Compositions and methods for delivering nucleobase editing systems

Abstract

[0003] The present invention provides compositions and methods for delivering first and second polynucleotides, each encoding a fragment of an A-to-G base editor fusion protein comprising one or more deaminases (e.g., adenosine deaminase) and nCas9, where the first polynucleotide encodes an N-terminal fragment of nCas9 fused to intein-N of the split-intein pair, and the second polynucleotide encodes a C-terminal fragment of nCas9 fused to intein-C of the split-intein pair. Methods are also provided for delivering these fragments together with an sgRNA (e.g., AAV delivery) into a cell, where the fragments are linked together by the split-intein system, thereby reconstituting a functional base editing system in the cell.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 728,703, filed September 7, 2018, and U.S. Provisional Patent Application No. 62 / 779,404, filed December 13, 2018, the entire contents of which are incorporated herein by reference.

[0002] The discovery of Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) has revolutionized the field of molecular biology. Much of this enthusiasm has centered on the clinical potential of CRISPR / Cas9 to treat human diseases and edit the human genome. Disease-causing mutations could be repaired using CRISPR or CRISPR-based systems. One challenge to achieve this goal is to deliver the necessary elements for genome editing. For example, with respect to CRISPR / Cas9, SpCas9 and sgRNAs can be encoded in a DNA plasmid vector and delivered via adeno-associated virus (AAV). However, because AAV has a small packaging capacity, it is difficult to include other elements (e.g., polypeptide domains, promoters, reporters, fluorescent tags, multiple sgRNAs, or DNA templates for HDR) to help achieve delivery of CRISPR / Cas9 components into cells and / or to fulfill the desired gene editing objectives. Summary of the Invention

[0003] In some embodiments, a first polynucleotide encodes a fusion protein comprising a deaminase and an N-terminal fragment of Cas9, wherein the N-terminal fragment of Cas9 begins at the N-terminus of Cas9 and is sequenced between A292 and G36 of Cas9 in the numbering of SEQ ID NO:2. 5and (b) a second polynucleotide encoding a C-terminal fragment of Cas9, the C-terminal fragment of Cas9 being a contiguous sequence ending at position A292 to G36 of Cas9 in the numbering of SEQ ID NO:2. 5 and ending at the C-terminus of Cas9, wherein the C-terminal fragment of Cas9 is fused to the split intein-C.

[0004] In some embodiments, (a) a first polynucleotide encoding an N-terminal fragment of Cas9, wherein the N-terminal fragment of Cas9 begins at the N-terminus of Cas9 and is between A292 and G36 of Cas9 according to the numbering in SEQ ID NO:2. 5 and (b) a second polynucleotide encoding a fusion protein comprising a C-terminal fragment of Cas9 and a deaminase, wherein the C-terminal fragment of Cas9 is a contiguous sequence that terminates at position A292 to G36 of Cas9 in the numbering of SEQ ID NO:2. 5 and ending at the C-terminus of Cas9, wherein the C-terminal fragment of Cas9 is fused to the split intein-C.

[0005] In some embodiments, a first polynucleotide encodes a fusion protein comprising a deaminase and an N-terminal fragment of Cas9, wherein the N-terminal fragment of Cas9 begins at the N-terminus of Cas9 and is between A292 and G36 of Cas9 in the numbering of SEQ ID NO:2. 5, F445 to K483, or E565 to T637 of Cas9, wherein the N-terminal fragment of Cas9 is fused to the split intein-N; and (b) a second polynucleotide encoding a C-terminal fragment of Cas9, wherein the C-terminal fragment of Cas9 is A292 to G36 of Cas9, as numbered in SEQ ID NO:2. 5 , F445-K483, or E565-T637, and terminating at the C-terminus of Cas9, wherein the N-terminal residue of the C-terminal fragment of Cas9 is Cys substituted for Ala, Ser, or Thr, and wherein the C-terminal fragment of Cas9 is fused to a split intein-C.

[0006] In some embodiments, (a) a first polynucleotide encoding an N-terminal fragment of Cas9, wherein the N-terminal fragment of Cas9 begins at the N-terminus of Cas9 and is between A292 and G36 of Cas9 according to the numbering in SEQ ID NO:2. 5 , F445 to K483, or E565 to T637 of Cas9, wherein the N-terminal fragment of Cas9 is fused to the split intein-N; and (b) a second polynucleotide encoding a fusion protein comprising a C-terminal fragment of Cas9 and a deaminase, wherein the C-terminal fragment of Cas9 is a contiguous sequence ending at positions A292 to G36 of Cas9 in the numbering of SEQ ID NO:2. 5 , F445-K483, or E565-T637, and ending at the C-terminus of Cas9, wherein the N-terminal residue of the C-terminal fragment of Cas9 is Cys substituted for Ala, Ser, or Thr, and wherein the C-terminal fragment of Cas9 is fused to a split intein-C.

[0007] In some embodiments, the N-terminal fragment of Cas9 includes amino acids 302, 309, 312, 354, 455, 459, 462, 465, 471, 473, 576, 588, or 589 in the numbering of SEQ ID NO:2. In some embodiments, the C-terminal fragment of Cas9 or the N-terminal fragment of Cas9 includes Ala / Cys, Ser / Cys, or Thr / Cys mutations at residues corresponding to amino acids S303, T310, T313, S355, A456, S460, A463, T466, S469, T472, T474, C574, S577, A589, or S590 in the numbering of SEQ ID NO:2. In some embodiments, the composition further includes a single guide RNA (sgRNA) or a polynucleotide encoding the same. In some embodiments, the first and second polynucleotides are linked. In some embodiments, the first and second polynucleotides are expressed separately. In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is wild-type TadA or TadA7.10. In some embodiments, the deaminase is a TadA dimer. In some embodiments, the TadA dimer comprises wild-type TadA and TadA 7.10. In some embodiments, the fusion protein comprises a nuclear localization signal (NLS). In some embodiments, the N-terminal fragment of Cas9 or the C-terminal fragment of Cas9 is linked to an NLS. In some embodiments, the NLS is a bipartite NLS. In some embodiments, the N-terminal fragment of Cas9 and the fusion protein are linked to form a base editor protein comprising a deaminase and SpCas9. In some embodiments, the C-terminal fragment of Cas9 and the fusion protein are linked to form a base editor protein comprising a deaminase and SpCas9. In some embodiments, SpCas9 has nickase activity or is catalytically inactive.

[0008] In some embodiments, provided herein is a composition comprising the fusion protein disclosed herein and the N-terminal fragment of Cas9. In some embodiments, provided herein is a composition comprising the fusion protein disclosed herein and the C-terminal fragment of Cas9. In some embodiments, the N-terminal fragment of Cas9 or the C-terminal fragment of Cas9 and the deaminase are linked by a linker. In some embodiments, the linker is a peptide linker.

[0009] In some embodiments, the present specification provides a vector comprising the first and second polynucleotides disclosed herein.In some embodiments, the vector comprises a promoter.In some embodiments, the promoter is a constitutive promoter.In some embodiments, the constitutive promoter is a CMV or CAG promoter.In some embodiments, the vector is selected from the group consisting of retroviral vector, adenoviral vector, lentiviral vector, herpes virus vector, and adeno-associated virus vector.In some embodiments, the vector is an adeno-associated virus vector.

[0010] In some embodiments, provided herein is a cell comprising the composition disclosed herein or the vector disclosed herein. In some embodiments, the cell is a mammalian cell.

[0011] In some aspects, provided herein are reconstituted A→G base editor proteins comprising a Cas9 domain that includes an Ala / Cys, Ser / Cys, or Thr / Cys mutation, in some embodiments, the mutation is at a residue corresponding to amino acid S303, T310, T313, S355, A456, S460, A463, T466, S469, T472, T474, C574, S577, A589, or S590 of SpCas9.

[0012] In some embodiments, (a) starting at the N-terminus of Cas9, the sequence is A292 to G36 of Cas9 as numbered in SEQ ID NO:2.5 (b) an N-terminal fragment of Cas9, which is a contiguous sequence ending at position A292 to G36 of Cas9 in the numbering of SEQ ID NO: 2, fused to a split intein-N; 5 and a C-terminal fragment of Cas9, wherein the C-terminal fragment is a contiguous sequence beginning at position A and ending at the C-terminus of Cas9, fused to a split intein-C.

[0013] In some embodiments, provided herein are compositions comprising one or more polynucleotides encoding (a) an N-terminal fragment of Cas9, the N-terminal fragment being a contiguous sequence beginning at the N-terminus of Cas9 and ending at amino acid 302, 309, 312, 354, 455, 459, 462, 465, 471, 473, 576, 588, or 589 of Cas9, as numbered in SEQ ID NO:2, fused to a split intein-N; and (b) a C-terminal fragment of Cas9, the C-terminal fragment being a contiguous sequence beginning at amino acid 303, 310, 313, 355, 456, 460, 463, 466, 472, 474, 577, 589, or 590 of Cas9, as numbered in SEQ ID NO:2, and ending at the C-terminus of Cas9, fused to a split intein-C.

[0014] In some embodiments, the N-terminal fragment of Cas9 or the C-terminal fragment of Cas9 is linked to a nuclear localization signal (NLS). In some embodiments, the N-terminal fragment of Cas9 and the C-terminal fragment of Cas9 are both linked to an NLS. In some embodiments, the NLS is a bipartite NLS. In some embodiments, the N-terminal fragment of Cas9 and the C-terminal fragment of Cas9 are linked to form SpCa9. In some embodiments, SpCas9 has nickase activity or is catalytically inactive.

[0015] In some embodiments, provided herein is a composition comprising in (a) an N-terminal fragment of Cas9 and in (b) a C-terminal fragment of Cas9 as disclosed herein.

[0016] In some embodiments, the present specification provides a vector comprising one or more polynucleotides disclosed herein.In some embodiments, the vector comprises a promoter.In some embodiments, the promoter is a constitutive promoter.In some embodiments, the constitutive promoter is a CMV or CAG promoter.In some embodiments, the vector is selected from the group consisting of retroviral vector, adenoviral vector, lentiviral vector, herpes virus vector, and adeno-associated virus vector.In some embodiments, the vector is an adeno-associated virus vector.

[0017] In some embodiments, provided herein is a cell comprising the composition disclosed herein or the vector disclosed herein. In some embodiments, the cell is a mammalian cell.

[0018] In some embodiments, provided herein are Cas9 variant polypeptides that include Ala / Cys, Ser / Cys, or Thr / Cys mutations. In some embodiments, provided herein are Cas9 variant polypeptides that include a Cys residue at amino acid 303, 310, 313, 355, 456, 460, 463, 466, 472, or 474.

[0019] In some embodiments, provided herein is a method for delivering a base editor system to a cell, the method comprising: delivering a cell to a target cell a first polynucleotide encoding a fusion protein comprising a deaminase and an N-terminal fragment of Cas9, wherein the N-terminal fragment of Cas9 begins at the N-terminus of Cas9 and is between A292 and G36 of Cas9 as numbered in SEQ ID NO:2; 5 wherein the N-terminal fragment of Cas9 is fused to the split intein-N; (b) a second polynucleotide encoding a C-terminal fragment of Cas9, wherein the C-terminal fragment of Cas9 is a contiguous sequence ending at position A292 to G36 of Cas9 in the numbering of SEQ ID NO:2.5 and terminating at the C-terminus of Cas9, wherein the C-terminal fragment of Cas9 is fused to the split intein-C; and (c) a single guide RNA (sgRNA) or a polynucleotide encoding the same.

[0020] In some embodiments, provided herein is a method for delivering a base editor system to a cell, the method comprising: administering to the cell (a) a first polynucleotide encoding a fusion protein comprising an N-terminal fragment of Cas9, the N-terminal fragment of Cas9 beginning at the N-terminus of Cas9 and ranging from A292 to G36 of Cas9 as numbered in SEQ ID NO:2; 5 (b) a second polynucleotide encoding a C-terminal fragment of Cas9 and a deaminase, wherein the C-terminal fragment of Cas9 is a contiguous sequence terminating at position A292 to G36 of Cas9 in the numbering of SEQ ID NO:2. 5 and terminating at the C-terminus of Cas9, wherein the C-terminal fragment of Cas9 is fused to the split intein-C; and (c) a single guide RNA (sgRNA) or a polynucleotide encoding the same.

