base editor or a nucleic acid molecule encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid molecule encoding the guide polynucleotide for preparing a medicament for treating a neurological disorder in a subject in need thereof, The adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, and wherein the guide polynucleotide directs the adenosine base editor to effect a single nucleobase modification at a splice site of a target gene associated with the neurological disorder in the subject, thereby treating the neurological disorder in the subject.

2. A use of (i) a base editor or a nucleic acid molecule encoding the base editor and (ii) a guide polynucleotide or a nucleic acid molecule encoding the guide polynucleotide for the preparation of a medicament for treating amyotrophic lateral sclerosis in a subject in need thereof, The base editor comprises a programmable DNA binding domain and a deaminase domain, and wherein the guide polynucleotide guides the base editor to achieve a single nucleobase modification located at a splice site of a superoxide dismutase 1 gene of the subject, thereby treating amyotrophic lateral sclerosis in the subject.

3. A use of (i) a base editor or a nucleic acid sequence encoding the base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide for the preparation of a medicament for treating amyotrophic lateral sclerosis in a subject in need thereof, The base editor comprises a programmable DNA binding domain and a deaminase domain. wherein the guide polynucleotide directs the base editor to effect a single nucleobase modification of a superoxide dismutase 1 gene located in the subject, and wherein the single nucleobase modification results in a premature stop codon in the superoxide dismutase 1 gene, thereby treating amyotrophic lateral sclerosis in the subject.

4. A use of (i) a base editor or a nucleic acid molecule encoding the base editor and (ii) a guide polynucleotide or a nucleic acid molecule encoding the guide polynucleotide for preparing a medicament for treating spinal bulbar muscular atrophy in a subject, The base editor comprises a programmable DNA binding domain and a deaminase domain, and wherein the guide polynucleotide guides the base editor to achieve a single nucleobase modification located at a splice site of an androgen receptor gene in the subject, thereby treating spinal bulbar muscular atrophy in the subject.

5. A use of (i) a base editor or a nucleic acid sequence encoding the base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide for preparing a medicament for treating spinal bulbar muscular atrophy in a subject, The base editor comprises a programmable DNA binding domain and a deaminase domain. wherein the guide polynucleotide directs the base editor to effect a single nucleobase modification located in an androgen receptor gene of the subject, and wherein the single nucleobase modification results in a premature stop codon in the androgen receptor gene, thereby treating spinal bulbar muscular atrophy in the subject.

6. A use of (i) an adenosine base editor or a nucleic acid molecule encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid molecule encoding the guide polynucleotide for preparing a drug for modifying a target gene associated with a neurological disease or a regulatory element thereof, wherein the target gene or its regulatory element is modified by contact with the drug, The adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, and wherein the guide polynucleotide directs the adenosine base editor to effect a single nucleobase change at a splice site of the target gene.

7. A use of (i) a base editor or a nucleic acid molecule encoding the base editor and (ii) a guide polynucleotide or a nucleic acid molecule encoding the guide polynucleotide for preparing a drug for regulating superoxide dismutase 1 gene expression, wherein the drug contacts the superoxide dismutase 1 gene or its regulatory element to regulate the expression of the superoxide dismutase 1 gene, The base editor comprises a programmable DNA binding domain and a deaminase domain, and The guide polynucleotide directs the base editor to effect a single nucleobase change at a splice site of a superoxide dismutase 1 gene.

8. Use of (i) a base editor or a nucleic acid molecule encoding the base editor and (ii) a guide polynucleotide or a nucleic acid molecule encoding the guide polynucleotide in the preparation of a drug for modifying a superoxide dismutase 1 gene, wherein the drug contacts the superoxide dismutase 1 gene or its regulatory element to modify the superoxide dismutase 1 gene, wherein the guide polynucleotide directs the base editor to effect a single nucleobase change in a superoxide dismutase 1 gene of the subject, and wherein the single nucleobase change results in a premature stop codon in the superoxide dismutase 1 gene.

9. A use of (i) a base editor or a nucleic acid molecule encoding the base editor and (ii) a guide polynucleotide or a nucleic acid molecule encoding the guide polynucleotide for preparing a drug for regulating androgen receptor gene expression, wherein the drug contacts the androgen receptor gene or its regulatory element to regulate the expression of the androgen receptor gene, The base editor comprises a programmable DNA binding domain and a deaminase domain, and The guide polynucleotide directs the base editor to effect a single nucleobase change at a splice site of the androgen receptor gene.

10. A use of (i) a base editor or a nucleic acid molecule encoding the base editor and (ii) a guide polynucleotide or a nucleic acid molecule encoding the guide polynucleotide for preparing a drug for modifying an androgen receptor gene, wherein the drug contacts the androgen receptor gene or its regulatory element to modify the androgen receptor gene, The base editor comprises a programmable DNA binding domain and a deaminase domain. wherein the guide polynucleotide directs the base editor to effect a single nucleobase change in an androgen receptor gene in the subject, and wherein the single nucleobase change results in a premature stop codon in the androgen receptor gene.

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