[0021] In some embodiments, provided herein is a method for delivering a base editor system to a cell, the method comprising: delivering a cell to a host cell a first polynucleotide encoding a fusion protein comprising a deaminase and an N-terminal fragment of Cas9, the N-terminal fragment of Cas9 beginning at the N-terminus of Cas9 and spanning A292 to G36 of Cas9 as numbered in SEQ ID NO:2; 5and (b) a second polynucleotide encoding a C-terminal fragment of Cas9, the C-terminal fragment of Cas9 being a contiguous sequence ending at positions A292 to G36 of Cas9 in the numbering of SEQ ID NO:2. 5 , a contiguous sequence beginning at positions F445 to K483, or E565 to T637, and terminating at the C-terminus of Cas9, wherein the N-terminal residue of the C-terminal fragment of Cas9 is Cys substituted for Ala, Ser, or Thr, and the C-terminal fragment of Cas9 is fused to a split intein-C; a second polynucleotide; and (c) a single guide RNA (sgRNA) or a polynucleotide encoding the same.

[0022] In some embodiments, provided herein is a method for delivering a base editor system to a cell, the method comprising: delivering to the cell (a) a first polynucleotide encoding a fusion protein comprising an N-terminal fragment of Cas9, wherein the N-terminal fragment of Cas9 begins at the N-terminus of Cas9 and is between A292 and G36 of Cas9 as numbered in SEQ ID NO:2; 5 , F445 to K483, or E565 to T637 of Cas9, wherein the N-terminal fragment of Cas9 is fused to the split intein-N; (b) a second polynucleotide encoding a C-terminal fragment of Cas9 and a deaminase, wherein the C-terminal fragment of Cas9 is A292 to G36 of Cas9 in the numbering of SEQ ID NO:2. 5 , a contiguous sequence beginning at positions F445 to K483, or E565 to T637, and terminating at the C-terminus of Cas9, wherein the N-terminal residue of the C-terminal fragment of Cas9 is Cys substituted for Ala, Ser, or Thr, and wherein the C-terminal fragment of Cas9 is fused to a split intein-C; and (c) a single guide RNA (sgRNA) or a polynucleotide encoding the same.

[0023] In some embodiments, the sgRNA is complementary to the target polynucleotide. In some embodiments, the target polynucleotide is present in the genome of an organism. In some embodiments, the organism is an animal, a plant, or a bacterium. In some embodiments, the first polynucleotide, the second polynucleotide, and / or the polynucleotide encoding them are contacted with the cell via a vector. In some embodiments, the vector is selected from the group consisting of a retroviral vector, an adenoviral vector, a lentiviral vector, a herpes virus vector, and an adeno-associated virus vector. In some embodiments, the vector is an adeno-associated virus vector. In some embodiments, the C-terminal fragment of Cas9 or the N-terminal fragment of Cas9 comprises an Ala / Cys, Ser / Cys, or Thr / Cys mutation at a residue corresponding to amino acid S303, T310, T313, S355, A456, S460, A463, T466, S469, T472, T474, C574, S577, A589, or S590 in the numbering of SEQ ID NO:2. In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is TadA or a variant thereof. In some embodiments, the deaminase is wild-type TadA or Tad7.10. In some embodiments, the deaminase is a TadA dimer. In some embodiments, the TadA dimer comprises wild-type TadA and TadA7.10. In some embodiments, the N-terminal fragment of Cas9 or the C-terminal fragment of Cas9 comprises an NLS. In some embodiments, the N-terminal fragment of Cas9 and the C-terminal fragment of Cas9 both comprise an NLS. In some embodiments, the NLS is a bipartite NLS. In some embodiments, the N-terminal fragment of Cas9 and the C-terminal fragment of Cas9 are linked to form SpCa9. In some embodiments, SpCas9 has nickase activity or is catalytically inactive.

[0024] In some embodiments, a polynucleotide encoding a fusion protein is provided herein, wherein the fusion protein comprises a deaminase and an N-terminal fragment of Cas9, the N-terminal fragment of Cas9 beginning at the N-terminus of Cas9 and spanning A292 to G36 of Cas9 in the numbering in SEQ ID NO:2. 5 and the N-terminal fragment of Cas9 is fused to the split intein-N. In some embodiments, provided herein is a polynucleotide encoding a fusion protein, wherein the fusion protein comprises a deaminase and an N-terminal fragment of Cas9, wherein the N-terminal fragment of Cas9 is a contiguous sequence beginning at the N-terminus of Cas9 and terminating at positions F445 to K483 of Cas9 as numbered in SEQ ID NO:2, and the N-terminal fragment of Cas9 is fused to the split intein-N. In some embodiments, provided herein is a polynucleotide encoding a fusion protein, wherein the fusion protein comprises a deaminase and an N-terminal fragment of Cas9, wherein the N-terminal fragment of Cas9 is a contiguous sequence beginning at the N-terminus of Cas9 and terminating at positions E565 to T637 of Cas9 as numbered in SEQ ID NO:2, and the N-terminal fragment of Cas9 is fused to the split intein-N. In some embodiments, a polynucleotide encoding a fusion protein is provided herein, wherein the fusion protein comprises a deaminase and a C-terminal fragment of Cas9, the C-terminal fragment of Cas9 being A292 to G36 of Cas9 in the numbering in SEQ ID NO:2. 5and terminating at the C-terminus of Cas9, wherein the C-terminal fragment of Cas9 is fused to the split intein-C. In some embodiments, provided herein is a polynucleotide encoding a fusion protein, wherein the fusion protein comprises a deaminase and a C-terminal fragment of Cas9, wherein the C-terminal fragment of Cas9 is a contiguous sequence beginning at positions F445-K483 of Cas9 in the numbering of SEQ ID NO:2 and terminating at the C-terminus of Cas9, wherein the C-terminal fragment of Cas9 is fused to the split intein-C. In some embodiments, provided herein is a polynucleotide encoding a fusion protein, wherein the fusion protein comprises a deaminase and a C-terminal fragment of Cas9, wherein the C-terminal fragment of Cas9 is a contiguous sequence beginning at positions E565-T637 of Cas9 in the numbering of SEQ ID NO:2 and terminating at the C-terminus of Cas9, wherein the C-terminal fragment of Cas9 is fused to the split intein-C. In one embodiment, a polynucleotide encoding a fusion protein is provided herein, wherein the fusion protein comprises a deaminase and a C-terminal fragment of Cas9, the C-terminal fragment of Cas9 being A292 to G36 of Cas9 according to the numbering in SEQ ID NO:2. 5 , F445-K483, or E565-T637, and terminating at the C-terminus of Cas9, wherein the N-terminal residue of the Cas9 C-terminal fragment is Cys substituted for Ala, Ser, or Thr, and the Cas9 C-terminal fragment is fused to split intein C.

[0025] In some embodiments, the C-terminal fragment of Cas9 or the N-terminal fragment of Cas9 comprises an Ala / Cys, Ser / Cys, or Thr / Cys mutation. In some embodiments, the mutation is at a residue corresponding to amino acid S303, T310, T313, S355, A456, S460, A463, T466, S469, T472, T474, C574, S577, A589, or S590 in the numbering in SEQ ID NO:2. In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is TadA or a variant thereof. In some embodiments, the deaminase is wild-type TadA or Tad7.10. In some embodiments, the fusion protein comprises two deaminases linked together. In some embodiments, the fusion protein comprises both wild-type TadA and TadA7.10. In some embodiments, the fusion protein comprises an NLS. In some embodiments, the NLS is a bipartite NLS. In some embodiments, the N-terminal fragment of Cas9 or the C-terminal fragment of Cas9 comprises the amino acid sequence of SpCas9. In some embodiments, the N-terminal fragment of Cas9 or the C-terminal fragment of Cas9 comprises one or more amino acid substitutions associated with reduced nuclease activity.

[0026] In some embodiments, provided herein is an N-terminal fragment of a Cas9 protein including up to amino acid 302, 309, 312, 354, 455, 459, 462, 465, 471, or 473 fused to a split intein-N. In some embodiments, provided herein is a C-terminal protein fragment of a Cas9 protein, wherein the N-terminal amino acid of the C-terminal fragment is a Cys substitution at amino acid 303, 310, 313, 355, 456, 460, 463, 466, 472, or 474, fused to a split intein-C. In some embodiments, provided herein is a polynucleotide encoding a fragment of an A→G base editor fusion protein, the fusion protein comprising one or more deaminases and an N-terminal fragment of Cas9, the N-terminal fragment fused to a split intein-N. In some embodiments, provided herein is a polynucleotide encoding a fragment of an A→G base editor fusion protein, the fusion protein comprising one or more deaminases and a C-terminal fragment of Cas9, the C-terminal fragment being fused to a split intein-C. In some embodiments, provided herein is a protein fragment of an A→G base editor fusion protein, the fusion protein comprising one or more deaminases and an N-terminal fragment of Cas9, the N-terminal fragment being fused to a split intein-N. In some embodiments, provided herein is a protein fragment of an A→G base editor fusion protein, the fusion protein comprising one or more deaminases and a C-terminal fragment of Cas9, the C-terminal fragment being fused to a split intein-C.

[0027] In some embodiments, provided herein are compositions comprising first and second polynucleotides, each encoding a fragment of an A→G base editor fusion protein comprising one or more deaminases and Cas9, where the first polynucleotide encodes an N-terminal fragment of Cas9 fused to a split intein-N and the second polynucleotide encodes a C-terminal fragment of Cas9 fused to a split intein-C. In some embodiments, provided herein are compositions comprising N- and C-terminal fragments of an A→G base editor fusion protein comprising one or more deaminases and Cas9, where the N-terminal fragment comprises a fragment of SpCas9 fused to a split intein-N and the C-terminal fragment comprises the remainder of SpCas9 fused to a split intein-C.

[0028] In some embodiments, provided herein are methods for delivering a base editor system to a cell, the methods comprising contacting a cell with first and second polynucleotides each encoding a fragment of an A→G base editor fusion protein comprising one or more deaminases and Cas9, where the first polynucleotide encodes an N-terminal fragment of Cas9 fused to a split intein-N, the second polynucleotide encodes a C-terminal fragment of Cas9 fused to a split intein-C, and either the first or second polynucleotide encodes a single guide RNA. In some embodiments, provided herein are methods for delivering a base editor system to a cell, the methods comprising contacting a cell with an N- and C-terminal fragment of an A→G base editor fusion protein comprising one or more deaminases and SpCas9, and a guide RNA, where the N-terminal fragment comprises a fragment of SpCas9 fused to a split intein-N, and the C-terminal fragment comprises the remainder of SpCas9 fused to a split intein-C. In some embodiments, provided herein are methods for editing a target polynucleotide in a cell, the method comprising contacting a cell with first and second polynucleotides, each encoding a fragment of an A→G Base Editor fusion protein comprising one or more deaminases and Cas9, where the first polynucleotide encodes an N-terminal fragment of Cas9 fused to a split intein-N, the second polynucleotide encodes a C-terminal fragment of Cas9 fused to a split intein-C, and either the first or second polynucleotide encodes a single guide RNA, and expressing the encoded proteins and single guide RNA in the cell.

[0029] Other features and advantages of the invention will become apparent from the detailed description and claims.

[0030] [Definition] 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 those skilled in the art with general definitions of many terms used in the present invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified.

[0031] "Adenosine deaminase" refers to a polypeptide or fragment thereof that can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine or deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) can be from any organism, such as bacteria. In some embodiments, the deaminase or deaminase domain is a variant of a natural deaminase from an organism. In some embodiments, the deaminase or deaminase domain is not naturally occurring. For example, in some embodiments, the deaminase or deaminase domain has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a naturally occurring deaminase. In some embodiments, the adenosine deaminase is derived from a bacterium, such as, for example, E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentus. In some embodiments, the adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase is E. coli TadA (ecTadA) deaminase or a fragment thereof.

[0032] For example, a truncated ecTadA may have one or more N-terminal amino acids deleted compared to full-length ecTadA. In some embodiments, a truncated ecTadA may have 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 deleted compared to full-length ecTadA. In some embodiments, a truncated ecTadA may have 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 deleted compared to full-length ecTadA. In some embodiments, the ecTadA deaminase does not include an N-terminal methionine. In some embodiments, the TadA deaminase is an N-terminal truncated TadA. In certain embodiments, the TadA is any one of the TadAs described in PCT / US2017 / 045381, the entirety of which is incorporated herein by reference.

[0033] In certain embodiments, the adenosine deaminase comprises the following amino acid sequence: MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPT AHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKT GAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD This is called the "TadA reference sequence."

[0034] In some embodiments, the TadA deaminase is a full-length E. coli TadA deaminase. For example, in certain embodiments, the adenosine deaminase comprises the following amino acid sequence: MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEG WNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEI KAQKKAQSSTD

[0035] However, it should be understood that additional adenosine deaminases useful in the present application will be apparent to those skilled in the art and are within the scope of the present disclosure.For example, the adenosine deaminase may be a homolog of adenosine deaminase acting on tRNA (AD AT).Exemplary AD AT homologs include, but are not limited to:

[0036] Staphylococcus aureus TadA: MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAH AEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCS GS LMNLLQQS NFNHRAIVDKG VLKE AC S TLLTTFFKNLRANKKS TN

[0037] Bacillus subtilis TadA: MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEML VIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGC S GTLMN LLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE

[0038] Salmonella typhimurium (S. typhimurium) TadA: MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEG WNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIG RVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIK ALKKADRAEGAGPAV

[0039] Shewanella putrefaciens (S. putrefaciens) TadA: MDE YWMQVAMQM AEKAEAAGE VPVGA VLVKDGQQIATGYNLS IS QHDPT AHAEI LCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGT VVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE

[0040] Haemophilus influenzae F3031 (H. influenzae) TadA: MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQS DPT ΑΗ AEIIALRNG AKNIQN YRLLNS TLY VTLEPCTMC AG AILHS RIKRLVFG AS D YK TGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSD K

[0041] Caulobacter crescentus (C. crescentus) TadA: MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAH DPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADD PKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI

[0042] Geobacter sulfurreducens (G. sulfurreducens) TadA: MSSLKKTPIRDDAYWMGKAIREAAKAAAARDEVPIGAVIVRDGAVIGRGHNLREGSN DPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDP KGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALF IDERKVPPEP

[0043] TadA7.10 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0044] By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

[0045] By "alteration" is meant a change in the structure, expression level or activity of a gene or polypeptide as detected by standard art known methods as described herein. As used herein, alteration (e.g., increase or decrease) includes a 10% change in expression level, a 25% change, a 40% change, a 50% or greater change in expression level.

[0046] "Analog" refers to a molecule that is not identical but has similar functional or structural characteristics. For example, a polypeptide analog has certain sequence modifications that enhance the function of the analog compared to the native polypeptide while retaining at least some of the biological activity of the corresponding native polypeptide. Such modifications can increase the protease resistance, membrane permeability, or half-life of the analog, for example, without changing the polynucleotide binding activity. In another example, a polynucleotide analog retains the biological activity of the corresponding native polynucleotide while having certain modifications that enhance the function of the analog compared to the native polynucleotide. Such modifications can increase the affinity of the polynucleotide for DNA, half-life, and / or nuclease resistance, and the analog can include non-natural nucleotides or amino acids.

[0047] "Base editor (BE)" or "nucleobase editor (NBE)" refers to an agent that binds to a polynucleotide and has nucleobase modifying activity. In one embodiment, the agent is a fusion protein that includes a domain with base editing activity, i.e., a domain that can modify a base (e.g., A, T, C, G, U) in a nucleic acid molecule (e.g., DNA). In some embodiments, the domain with base editing activity can deaminate a base in a nucleic acid molecule. In an embodiment, the base editor can deaminate a base in a DNA molecule. In an embodiment, the base editor can deaminate a cytosine (C) or adenosine in DNA. In an embodiment, the base editor can deaminate cytosine (C) and adenosine (A) in DNA. In an embodiment, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the base editor is an adenosine base editor (ABE) and a cytidine base editor (CBE). In some embodiments, the base editor is a nuclease-inactive Cas9 (dCas9) fused to an adenosine deaminase. In some embodiments, the Cas9 is a circular permutant Cas9 (e.g., spCas9 or saCas9). Circular permutant Cas9s are known in the art and are described, for example, in Oakes et al., Cell 176, 254-267, 2019. In some embodiments, the base editor is fused to an inhibitor of base excision repair, such as a UGI 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. In other embodiments, the base editor is an abasic base editor.

[0048] The nucleobase component and the polynucleotide programmable nucleotide binding component of the base editor system can be covalently or non-covalently linked to each other. For example, in some embodiments, the deaminase domain can be targeted to a target nucleotide sequence by the polynucleotide programmable nucleotide binding domain. In some aspects, the polynucleotide programmable nucleotide binding domain can be fused or linked to the deaminase domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the deaminase domain to a target nucleotide sequence by interacting or binding non-covalently with the deaminase domain. For example, in some embodiments, the nucleobase editing component, e.g., the deaminase component, can include an additional heterologous moiety or domain that can interact, associate, or form a complex with an additional heterologous moiety or domain that is part of the polynucleotide programmable nucleotide binding domain. In some embodiments, the additional heterologous moiety can bind, interact, associate, or form a complex with a polypeptide. In some embodiments, the additional heterologous moiety can bind, interact, associate, or form a complex with a polynucleotide. In some embodiments, the additional heterologous moiety can bind to a guide polynucleotide. In some embodiments, the additional heterologous moiety can be attached to a polypeptide linker. In some embodiments, the additional heterologous moiety can be attached to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a steryl alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0049] The base editor system may further comprise a guide polynucleotide component. It should be understood that the components of the base editor system may be bound to each other via covalent bonds, non-covalent interactions, or any combination of those bonds and interactions. In some embodiments, the deaminase domain may be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the nucleic acid base editing component of the base editor system, e.g., the deaminase component, may comprise an additional heterologous moiety or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) that may interact, bind, or form a complex with a portion or segment (e.g., a polynucleotide motif) of the guide polynucleotide. In some embodiments, the additional heterologous moiety or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) may be fused or linked to the deaminase domain. In some embodiments, the additional heterologous moiety may bind, interact, associate, or form a complex with a polypeptide. In some embodiments, the additional heterologous moiety may bind, interact, associate, or form a complex with a polynucleotide. In some embodiments, the additional heterologous moiety may bind to a guide polynucleotide. In some embodiments, the additional heterologous moiety can be attached to a polypeptide linker. In some embodiments, the additional heterologous moiety can be attached to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0050] In some embodiments, the base editor system may include one or more proteins, fusion proteins, polypeptides, or polynucleotides encoding them. In some embodiments, the base editor system may include a first polynucleotide encoding a fusion protein comprising a deaminase and an N-terminal fragment of napDNAbp, and a second polynucleotide encoding a C-terminal fragment of napDNAbp. For example, in certain embodiments, the N-terminal fragment of napDNAbp may be fused to intein-N and the C-terminal fragment of napDNAbp may be fused to intein-C, such that the N-terminal fragment and the C-terminal fragment of napDNAbp are reconstituted to form the base editor protein.

[0051] In certain embodiments, the base editor system can further comprise an inhibitor of a base excision repair (BER) component. In certain embodiments, the base editor system can further comprise an inhibitor of a base excision repair (BER) component. It should be understood that the components of the base editor system can be bound to each other via covalent bonds, non-covalent interactions, or any combination of those bonds and interactions. The inhibitor of the BER component can comprise a base excision repair inhibitor. In certain embodiments, the inhibitor of base excision repair can be a uracil DNA glycosylase inhibitor (UGI). In certain embodiments, the inhibitor of base excision repair can be an inosine base excision repair inhibitor. In certain embodiments, the inhibitor of base excision repair can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain. In certain embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to an inhibitor of base excision repair. In certain embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain and an inhibitor of base excision repair. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the base excision repair inhibitor to the target nucleotide sequence by non-covalently interacting with the base excision repair inhibitor or by associating with the base excision repair inhibitor.For example, in some embodiments, the inhibitor of base excision repair component can include an additional heterologous moiety or domain that can interact, associate, or complex with the additional heterologous moiety or domain that is part of the polynucleotide programmable nucleotide binding domain.In some aspects, the base excision repair inhibitor can be targeted to the target nucleotide sequence by the guide polynucleotide.For example, in some embodiments, the inhibitor of base excision repair may include an additional heterologous moiety or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) that can interact, associate, or complex with a portion or segment of the guide polynucleotide (e.g., a polynucleotide motif). In some embodiments, the additional heterologous moiety or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) of the guide polynucleotide may be fused or linked to the inhibitor of base excision repair. In some embodiments, the additional heterologous moiety can bind, interact, associate, or complex with the polynucleotide. In some embodiments, the additional heterologous moiety can be linked to the guide polynucleotide. In some embodiments, the additional heterologous moiety can be linked to a polypeptide linker. In some embodiments, the additional heterologous moiety can be linked to a polynucleotide linker. The additional heterologous moiety may be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0052] "Base editing activity" means acting to chemically change a base in a polynucleotide. In one embodiment, a first base is converted to a second base. In another embodiment, a base is excised from a polynucleotide. In one embodiment, the base editing activity is a cytidine deaminase activity, e.g., converting a target C·G to T·A. In another embodiment, the base editing activity is an adenosine deaminase activity, e.g., converting A·T to G·C.

[0053] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease that includes the Cas9 protein or a fragment thereof (e.g., a protein that includes an active, inactive, or partially active DNA cleavage domain of Cas9, and / or a gRNA-binding domain of Cas9). Cas9 nuclease is sometimes referred to as casn1 nuclease or CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. CRISPR is an adaptive immune system that provides defense against mobile genetic elements (viruses, transposable elements, conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of the pre-crRNA requires a transcoding small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. The tracrRNA guides the processing of the pre-crRNA by ribonuclease 3. Cas9 / crRNA / tracrRNA then endonucleolytically cleaves the linear or circular dsDNA target that is complementary to the spacer. The target strand that is not complementary to the crRNA is first endonucleolytically cleaved and then exonucleolytically trimmed 3'-5'. Single guide RNAs ("sgRNAs", or simply "gRNAs") can be engineered to incorporate both crRNA and tracrRNA aspects into a single RNA species. See, for example, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821(2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif (the PAM or protospacer adjacent motif) in the CRISPR repeats, helping to distinguish self from non-self.The sequence and structure of Cas9 nuclease are well known to those skilled in the art (e.g. “Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C, Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma CM., Gonzales K., Chao See, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E., Nature 471:602-607(2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821(2012), the entire contents of which are incorporated by reference herein. Cas9 orthologs have been described in a variety of species, including, but not limited to, S. pyogenes and S. thermophilus.Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, including Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference.

[0054] Nuclease-inactivated Cas9 protein may be interchangeably referred to as "dCas9" protein (for nuclease-"dead" Cas9) or catalytically inactive Cas9. Methods for generating Cas9 protein (or fragments thereof) with an inactive DNA cleavage domain are known (see, e.g., Jinek et al, Science. 337:816-821(2012); Qi et al, "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression"(2013) Cell. 28; 152(5): 1173-83, the contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, and the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can suppress the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al, Science. 337:816-821(2012); Qi et al, Cell. 28;152(5): 1173-83 (2013)). In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase, referred to as 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 of the following two Cas9 domains: (1) the gRNA binding domain of Cas9; (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as "Cas9 variants." The Cas9 variants share homology with Cas9 or a fragment thereof.For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to a wild-type Cas9. In some embodiments, the Cas9 mutant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In certain embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of the corresponding wild-type Cas9.

[0055] In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length. In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_17053.1, nucleotide and amino acid sequences are as follows). TIFF0007657711000001.tif161169 (single underline: HNH domain; double underline: RuvC domain)

[0056] In certain embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence: TIFF0007657711000002.tif157169 (single underline: HNH domain; double underline: RuvC domain)

[0057] 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). TIFF0007657711000003.tif156168 (single underline: HNH domain; double underline: RuvC domain)

[0058] In some embodiments, Cas9 is Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Cas9 from 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 any other organism.

[0059] In some embodiments, the dCas9 corresponds to or comprises a portion or all of a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises D10A and H840A mutations or corresponding mutations in another Cas9. In some embodiments, the dCas9 comprises the following amino acid sequence of dCas9 (D10A and H840A): TIFF0007657711000004.tif157168 (single underline: HNH domain; double underline: RuvC domain)

[0060] In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 in the amino acid sequence provided above, or the residue at the corresponding position in any of the amino acid sequences provided herein, remains a histidine.

[0061] In other embodiments, dCas9 variants are provided that have mutations other than D10A and H840A, e.g., that result in nuclease-inactivated Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs of dCas9 are provided that have at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity. In some embodiments, variants of dCas9 are provided that have amino acid sequences that are shorter or longer by about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids or more.

[0062] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but only one or more fragments thereof.

[0063] Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and further suitable sequences of Cas9 domains and fragments will be apparent to those of skill in the art.

[0064] In some embodiments, Cas9 is Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); or Neisseria meningitidis (NCBI Ref: YP_002342100.1).

[0065] It should be understood that additional Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including variants and homologs thereof, are within the scope of this disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is 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.

[0066] Exemplary catalytically inactive Cas9 (dCas9):

[0067] Exemplary catalytic Cas9 nickases (nCas9):

[0068] Exemplary catalytically active Cas9:

[0069] In some embodiments, Cas9 refers to Cas9 from Archaea (e.g., Nanoarchaea), which constitutes the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, Cas protein refers to CasX or CasY, e.g., as described in Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genome-resolved metagenomics, many CRISPR-Cas systems have been identified, including the first reported Cas9 in the Archaea domain of life. This divergent Cas9 protein was discovered as part of an active CRISPR-Cas system in the little-studied Nanoarchaea. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, have been discovered, which are among the most compact systems discovered to date. In some embodiments, Cas9 represents CasX or a variant of CasX. In some embodiments, Cas9 represents CasY or a variant of CasY. It should be understood that other RNA-guided DNA binding proteins may also be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0070] In some embodiments, napDNAbp is a Cas9 domain, such as nuclease-active Cas9, Cas9 nickase (nCas9), or nuclease-inactive Cas9 (dCas9).Non-limiting examples of nucleic acid programmable DNA binding proteins include Cas9 (e.g., dCas9 and nCas9), type II Cas effector proteins, type V Cas effector proteins, type VI Cas effector proteins, CARF, DinG, homologs thereof, or modified or engineered versions thereof.Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, even if not 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; 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).

[0071] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein can be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp is a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to any CasX or CasY protein described herein. It is understood that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.

[0072] CasX (uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53) >tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx protein OS = Sulfolobus islandicus (strain HVE10 / 4) GN = SiH_0402 PE=4 SV=1 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTSNIILPLSGNDKNPWTETLKCYNFPTTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0073] >tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS = Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4 SV=1 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTSNIILPLSGNDKNPWTETLKCYNFPTTTVALSEVFKNFSQVKECEEVSAPSFVKPFEYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0074] CasY (ncbi.nlm.nih.gov / protein / APG80656.1) >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacterium]

[0075] The term "CRISPR-Cas domain" or "CRISPR-Cas DNA binding domain" refers to an RNA-guided protein comprising a CRISPR-associated (Cas) protein or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of a Cas protein, and / or a gRNA-binding domain of a Cas protein). CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In some CRISPR systems, a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas protein are required for correct processing of pre-crRNA. The tracrRNA serves as a guide for ribonuclease 3-assisted processing of pre-crRNA. Cas9 / crRNA and / or tracrRNA then endonucleolytically cleave linear or circular dsDNA targets complementary to the spacer. In nature, both RNAs may be required for DNA binding and cleavage. However, single guide RNAs ("sgRNAs" or simply "gNRAs") can be engineered to incorporate both crRNA and tracrRNA aspects into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas proteins recognize short motifs (PAM or protospacer adjacent motifs) in CRISPR repeats and help distinguish self from non-self. CRISPR-Cas proteins include, but are not limited to, Cas9, CasX, CasY, Cpf1, C2c1, and C2c3 or active fragments thereof. Additional suitable CRISPR-Cas proteins and sequences will be apparent to those of skill in the art based on this disclosure.

[0076] Nuclease-inactivated CRISPR-Cas proteins can be interchangeably referred to as "dCas" proteins (for nuclease "dead" Cas) or catalytically inactive Cas. Methods for generating Cas 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 which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains suppress the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28;152(5):1173-83 (2013)). In some embodiments, the Cas nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., the Cas is a nickase, referred to as a "nCas" protein (for "nickase" Cas). Cas variants share homology with CRISPR-Cas proteins or fragments thereof. For example, a Cas variant has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to a wild-type CRISPR-Cas protein.In some embodiments, the Cas variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to the wild-type CRISPR-Cas protein. In some embodiments, the Cas variant comprises a fragment of a CRISPR-Cas protein (e.g., a gRNA programmable DNA binding domain or a DNA cleavage domain), such that the fragment has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to a corresponding fragment of a wild-type CRISPR-Cas protein. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of the corresponding wild-type CRISPR-Cas protein. In some embodiments, the fragment is at least 100 amino acids in length. In certain embodiments, fragments are at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0077] In this disclosure, "comprises," "comprising," "containing," "having," and the like can have the meanings given to them in U.S. Patent Law, and can mean "includes," "including," and the like, and "consisting essentially of" or "consists essentially" likewise have the meanings given to them in U.S. Patent Law, and the terms are open-ended, allowing for other entities than those described, but excluding prior art embodiments, so long as the basic or novel characteristics of the described entity are not altered by the other entities than those described.

[0078] "Cytidine deaminase" refers to a polypeptide or fragment thereof capable of catalyzing a deamination reaction that converts an amino group to a carbonyl group. In one embodiment, the cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine. PmCDA1 from Petromyzon marinus (Petromyzon marinus cytosine deaminase 1, "PmCDA1"), AID (Activation-Induced Cytidine Deaminase; AICDA) from mammals (e.g., human, pig, cow, horse, monkey, etc.), and APOBEC are exemplary cytidine deaminases.

[0079] The nucleotide sequence and amino acid sequence of PmCDA1 and the nucleotide sequence and amino acid sequence of the CDS of human AID are shown below.

[0080] >tr|A5H718|A5H718_PETMA Cytosine deaminase OS=Petromyzon marinus OX=7757 PE=2 SV=1 MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSG TERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLK IWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKT LKRAEKRRSELSIMIQVKILHTTKSPAV

[0081] >EF094822.1 Petromyzon marinus isolate PmCDA.21 cytosine deaminase mRNA, complete cds TGACACGACACAGCCGTGTATATGAGGAAGGGTAGCTGGATGGGGGGGGGGGGAATACGTTCAGAGAGGA CATTAGCGAGCGTCTTGTTGGTGGCCTTGAGTCTAGACACCTGCAGACATGACCGACGCTGAGTACGTGA GAATCCATGAGAAGTTGGACATCTACACGTTTAAGAAACAGTTTTTCAACAACAAAAAATCCGTGTCGCA TAGATGCTACGTTCTCTTTGAATTAAAACGACGGGGTGAACGTAGAGCGTGTTTTTGGGGCTATGCTGTG AATAAACCACAGAGCGGGACAGAACGTGGAATTCACGCCGAAATCTTTAGCATTAGAAAAGTCGAAGAAT ACCTGCGCGACAACCCCGGACAATTCACGATAAATTGGTACTCATCCTGGAGTCCTTGTGCAGATTGCGC TGAAAAGATCTTAGAATGGTATAACCAGGAGCTGCGGGGGAACGGCCACACTTTGAAAATCTGGGCTTGC AAACTCTATTACGAGAAAAATGCGAGGAATCAAATTGGGCTGTGGAACCTCAGAGATAACGGGGTTGGGT TGAATGTAATGGTAAGTGAACACTACCAATGTTGCAGGAAAATATTCATCCAATCGTCGCACAATCAATT GAATGAGAATAGATGGCTTGAGAAGACTTTGAAGCGAGCTGAAAAACGACGGAGCGAGTTGTCCATTATG ATTCAGGTAAAAATACTCCACACCACTAAGAGTCCTGCTGTTTAAGAGGCTATGCGGATGGTTTTC

[0082] >tr|Q6QJ80|Q6QJ80_HUMAN Activation-induced cytidine deaminase OS=Homo sapiens OX=9606 GN=AICDA PE=2 SV=1 MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELL FLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRK AEPEGLRRLHRAGVQIAIMTFKAPV

[0083] >NG_011588.1:5001-15681 Homo sapiens activation induced cytidine deaminase (AICDA), RefSeqGene (LRG_17) on chromosome 12 AGAGAACCATCATTAATTGAAGTGAGATTTTTCTGGCCTGAGACTTGCAGGGAGGCAAGAAGACACTCTG GACACCACTATGGACAGGTAAAGAGGCAGTCTTCTCGTGGGTGATTGCACTGGCCTTCCTCTCAGAGCAA ATCTGAGTAATGAGACTGGTAGCTATCCCTTTCTCTCATGTAACTGTCTGACTGATAAGATCAGCTTGAT CAATATGCATATATTTTTTGATCTGTCTCCTTTTCTTCTATTCAGATCTTATACGCTGTCAGCCCAAT TCTTTCTGTTTCAGACTTCTCTTGATTTCCCTCTTTTCATGTGGCAAAAGAAGTAGTGCGTACAATGTA CTGATTCGTCCTGAGATTTGTACCATGGTTGAAACTAATTTATGGTAATAATATTAACATAGCAAATCTT TAGAGACTCAAATCATGAAAAGGTAATAGCAGTACTGTACTAAAAACGGTAGTGCTAATTTTCGTAATAA TTTTGTAAATATTCAAACAGTAAAACACTTGAAGACACACTTTCCTAGGGAGGCGTTTACTGAAATAATTT AGCTATAGTAAGAAAATTTGTAATTTTAGAAATGCCAAGCATTCTAAATTAATTGCTTGAAAGTCACTAT GATTGTGTCCATTATAAGGAGACAAATTCATTCAAGCAAGTTATTTAATGTTAAAGGCCCAATTGTTAGG CAGTTAATGGCACTTTTACTATTAACTAATCTTTCCATTTGTTCAGACGTAGCTTAACTTACCTCTTAGG TGTGAATTTGGTTAAGGTCCTCATAATGTCTTTATGTGCAGTTTTTGATAGGTTATTGTCATAGAACTTA TTCTATTCCTACATTTTATGATTACTATGGATGTATGAGAATAACACCTAATCCTTTACTTTACCTCAAT TTAACTCCTTTATAAAGAACTTACATTACAGAATAAAGATTTTTTAAAAAATATTTTTTGTAGGACA GGGTCTTAGCCCAGCGAGGCTGGTCTCTAAGTCCTGGCCCAAGCGATCCTCCTGCCTGGGCCTCCTAAAA GTGCTGGAATTATAGACATGAGCCATCACATCCAATATACAGAATAAAGATTTTTAATGGAGGATTTAAT GTTCTTCAGAAAATTTTCTTGAGGTCAGACAATGTCAAATGTCTCCTCAGTTTACACTGAGATTTTGAAA ACAAGTCTGAGCTATAGGTCCTTGTGAAGGGTCCATTGGAAATACTTGTTCAAAGTAAAATGGAAAGCAA AGGTAAAATCAGCAGTTGAAATTCAGAGAAAGACAGAAAAGGAGAAAAGATGAAATTCAACAGGACAGAA GGGAAATATATTATCATTAAGGAGGACAGTATCTGTAGAGCTCATTAGTGATGGCAAAATGACTTGGTCA GGATTATTTTTAACCCGCTTGTTTCTGGTTTGCACGGCTGGGGATGCAGCTAGGGTTCTGCCTCAGGGAG CACAGCTGTCCAGAGCAGCTGTCAGCCTGCAAGCCTGAAACACTCCCTCGGTAAAGTCCTTCCTACTCAG GACAGAAATGACGAGAACAGGGAGCTGGAAACAGGCCCCTAACCAGAGAAGGGAAGTAATGGATCAACAA AGTTAACTAGCAGGTCAGGATCACGCAATTCATTTCACTCTGACTGGTAACATGTGACAGAAACAGTGTA GGCTTATTGTATTTTCATGTAGAGTAGGACCCAAAAATCCACCCAAAGTCCTTTATCTATGCCACATCCT TCTTATCTATACTTCCAGGACACTTTTTCTTCCTTATGATAAGGCTCTCTCTCTCTCCACACACACACAC ACACACACACACACACACACACACACACACACAAACACACACCCCGCCAACCAAGGTGCATGTAAAAAGA TGTAGATTCCTCTGCCTTTCTCATCTACACAGCCCAGGAGGGTAAGTTAATATAAGAGGGATTTATTGGT AAGAGATGATGCTTAATCTGTTTAACACTGGGCCTCAAAGAGAGAATTTCTTTTCTTCTGTACTTATTAA GCACCTATTATGTGTTGAGCTTATATATACAAAGGGTTATTATATGCTAATATAGTAATAGTAATGGTGG TTGGTACTATGGTAATTACCATAAAAATTATTATCCTTTTAAAATAAAGCTAATTATTATTGGATCTTTT TTAGTATTCATTTTATGTTTTTTATGTTTTTGATTTTTTAAAAGACAATCTCACCCTGTTACCCAGGCTG GAGTGCAGTGGTGCAATCATAGCTTTCTGCAGTCTTGAACTCCTGGGCTCAAGCAATCCTCCTGCCTTGG CCTCCCAAAGTGTTGGGATACAGTCATGAGCCACTGCATCTGGCCTAGGATCCATTTAGATTAAAATATG CATTTTAAATTTTAAAATAATATGGCTAATTTTTACCTTATGTAATGTGTATACTGGCAATAAATCTAGT TTGCTGCCTAAAGTTTAAAGTGCTTTCCAGTAAGCTTCATGTACGTGAGGGGAGACATTTAAAGTGAAAC AGACAGCCAGGTGTGGTGGCTCACGCCTGTAATCCCAGCACTCTGGGAGGCTGAGGTGGGTGGATCGCTT GAGCCCTGGAGTTCAAGACCAGCCTGAGCAACATGGCAAAACGCTGTTTCTATAACAAAAATTAGCCGGG CATGGTGGCATGTGCCTGTGGTCCCAGCTACTAGGGGGCTGAGGCAGGAGAATCGTTGGAGCCCAGGAGG TCAAGGCTGCACTGAGCAGTGCTTGCGCCACTGCACTCCAGCCTGGGTGACAGGACCAGACCTTGCCTCA AAAAAATAAGAAGAAAAATTAAAAATAAATGGAAACAACTACAAAGAGCTGTTGTCCTAGATGAGCTACT TAGTTAGGCTGATATTTTGGTATTTAACTTTTAAAGTCAGGGTCTGTCACCTGCACTACATTATTAAAAT ATCAATTCTCAATGTATATCCACACAAAGACTGGTACGTGAATGTTCATAGTACCTTTATTCACAAAACC CCAAAGTAGAGACTATCCAAATATCCATCAACAAGTGAACAAATAAACAAAATGTGCTATATCCATGCAA TGGAATACCACCCTGCAGTACAAAGAAGCTACTTGGGGATGAATCCCAAAGTCATGACGCTAAATGAAAG AGTCAGACATGAAGGAGGAGATAATGTATGCCATACGAAATTCTAGAAAATGAAAGTAACTTATAGTTAC AGAAAGCAAATCAGGGCAGGCATAGAGGCTCACACCTGTAATCCCAGCACTTTGAGAGGCCACGTGGGAA GATTGCTAGAACTCAGGAGTTCAAGACCAGCCTGGGCAACACAGTGAAACTCCATTCTCCACAAAAATGG GAAAAAAAGAAAGCAAATCAGTGGTTGTCCTGTGGGGAGGGGAAGGACTGCAAAGAGGGAAGAAGCTCTG GTGGGGTGAGGGTGGTGATTCAGGTTCTGTATCCTGACTGTGGTAGCAGTTTGGGGTGTTTACATCCAAA AATATTCGTAGAATTATGCATCTTAAATGGGTGGAGTTTACTGTATGTAAATTATACCTCAATGTAAGAA AAAATAATGTGTAAGAAAACTTTCAATTCTCTTGCCAGCAAACGTTATTCAAATTCCTGAGCCCTTTACT TCGCAAATTCTCTGCACTTCTGCCCCGTACCATTAGGTGACAGCACTAGCTCCACAAATTGGATAAATGC ATTTCTGGAAAAGACTAGGGACAAAATCCAGGCATCACTTGTGCTTTCATATCAACCATGCTGTACAGCT TGTGTTGCTGTCTGCAGCTGCAATGGGGACTCTTGATTTCTTTAAGGAAACTTGGGTTACCAGAGTATTT CCACAAATGCTATTCAAATTAGTGCTTATGATATGCAAGACACTGTGCTAGGAGCCAGAAAACAAAGAGG AGGAGAAATCAGTCATTATGTGGGAACAACATAGCAAGATATTTAGATCATTTTGACTAGTTAAAAAAGC AGCAGAGTACAAAATCACACATGCAATCAGTATAATCCAAATCATGTAAATATGTGCCTGTAGAAAGACT AGAGGAATAAACACAAGAATCTTAACAGTCATTGTCATTAGACACTAAGTCTAATTATTATTATTAGACA CTATGATATTTGAGATTTAAAAAATCTTTAATATTTTAAAATTTAGAGCTCTTCTATTTTTCCATAGTAT TCAAGTTTGACAATGATCAAGTATTACTCTTTCTTTTTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTTT TGGTCTTGTTGCCCATGCTGGAGTGGAATGGCATGACCATAGCTCACTGCAACCTCCACCTCCTGGGTTC AAGCAAAGCTGTCGCCTCAGCCTCCCGGGTAGATGGGATTACAGGCGCCCACCACCACACTCGGCTAATG TTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAGAGG ATCCACCTGCCTCAGCCTCCCAAAGTGCTGGGATTACAGATGTAGGCCACTGCGCCCGGCCAAGTATTGC TCTTATACATTAAAAAACAGGTGTGAGCCACTGCGCCCAGCCAGGTATTGCTCTTATACATTAAAAAATA GGCCGGTGCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAAGCCAAGGCGGGCAGAACACCCGAGGT CAGGAGTCCAAGGCCAGCCTGGCCAAGATGGTGAAACCCCGTCTCTATTAAAAATACAAACATTACCTGG GCATGATGGTGGGCGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGGATCCGCGGAGCCTGGCA GATCTGCCTGAGCCTGGGAGGTTGAGGCTACAGTAAGCCAAGATCATGCCAGTATACTTCAGCCTGGGCG ACAAAGTGAGACCGTAACAAAAAAAAAAAAATTTAAAAAAAGAAATTTAGATCAAGATCCAACTGTAAAA AGTGGCCTAAACACCACATTAAAGAGTTTGGAGTTTATTCTGCAGGCAGAAGAGAACCATCAGGGGGTCT TCAGCATGGGAATGGCATGGTGCACCTGGTTTTTGTGAGATCATGGTGGTGACAGTGTGGGGAATGTTAT TTTGGAGGGACTGGAGGCAGACAGACCGGTTAAAAGGCCAGCACAACAGATAAGGAGGAAGAAGATGAGG GCTTGGACCGAAGCAGAGAAGAGCAAACAGGGAAGGTACAAATTCAAGAAATATTGGGGGGTTTGAATCA ACACATTTAGATGATTAATTAAATATGAGGACTGAGGAATAAGAAATGAGTCAAGGATGGTTCCAGGCTG CTAGGCTGCTTACCTGAGGTGGCAAAGTCGGGAGGAGTGGCAGTTTAGGACAGGGGGCAGTTGAGGAATA TTGTTTTGATCATTTTGAGTTTGAGGTACAAGTTGGACACTTAGGTAAAGACTGGAGGGGAAATCTGAAT ATACAATTATGGGGACTGAGGAACAAGTTTTTTATTTTTTGTTTCGTTTTCTTGTTGAAGAACAAATTT AATTGTAATCCCAAGTCATCAGCATCTAGAAGACAGTGGCAGGAGGTGACTGTCTTGTGGGTAAGGGTTT GGGGTCCTTGATGAGTATCTCTCAATTGGCCTTAAATATAAGCAGGAAAAGGAGTTTATGATGGATTCCA GGCTCAGCAGGGCTCAGGGAGGGCTCAGGCAGCCAGCAGAGGAAGTCAGAGCATCTTCTTTGGTTTAGCCCC AAGTAATGACTTCCTTAAAAAAGCTGAAGGAAAATCCAGAGTGACCAGATTATAAACTGTACTCTTGCATT TTCTCTCCCTCCTTCACCCACAGCCTCTTGATCGAACCGGAGGAAGTTTCTTTACCAATTCAAAAATGTC CGCTGGGCTAAGGGTCGGCGTGAGACCTACCTGTGCTACGTAGTGAAGAGGCGTGACAGTGCTACATCCT TTTCACTGGACTTTGGTTATCTTCGCAATAAGGTATCAATTAAAGTCGGCTTTGCAAGCAGTTTAATGGT CAACTGTGAGTGCTTTAGAGCCACCTGCTGATGGTATTACTTCCATCCTTTTTTGGCATTTGTGTCTCT ATCACATTCCTCAAATCCTTTTTTTTATTTCTTTTTCCATGTCCATGCACCCATATTAGACATGGCCCAA AATATGTGATTTAATTCCTCCCCAGTAATGCTGGGCACCCTAATACCACTCCTTCCTTCAGTGCCAAGAA CAACTGCTCCCAAACTGTTTACCAGCTTTCCTCAGCATCTGAATTGCCTTTGAGATTAATTAAGCTAAAA GCATTTTTATATGGGAGAATATTATCAGCTTGTCCAAGCAAAAATTTTAAATGTGAAAAACAAATTGTGT CTTAAGCATTTTTGAAAATTAAGGAAGAAGAATTTGGGAAAAAATTAACGGTGGCTCAATTCTGTCTTCC AAATGATTTCTTTTCCCTCCTACTCACATGGGTCGTAGGCCAGTGAATACATTCAACATGGTGATCCCCA GAAAACTCAGAGAAGCCTCGGCTGATGATTAATTAAATTGATCTTTCGGCTACCCGAGAGAATTACATTT CCAAGAGACTTCTTCACCAAAATCCAGATGGGTTTACATAAACTTCTGCCCACGGGTATCTCCTCTCTCC TAACACGCTGTGACGTCTGGGCTTGGTGGAATCTCAGGGAAGCATCCGTGGGGTGGAAGGTCATCGTCTG GCTCGTTGTTTGATGGTTATATTACCATGCAATTTTCTTTGCCTACATTTGTATTGAATACATCCCAATC TCCTTCCTATTCGGTGACATGACACATTCTATTTCAGAAGGCTTTGATTTTATCAAGCACTTTCATTTAC TTCTCATGGCAGTGCCTATTACTTCTCTTACAATACCCATCTGTCTGCTTTACCAAAATCTATTTCCCCT TTTCAGATCCTCCCAAATGGTCCTCATAAACTGTCCTGCCTCCACCTAGTGGTCCAGGTATATTTCCACA ATGTACATCAACAGGCACTTCTAGCCATTTTCCTTCCAAAAGGTGCAAAAAGCAACTTCATAAACACA AATTAAATCTTCGGTGAGGTAGTGTGATGCTGCTTCCTCCCAACTCAGCGCACTTCGTCTTCCTCATTCC ACAAAAACCCATAGCCTTCCTTCACTCTGCAGGACTAGTGCTGCCAAGGGTTCAGCTCTACCTACTGGTG TGCTCTTTTGAGCAAGTTGCTTAGCCTCTCTGTAACAAGGACAATAGCTGCAAGCATCCCCAAAGATC ATTGCAGGAGACAATGACTAAGGCTACCAGAGCCGCAATAAAAGTCAGTGAATTTTAGCGTGGTCCTCTC TGTCTCTCCAGAACGGCTGCCACGTGGAATTGCTCTTCCTCCGCTACATCTCGGACTGGGACCTAGACCC TGGCCGCTGCTACCGCGTCACCTGGTTCACCTCCTGGAGCCCCTGCTACGACTGTGCCCGACATGTGGCC GACTTTCTGCGAGGGAACCCCAACCTCAGTCTGAGGATCTTCACCGCGCGCCTCTACTTCTGTGAGGACC GCAAGGCTGAGCCCGAGGGGCTGCGGCGGCTGCACCGCGCCGGGTGCAAATAGCCATCATGACCTTCAA AGGTGCGAAAGGGCCTTCCGCGCAGGCGCAGTGCAGCAGCCCGCATTCGGGATTGCGATGCGGAATGAAT GAGTTAGTGGGGAAGCTCGAGGGGAAAGTGGGCGGGGATTCTGGTTCACCTCTGGAGCCGAAATTAAA GATTAGAAGCAGAGAAAAGAGTGAATGGCTCAGAGACAAGGCCCCGAGGAAATGAGAAAATGGGGCCAGG GTTGCTTCTTTCCCCTCGATTTGGAACCTGAACTGTCTTCTACCCCCATATCCCCGCCTTTTTTTCCTTT TTTTTTTTTTGAAGATTATTTTTACTGCTGGAATACTTTTGTAGAAAACCACGAAAGAACTTTCAAAGCC TGGGAAGGGCTGCATGAAAATTCAGTTCGTCTCTCCAGACAGCTTCGGCGCATCCTTTTGGTAAGGGGCT TCCTCGCTTTTTAAATTTTCTTTCTTTCTCTACAGTCTTTTTTGGAGTTTCGTATATTTCTTATATTTTC TTATTGTTCAATCACTCTCAGTTTTCATCTGATGAAAACTTTATTTCTCCTCCACATCAGCTTTTTCTTC TGCTGTTTCACCATTCAGAGCCCTCTGCTAAGGTTCCTTTTCCCTCCCTTTTCTTTCTTTTGTTGTTTCA CATCTTTAAATTTCTGTCTCTCCCCAGGGTTGCGTTTCCTTCCTGGTCAGAATTCTTTTCTCCTTTTTTT TTTTTTTTTTTTTTTTTTTTAAACAAACAAACAAAAAACCCAAAAAAACTCTTTCCCAATTTACTTTCTT CCAACATGTTACAAAGCCATCCACTCAGTTTAGAAGACTCTCCGGCCCCACCGACCCCCAACCTCGTTTT GAAGCCATTCACTCAATTTGCTTCTCTCTTTCTCTACAGCCCCTGTATGAGGTTGATGACTTACGAGACG CATTTCGTACTTTGGGACTTTGATAGCAACTTCCAGGAATGTCACACACGATGAAATATCTCTGCTGAAG ACAGTGGATAAAAAACAGTCCTTCAAGTCTTCTCTGTTTTTATTCTTCAACTCTCACTTTCTTAGAGTTT ACAGAAAAAATATTTATATACGACTCTTTAAAAAGATCTATGTCTTGAAAATAGAGAAGGAACACAGGTC TGGCCAGGGACGTGCTGCAATTGGTGCAGTTTTGAATGCAACATTGTCCCCTACTGGGAATAACAGAACT GCAGGACCTGGGAGCATCCTAAAGTGTCAACGTTTTTCTATGACTTTTAGGTAGGATGAGAGCAGAAGGT AGATCCTAAAAAGCATGGTGAGAGGATCAAATGTTTTTATATCAACATCCTTTATTATTTGATTCATTTG AGTTAACAGTGGTGTTAGTGATAGATTTTTCTATTCTTTTCCCTTGACGTTTACTTTCAAGTAACACAAA CTCTTCCATCAGGCCATGATCTATAGGACCTCCTAATGAGAGTATCTGGGTGATTGTGACCCCAAACCAT CTCTCCAAAGCATTAATATCCAATCATGCGCTGTATGTTTTAATCAGCAGAAGCATGTTTTTATGTTTGT ACAAAAGAAGATTGTTATGGGTGGGGATGGAGGTATAGACCATGCATGGTCACCTTCAAGCTACTTTAAT AAAGGATCTTAAAATGGGCAGGAGGACTGTGAACAAGACACCCTAATAATGGGTTGATGTCTGAAGTAGC AAATCTTCTGGAAACGCAAACTCTTTTAAGGAAGTCCCTAATTTAGAAACACCCACAAACTTCACATATC ATAATTAGCAAACAATTGGAAGGAAGTTGCTTGAATGTTGGGGAGAGGAAAATCTATTGGCTCTCGTGGG TCTCTTCATCTCAGAAATGCCAATCAGGTCAAGGTTTGCTACATTTTGTATGTGTGTGATGCTTCTCCCA AAGGTATATTAACTATATAAGAGAGTTGTGACAAAACAGAATGATAAAGCTGCGAACCGTGGCACACGCT CATAGTTCTAGCTGCTTGGGAGGTTGAGGAGGGAGGATGGCTTGAACACAGGTGTTCAAGGCCAGCCTGG GCAACATAACAAGATCCTGTCTCTCAAAAAAAAAAAAAAAAAAAAGAAAGAGAGAGGGCCGGGCGTGGTG GCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGCCGGGCGGATCACCTGTGGTCAGGAGTTTGAGA CCAGCCTGGCCAACATGGCAAAACCCCGTCTGTACTCAAAATGCAAAAATTAGCCAGGCGTGGTAGCAGG CACCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCAGGAGGTGGAGGTTGCA GTAAGCTGAGATCGTGCCGTTGCACTCCAGCCTGGGCGACAAGAGCAAGACTCTGTCTCAGAAAAAAAAA AAAAAAAGAGAGAGAGAGAGAAAGAGAACAATATTTGGGAGAGAAGGATGGGGAAGCATTGCAAGGAAAT TGTGCTTTATCCAACAAAATGTAAGGAGCCAATAAGGGATCCCTATTTGTCTCTTTTGGTGTCTATTTGT CCCTAACAACTGTCTTTGACAGTGAGAAAAATATTCAGAATAACCATATCCCTGTGCCGTTATTACCTAG CAACCCTTGCAATGAAGATGAGCAGATCCACAGGAAAACTTGAATGCACAACTGTCTTATTTTAATCTTA TTGTACATAAGTTTGTAAAAGAGTTAAAAATTGTTACTTCATGTATTCATTTATATTTTATATTATTTTG CGTCTAATGATTTTTTATTAACATGATTTCCTTTTCTGATATATTGAAATGGAGTCTCAAAGCTTCATAA ATTTATAACTTTAGAAATGATTCTAATAACAACGTATGTAATTGTAACATTGCAGTAATGGTGCTACGAA GCCATTTCTCTTGATTTTTAGTAAACTTTTATGACAGCAAATTTGCTTCTGGCTCACTTTCAATCAGTTA AATAAATGATAAATAATTTTGGAAGCTGTGAAGATAAAATACCAAATAAAATAATATAAGGTGATTTAT ATGAAGTTAAAATAAAAAATCAGTATGATGGAATAAACTTG

[0084] Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) is an evolutionarily conserved family of cytidine deaminases. Members of this family are C to U editing enzymes. The N-terminal domain of APOBEC-like proteins is the catalytic domain, and the C-terminal domain is the pseudocatalytic domain. More specifically, the catalytic domain is a zinc-dependent cytidine deaminase domain, which is important for cytidine deamination. APOBEC family members include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (now referred to as "APOBEC3E"), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) deaminase. A number of engineered cytidine deaminases are commercially available, including, but not limited to, SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, VRER-BE3, VRER-BE3, YE1-BE3, EE-BE3, YE2-BE3, and YEE-BE3, available from Addgene (plasmids 85169, 85170, 85171, 85172, 85173, 85174, 85175, 85176, 85177).

[0085] Other exemplary deaminases that can be fused to Cas9 according to aspects of the present disclosure are provided below. It should be understood that in some embodiments, the active domain of the respective sequence can be used, for example, a domain without a localization signal (nuclear localization sequence, nuclear export signal, cytoplasmic localization signal).

[0086] Human AID: TIFF0007657711000005.tif30169 (underline: nuclear localization sequence; double underline: nuclear export signal)

[0087] Mouse AID: TIFF0007657711000006.tif29169 (Underline: Nuclear localization sequence; Double underline: Nuclear export signal)

[0088] Canine AID: TIFF0007657711000007.tif28169 (Underline: Nuclear localization sequence; Double underline: Nuclear export signal)

[0089] Bovine AID: TIFF0007657711000008.tif29169 (Underline: Nuclear localization sequence; Double underline: Nuclear export signal)

[0090] Rat AID TIFF0007657711000009.tif29169 (Underline: Nuclear localization sequence; Double underline: Nuclear export signal)

[0091] Mouse APOBEC-3 TIFF0007657711000010.tif45167 (Italic: Nucleic acid editing domain)

[0092] Rat APOBEC-3 : TIFF0007657711000011.tif45167 (Italic: Nucleic acid editing domain)

[0093] Rhesus macaque APOBEC-3 G: TIFF0007657711000012.tif41167 (Italic: Nucleic acid editing domain; Underline: Cytoplasmic localization signal)

[0094] Chimpanzee APOBEC-3 G: TIFF0007657711000013.tif38167 (Italic: Nucleic acid editing domain; Underline: Cytoplasmic localization signal)

[0095] Green monkey APOBEC-3G: TIFF0007657711000014.tif40167 (italics: nucleic acid editing domain; underline: cytoplasmic localization signal)

[0096] Human APOBEC-3G: TIFF0007657711000015.tif38168 (italics: nucleic acid editing domain; underline: cytoplasmic localization signal)

[0097] Human APOBEC-3F: TIFF0007657711000016.tif38169(italics: nucleic acid editing domain)

[0098] Human APOBEC-3B: TIFF0007657711000017.tif38169(italics: nucleic acid editing domain)

[0099] Rat APOBEC-3B: MQPQGLGPNAGMGPVCLGCSHRRPYSPIRNPLKKLYQQTFYFHFKNVRYAWGRKNNFLCYEVNGMDCALPVPLRQGVFRKQGHIHAELCFIYWFHDKV LRVLSPMEFKVTWYMSWSPCSKCAEQVARFLAAHRNLSLAIFSSRLYYYLRNPNYQQKLCRLIQEGVHVAAMDLPEFKKCWNKFVDNDGQPFRPWMRL RINFSFYDCKLQEIFSRMNLLREDVFYLQFNNSHRVKPVQNRYYRRKSYLCYQLERANGQEPLKGYLLYKKGEQHVEILFLEKMRSMELSQVRITCYLT WSPCPNCARQLAAFKKDHPDLILRIYTSRLYFWRKKFQKGLCTLWRSGIHVDVMDLPQFADCWTNFVNPQRPFRPWNELEKNSWRIQRRLRRIKESWGL

[0100] Bovine APOBEC-3B: DGWEVAFRSGTVLKAGVLGVSMTEGWAGSGHPGQGACVWTPGTRNTMNLLREVLFKQQFGNQPRVPAPYYRRKTYLCYQLKQRNDLLDRGCFRNKKQRHAERFIDKINSLDLNPSQSYKIICYITWSPCPNCANELVNFITRNNHLKLEIFASRLYFHWIKSFKMGLQDLQNAGISVAVMTHTEFEDCWEQFVDNQSRFPQPWDKLEQYSASIRRLQRILTAPI

[0101] Chimpanzee APOBEC-3B: MNPQIRNPMEWMYQRTFYYNFENEPILYGRSYTWLCYEVKIRRGHSNLLWDTGVFRGQMYSQPEHHAEMCFLSWFCGNQLSAYKCFQITWFVSWTPCPDCVAKLAKFLAEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYNEGQPFMPWYKFDDNYAFLHRTLKEIIRHLMDPDTFTFNNDPLVLRRHQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNL LCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGQVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQVRASSLCMVPHRPPPPPQSPGPCLPLCSEPPLGSLLPTGRPASLPFLLTASSFPPPASLPPLSSLSPGHLPVPSFHSLTSCSIQPPSSRIRETEGWASSKEGRDLG

[0102] Human APOBEC-3C: TIFF0007657711000018.tiff20169

[0103] Gorilla APOBEC3C TIFF0007657711000019.tif20169(italics: nucleic acid editing domain)

[0104] Human APOBEC-3A: TIFF0007657711000020.tif20169(italics: nucleic acid editing domain)

[0105] Rhesus macaque APOBEC-3A: TIFF0007657711000021.tif20169(italics: nucleic acid editing domain)

[0106] Bovine APOBEC-3A: TIFF0007657711000022.tif20169(italics: nucleic acid editing domain)

[0107] Human APOBEC-3H: TIFF0007657711000023.tif20169(italics: nucleic acid editing domain)

[0108] Rhesus macaque APOBEC-3H: MALLTAKTFSLQFNNKRRVNKPYYPRKALLCYQLTPQNGSTPTRGHLKNKKKDHAEIRFINKIKSMGLDETQCYQVTCYLTWSPCPSCAGELVDFIKAHRHLNLRIFASRLYYHWRPNYQEGLLLLGCGSQVPVEVMGLPEFTDCWENFVDHKEPPSFNPSEKLEELDKNSQAIKRRLERIKSRSVDVLENGLRSLQLGPVTPSSSIRNSR

[0109] Human APOBEC-3D: TIFF0007657711000024.tif37169(italics: nucleic acid editing domain)

[0110] Human APOBEC-1: MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR

[0111] Mouse APOBEC-1 : MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTSNHVEVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLYHHTDQRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHLWVKLYVLELYCIILGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLK

[0112] Rat APOBEC-1 : MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK

[0113] Human APOBEC-2: MAQKEEAAVATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPANFFKFQFRNVEYSSGRNKTFLCYVVEAQGKGGQVQASRGYLEDEHAAAHAEEAFFNTILPAFDPALRYNVTWYVSSSPCAACADRIIKTLSKTKNLRLLILVGRLFMWEEPEIQAALKKLKEAGCKLRIMKPQDFEYVWQNFVEQEEGESKAFQPWEDIQENFLYYEEKLADILK

[0114] Mouse APOBEC-2: MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVEYSSGRNKTFLCYVVEVQSKGGQAQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK

[0115] Rat APOBEC-2: MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVEYSSGRNKTFLCYVVEAQSKGGQVQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEYLWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK

[0116] Bovine APOBEC-2: MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPAHYFKFQFRNVEYSSGRNKTFLCYVVEAQSKGGQVQASRGYLEDEHATNHAEEAFFNSIMPTFDPALRYMVTWYVSSSPCAACADRIVKTLNKTKNLRLLILVGRLFMWEEPEIQAALRKLKEAGCRLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK

[0117] Petromyzon marinus CDA1 (pmCDAl) MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSFMIQVKILHTTKSPAV

[0118] Human APOBEC3G D316R D317R MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKFNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHFMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTC FTSWSPCFSCAQEMAKFISKKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISFTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN

[0119] Human APOBEC3G chain A MDPPTFTFNFNNEPWWGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISF TYSEFKHCWDTFVDHQGCPFQPWDGLD EHSQDLSGRLRAILQ

[0120] Human APOBEC3G chain A D120R D121R MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISFMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQ

[0121] The term "deaminase" or "deaminase domain" refers to a protein or fragment thereof that catalyzes a deamination reaction.

[0122] "Detecting" refers to identifying the presence, absence, or amount of the analyte to be detected. In one embodiment, a sequence variation in a polynucleotide or polypeptide is detected. In another embodiment, the presence of an indel is detected.

[0123] By "detectable label" is meant a composition that, when linked to a molecule of interest, renders it detectable via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (such as those commonly used in ELISA), biotin, digoxigenin, or haptens.

[0124] "Disease" refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue or organ. In certain embodiments, diseases suitable for treatment with the compositions of the invention are associated with point mutations, splicing events, premature stop codons, or misfolding events.

[0125] "DNA binding protein domain" refers to a polypeptide or fragment thereof that binds to DNA. In some embodiments, the DNA binding protein domain is a zinc finger or TALE domain with sequence-specific DNA binding activity. In other embodiments, the DNA binding protein domain is a domain of a CRISPR-Cas protein that binds to DNA (e.g., Cas9), including, for example, those that bind to protospacer adjacent motifs (PAMs). In some embodiments, the DNA binding protein domain forms a complex with a polynucleotide (e.g., a single guide RNA), and the complex binds to the DNA sequence specified by the gRNA and the protospacer adjacent motif. In some embodiments, the DNA binding protein domain includes a nickase activity (e.g., nCas9) or is catalytically inactive (e.g., dCas9, zinc finger domain, TALE). In yet other embodiments, the DNA binding protein domain is a catalytically inactive variant of the homing endonuclease I-SceI, or the DNA binding domain of the TALE protein AvrBs4. See, e.g., Gabsalilow et al., Nucleic Acids Research, Volume 41, Issue 7, 1 April 2013, Pages e83. In some embodiments, the DNA binding protein domain is fused to a domain with catalytic activity (e.g., FokI, MutH). In certain embodiments, the zinc finger domain is fused to the catalytic domain of the endonuclease FokI. In other embodiments, the TALE is fused to MutH, which contains site-specific DNA nicking activity.

[0126] The term "effective amount" as used herein refers to an amount of a biologically active agent sufficient to induce a desired biological response. In certain embodiments, an effective amount is an amount of two or more plasmids containing a portion of a base editor system sufficient to express an active base editing system in a cell transfected with the plasmid. As will be understood by those skilled in the art, the effective amount of an agent (e.g., a fusion protein) can vary depending on various factors, such as the desired biological response, e.g., the specific allele, genome, or target site to be edited, the cell or tissue to be targeted, and the agent used.

[0127] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. The portion comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. A fragment can comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0128] "Hybridization" refers to hydrogen bonding between complementary nucleobases, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that form hydrogen bonds to pair.

[0129] The term "base repair inhibitor" or "IBR" refers to a protein that can inhibit the activity of nucleic acid repair enzymes, such as base excision repair enzymes. In some embodiments, the IBR is an inhibitor of inosine base excision repair. Examples of base repair inhibitors include APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4 PDG, UDG, hSMUGL and hAAG inhibitors. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is catalytically inactive EndoV or catalytically inactive hAAG.

[0130] An "intein" is a fragment of a protein that can excise itself and link the remaining fragment (extein) with a peptide bond in a process known as protein splicing. Inteins are also called "protein introns." The process by which an intein excises itself and links the remaining portion of a protein is referred to herein as "protein splicing" or "intein-mediated protein splicing." In some embodiments, the inteins of a precursor protein (the intein-containing protein prior to intein-mediated protein splicing) are derived from two genes. Such inteins are referred to herein as split inteins (e.g., split intein-N and split intein-C). For example, in cyanobacteria, DnaE, ​​the catalytic subunit a of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c. The intein encoded by the dnaE-n gene may be referred to herein as "intein N." The intein encoded by the dnaE-c gene may be referred to herein as "intein C."

[0131] Other intein systems can also be used. For example, synthetic inteins based on dnaE intein, i.e., Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C) intein pairs have been described (e.g., Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5, incorporated herein by reference). Non-limiting examples of intein pairs that can be used according to the present disclosure include Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, and Cne Prp8 intein (e.g., as described in U.S. Patent No. 8,394,604, incorporated herein by reference).

[0132] Exemplary nucleotide and amino acid sequences of inteins are provided.

[0133] DnaE intein-N DNA:TGCCTGTCATACGAACCGAGATACTGACAGTAGAATATGGCCTTCTGCC AATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCG ATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGG GGAGAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAG GGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTA TAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTT CCTAAT

[0134] DnaE Intein-N Protein: CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDR GEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNL PN

[0135] DnaE intein-C DNA:ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGA TATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAG CTTCTAAT

[0136] Intein-C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN

[0137] Cfa-N DNA: TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCC TATTGGAAAGATTGTCGAAGAGGAGAATTGAATGCACAGTATATACTGTAG ACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGGCCAATCTGC GGCGACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACG AGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAA TAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTG CCA

[0138] Cfa-N protein: CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNR GEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGDLLKQVDGL P

[0139] Cfa-C DNA: ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAG GAAAGTAAAGATAATATCTCGAAAAGTCTTGGTACCCAAAATGTCTATG ATATTGGAGTGGAAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTA GCCAGCAAC

[0140] Cfa-C protein: MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLV ASN

[0141] To join the N-terminal portion of split Cas9 with the C-terminal portion of split Cas9, an intein-N and an intein-C can be fused to the N-terminal portion of split Cas9 and the C-terminal portion of split Cas9, respectively. For example, in some embodiments, an intein-N is fused to the C-terminus of the N-terminal portion of split Cas9, i.e., forming a structure of N--[N-terminal portion of split Cas9]-[intein-N]--C. In some embodiments, an intein-C is fused to the N-terminus of the C-terminal portion of split Cas9, i.e., forming a structure of N--[intein-C]--[C-terminal portion of split Cas9]-C. The mechanism of intein-mediated protein splicing for linking proteins (e.g., split Cas9) to which inteins are fused is known in the art, for example, as described in Shah et al., Chem Sci. 2014; 5(1):446-461, which is incorporated herein by reference. Methods for designing and using inteins are known in the art, for example, as described by WO2014004336, WO2017132580, US20150344549 and US20180127780, each of which is incorporated herein by reference in its entirety.

[0142] The terms "isolated," "purified," or "biologically pure" refer to a material that has been removed to various degrees from components that normally accompany it when found in its native state. "Isolated" refers to a degree of separation from the original source or surrounding environment. "Purified" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein has been sufficiently free of other materials such that the impurities do not substantially affect the biological properties of the protein or cause other deleterious consequences. That is, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For proteins that can undergo modifications, such as phosphorylation or glycosylation, different modifications can give rise to different isolated proteins that can be purified separately.

[0143] By "isolated polynucleotide" is meant a nucleic acid (e.g., DNA) that is free of the genes that flank the gene in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. Thus, the term includes recombinant DNA that is present as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments generated by PCR or restriction endonuclease digestion), for example, incorporated into a vector; incorporated into an autonomously replicating plasmid or virus; incorporated into the genomic DNA of a prokaryote or eukaryote; or as a separate molecule independent of other sequences. In addition, the term includes RNA molecules that are transcribed from a DNA molecule, as well as recombinant DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

[0144] By "isolated polypeptide" is meant a polypeptide of the invention separated from components that naturally accompany it. Typically, a polypeptide is isolated when it is at least 60% free, by weight, from proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0145] The term "linker" as used herein refers to a bond (e.g., a covalent bond), chemical group, or molecule that connects two molecules or moieties (e.g., two domains of a fusion protein). In some embodiments, the linker connects the gRNA binding domain of an RNA programmable nuclease, including a Cas9 nuclease domain, and the catalytic domain of a nucleic acid editing protein. In some embodiments, the linker connects a dCas9 and a nucleic acid editing protein. Typically, the linker is placed between or adjacent to two groups, molecules, or other moieties, and is linked to each other via a covalent bond, thus connecting 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 between 5 and 200 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90, 95, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 175, 180, 190, or 200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES, which may be referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker comprises the amino acid sequence (SGGS) n , (GGGS) n , (GGGGS) n , (G) n、 (EAAAK) n , (GGS) n , SGSETPGTSESATPES, or (XP) n In some embodiments, the amino acid sequence includes any one of the following: a) a nucleotide sequence, b) a nucleotide sequence, c) a nucleotide sequence, d) a nucleotide sequence, e) a nucleotide sequence, f ...

[0146] In some embodiments, the nucleobase editor domain is fused via a linker comprising the amino acid sequence of SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS. In some embodiments, the nucleobase editor domain is fused via a linker comprising the amino acid sequence SGSETPGTSESATPES, which may be referred to as an XTEN linker. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSS. In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS. In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0147] By "marker" is meant any protein or polynucleotide having an alteration in expression levels or activity that is associated with a disease or disorder.

[0148] As used herein, the term "mutation" refers to the replacement of a residue in a sequence, such as a nucleic acid or amino acid sequence, with another residue, or the deletion or insertion of one or more residues in a sequence. Mutations are typically described herein by identifying the original residue, then identifying the position of the residue in the sequence, and identifying the newly replaced residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art and are provided, for example, by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)).

[0149] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a compound, such as a nucleoside, a nucleotide, or a polymer of nucleotides, that contains a nucleobase and an acidic moiety. Typically, a polymeric nucleic acid, such as a nucleic acid molecule that contains 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 (e.g., a nucleotide and / or a nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide chain that contains three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably to refer to a polymer of nucleotides (e.g., a chain of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA as well as single-stranded and / or double-stranded DNA. Nucleic acids can occur naturally, for example, in the context of a genome, a transcript, an mRNA, a tRNA, a rRNA, a siRNA, a snRNA, a plasmid, a cosmid, a chromosome, a chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule can be, for example, a non-naturally occurring molecule, recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids, or non-naturally occurring molecules that contain non-naturally occurring nucleotides or nucleosides. Additionally, the terms "nucleic acid", "DNA", "RNA", and / or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. In the case of chemically synthesized molecules, the nucleic acid can include, where appropriate, nucleoside analogs, e.g., chemically modified bases or sugars, and analogs having backbone modifications. Nucleic acid sequences are shown in the 5' to 3' direction unless otherwise indicated.In certain embodiments, nucleic acids are selected from the group consisting of natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5- -methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanine, O(6)-methylguanine, and 2-thiocytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0150] The term "nuclear localization sequence", "nuclear localization signal" or "NLS" refers to an amino acid sequence that promotes the import of a protein into the cell nucleus. Nuclear localization sequences are known in the art and are described, for example, in Plank et al., International PCT Application PCT / EP 2000 / 011690, filed November 23, 2000, and published May 31, 2001 as WO / 2001 / 038547, the contents of which are incorporated herein by reference for disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS, for example, as described by Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4172. In certain embodiments, the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0151] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid that guides the napDNAbp to a specific nucleic acid sequence. For example, a Cas9 protein can bind to a guide RNA that guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, such as a nuclease-active Cas9, Cas9 nickase (nCas9), or 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, C2c1, and C2c3. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, even if not specifically listed in this disclosure.

[0152] As used herein, "obtaining," as in "obtaining an agent," includes synthesizing, purchasing, or otherwise acquiring the agent.

[0153] As used herein, a "patient" or "subject" refers to a subject, such as a mammalian subject, who has been diagnosed with or is suspected of having or developing a disease or disorder. In some embodiments, the term "patient" refers to a mammalian subject who has a higher than average likelihood of developing a disease or disorder. Exemplary patients can be humans, non-human primates, felines, dogs, pigs, cows, cats, horses, goats, sheep, rodents (e.g., mice, rabbits, rats, guinea pigs), and other mammals that can benefit from the treatments disclosed herein. Exemplary human patients can be male and / or female. A "patient in need thereof" or a "subject in need thereof" is referred to herein as a patient who has been diagnosed with or is suspected of having a disease or disorder.

[0154] The terms "RNA programmable nuclease" and "RNA-guided nuclease" are used with (e.g., bound to or associated with) one or more RNAs that are not targets for cleavage. In some embodiments, when an RNA programmable nuclease is complexed with an RNA, it may be referred to as a nuclease:RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA). A gRNA may exist as a complex with two or more RNAs or as a single RNA molecule. A gRNA that exists as a single RNA molecule may be referred to as a single guide RNA (sgRNA), although "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or as a complex with two or more molecules. Typically, a gRNA that exists as a single RNA species contains two domains: (1) a domain that shares homology with a target nucleic acid (e.g., directs binding of the Cas9 complex to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and contains a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to tracrRNA as provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNAs (e.g., those containing domain 2) can be found in U.S. Provisional Patent Application USSN61 / 874,682, entitled "Switchable Cas9 Nucleases and Uses Thereof," filed September 6, 2013, and U.S. Provisional Patent Application USSN61 / 874,746, entitled "Delivery System For Functional Nucleases," filed September 6, 2013, the entire contents of each of which are incorporated herein by reference. In some embodiments, the gRNA comprises two or more of domains (1) and (2), and may be referred to as an "extended gRNA."For example, the extended gRNA binds, e.g., to two or more Cas9 proteins and binds to a target nucleic acid in two or more different regions, as described herein. The gRNA comprises a nucleotide sequence complementary to a target site, which mediates binding of a nuclease / RNA complex to said target site and provides sequence specificity for the nuclease:RNA complex. In certain embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 (Csnl) from Streptococcus pyogenes (e.g., "Complete genome sequence of an Ml strain of Streptococcus pyogenes." Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C, Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl.Acad. trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma CM., Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011)).

[0155] The term "recombinant" as used herein with respect to a protein or nucleic acid refers to a protein or nucleic acid that does not occur in nature but is the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any naturally occurring sequence.

[0156] By "reduce" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0157] "Reference" refers to a standard or control condition. In one embodiment, the reference is the activity of a full-length nucleobase editor expressed in a single plasmid in the same cell under the same conditions as a nucleobase editor expressed in an intein-containing fragment for intein-dependent reassembly.

[0158] A "reference sequence" is a defined sequence used as the basis for sequence comparison. A reference sequence can be a subset or the entirety of a particular sequence; for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is generally at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is generally at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides, or any integer number therebetween or thereabout.

[0159] The term "single nucleotide polymorphism (SNP)" refers to a variation of a single nucleotide occurring at a specific position in the genome, where each variation is present to a degree that is recognizable in the population (e.g., >1%). For example, at a particular base position in the human genome, a C nucleotide can occur in most individuals, but in a small number of individuals, the position is occupied by an A. This means that there is an SNP at this particular position, and the variations of the two nucleotides, C or A, are alleles at this position. SNPs underlie differences in susceptibility to disease. The severity of disease and the body's response to treatment are also manifestations of genetic variation. SNPs can be present in the coding region of a gene, in the non-coding region of a gene, or in intergenic regions (regions between genes). In certain embodiments, SNPs in the coding sequence do not necessarily change the amino acid sequence of the protein produced due to the degeneracy of the genetic code. There are two types of SNPs in coding regions: synonymous SNPs and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, whereas non-synonymous SNPs change the amino acid sequence of the protein. There are two types of nonsynonymous SNPs: missense and nonsense. SNPs that are not in protein-coding regions can affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of noncoding RNA. Gene expression affected by this type of SNP is called eSNP (expressed SNP) and can be upstream or downstream of the gene. Single-nucleotide variants (SNVs) are single-nucleotide variations with unlimited frequency that can occur somatically. Somatic single-nucleotide variations can also be called single-base alterations.

[0160] By "specifically binds" is meant a nucleic acid molecule, polypeptide, or complex thereof (e.g., a nucleic acid programmable DNA binding domain and a guide nucleic acid), compound, or molecule that recognizes and binds to a polypeptide and / or nucleic acid molecule of the invention, but does not substantially recognize and bind to other molecules in a sample (e.g., a biological sample).

[0161] 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 do not need to be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence can typically hybridize with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules do not need to be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence can typically hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridize" refers to pairing to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or between parts thereof under various stringency conditions. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0162] For example, stringent salt concentrations are usually less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvents, such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions will usually include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, concentration of detergent (e.g., sodium dodecyl sulfate (SDS)), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are achieved by combining these various conditions as necessary. In one embodiment, hybridization occurs in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30° C. In another embodiment, hybridization occurs 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 occurs in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations of these conditions will be readily apparent to one of skill in the art.

[0163] In most applications, the washing steps following hybridization also vary in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As mentioned above, washing stringency can be increased by decreasing salt concentration or increasing temperature. For example, stringent salt concentrations for the washing steps are preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the washing steps usually 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 washing steps are performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing steps are performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps are performed in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 68° C. Further variations of these conditions will be readily apparent to those of skill in the art.Hybridization techniques are well known to those of skill 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.

[0164] "Split" means divided into two or more pieces.

[0165] "Split Cas9 protein" or "split-Cas9" refers to a Cas9 protein provided as an N-terminal fragment and a C-terminal fragment encoded by two separate nucleotide sequences. Polypeptides corresponding to the N-terminal and C-terminal portions of the Cas9 protein can be spliced ​​to form a "reconstituted" Cas9 protein. In certain embodiments, the Cas9 protein is split into two fragments within a disordered region of the protein, 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. The disordered region may be determined by one or more protein structure determination techniques known in the art, including, but not limited to, X-ray crystallography, NMR spectroscopy, electron microscopy (e.g., cryo-EM), and / or in silico protein modeling. In some embodiments, the protein comprises about amino acids A292-G36 of SpCas9. 5 , F445-K483, or any C, T, A, or S in the region between E565-T637, or at the corresponding positions in 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 "splitting" the protein.

[0166] By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). In one embodiment, such a sequence has at least 60%, 80%, or 85%, 90%, 95%, or even 99% identity at the amino acid level or nucleic acid to the sequence used for comparison.

[0167] Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine. In an exemplary approach to determine the degree of identity, the BLAST program can be used, e.g., -3 and e -100 A probability score between indicates closely related sequences.

[0168] "Subject" means a mammal, including, but not limited to, a human or non-human mammal, such as a cow, horse, dog, sheep, or cat. Subjects include livestock, farm animals that are kept to produce labor and provide goods such as food, including, but not limited to, cows, goats, chickens, horses, pigs, rabbits, and sheep.

[0169] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a nucleobase editor. In one embodiment, the target site is deaminated by a deaminase or a fusion protein comprising same (e.g., a cytidine or adenine deaminase).

[0170] Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins can, in principle, target any sequence specified by a guide RNA. Methods of using RNA-programmable nucleases such as Cas9 for site-specific cleavage (e.g., to modify genomes) are known in the art (e.g., Cong, L. et al., Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, WY et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013), the entire contents of each of which are incorporated herein by reference.

[0171] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0172] As used herein, the terms "treat," "treating," "treatment," and the like refer to alleviating or ameliorating a disorder and / or its associated symptoms. It will be understood that treating a disorder or condition does not require (nor is exclusionary) the complete elimination of the associated disorder, condition, or symptom.

[0173] The term "uracil glycosylase inhibitor" or "UGI" as used herein refers to a protein that can inhibit uracil-DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain comprises wild-type UGI or a modified version thereof. In some embodiments, the UGI protein provided herein comprises a fragment of UGI and a protein homologous to UGI or a UGI fragment. For example, in some embodiments, the UGI domain comprises a fragment of the amino acid sequence provided below. In some embodiments, the UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the exemplary UGI sequence provided herein. In some embodiments, the UGI comprises an amino acid sequence that is homologous to the amino acid sequence provided herein below, or an amino acid sequence that is homologous to a fragment of the amino acid sequence provided herein below. In some embodiments, a protein comprising UGI or a fragment of UGI, or a homolog of UGI or a fragment of UGI, is referred to as a "UGI variant." A UGI variant has homology to UGI or a fragment thereof. For example, a UGI variant has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or at least 99.9% identity to wild-type UGI or a UGI described herein.In some embodiments, the UGI variant comprises a fragment of UGI, which fragment has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or at least 99.9% identity to wild-type UGI or the corresponding fragment of UGI provided below. In some embodiments, UGI comprises the following amino acid sequence: >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLT SD APE YKPW ALVIQDS NGENKIKML

[0174] As used herein, unless specifically stated or clear from context, the term "or" is understood to be inclusive. Unless specifically stated or clear from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural.

[0175] Unless otherwise stated or clear from the context, the term "about" as used herein is understood to be within the normal tolerance in the art, for example, within 2 standard deviations of the mean value.About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0176] The recitation of a list of chemical groups in a definition of a variable herein includes a definition of that variable as any single group or combination of the listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0177] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein. [Brief description of the drawings]

[0178] [Figure 1] Figure 1 is a schematic diagram of the A to G base editor (ABE) fusion protein nucleobase editor, which contains two adenosine deaminase domains, wild-type (wt) TadA and evolved (evo) TadA, fused to the S. pyogenes (Sp) Cas9 nickase (nCas9) with a C-terminal bipartite nuclear localization signal (NLS). The diagram also identifies three regions of the unstructured Cas9 protein where the fusion protein can be split into N- and C-terminal fragments that can be reconstituted using the split-intein system (i.e., fusing intein-N and intein-C to the N- and C-terminal fragments, respectively).

[0179] [Diagram 2]Figure 2 provides three graphs quantifying the base editing activity of a base editing system that includes a nucleobase editor fusion protein (i.e., ABE) that reproduces the fusion protein described in Figure 1 and includes a spliced ​​nCas9. The ABE was split at the indicated amino acid positions (e.g., T310, T313, A456, S469, and C574 with respect to the SpCas9 amino acid sequence). The N- and C-terminal fragments of ABE were fused to intein-N and intein-C, respectively. These fragments and the indicated guide RNAs were expressed on separate plasmids in cultured HEK293 cells expressing a protein with the ABCA4 gene carrying the 5882G>A mutation, respectively. The base editing activity of the reconstituted ABE against the ABCA4 5882A>G target was compared to the activity of a control ABE. The base editing activity was dependent on the presence of both the N- and C-terminal fragments of ABE. No base editing activity was observed when only one of the N- or C-terminal fragments of ABE was expressed.

[0180] [Diagram 3] Figure 3 is a graph confirming base editing activity with the 21 nt guide described in Figure 2. Note that the experiment in Figure 3 was performed in a different format than the experiment in Figure 2. All reconstituted ABEs showed good base editing activity. This activity was dependent on the presence of both the N- and C-terminal fragments of nCas9. No activity was observed when only the N- or C-terminal fragments of nCas9 were expressed. The activity of the reconstituted ABEs was compared to that of the control ABE7.09 and ABE7.10 fusion proteins.

[0181] [Figure 4] Figure 4 is a graph quantifying the base editing activity described in Figure 2. In Figure 4, a 20 nucleotide (nt) guide RNA containing a hammerhead ribozyme (HRz) was used. The activity of the various reconstituted ABEs was compared to that of the control ABE7.09 and ABE7.10 fusion proteins.

[0182] [Diagram 5]5A-5D show the determination of the multiplicity of infection (MOI) for AAV2 coinfection of ARPE-19 cells. FIG. 5A is a graph showing 1:1 coinfection of AAV2 / CMV-mCherry and AAV2 / CMV-EmGFP at various viral loads (vg / cell). FIG. 5B shows fluorescent images detecting EmGFP (left) and mCherry (center), as well as an overlay image showing colocalization of EmGFP and mCherry (right). FIG. 5C is a graph showing the percentage of cells expressing mCherry at various viral loads (vg / cell) when infected with AAV2 / CMV-mCherry. FIG. 5D is a graph showing the percentage of cells expressing EmGFP at various viral loads (vg / cell) when infected with AAV2 / EmGFP.

[0183] [Figure 6] Figure 6 is a series of graphs showing that delivery of split editors into ARPE-19 cells via dual AAV2 infection results in high A>G conversion rates in ABCA4 5882A. Shown are multiplicities of infection (MOI) for dual infection: 20,000 vg / cell (top left); 30,000 vg / cell (top right); 40,000 vg / cell (bottom left); 60,000 vg / cell (bottom right). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0184] As described below, the present invention provides compositions and methods for delivering a base editing system. The present invention is based, at least in part, on the discovery that an A to G nucleobase editor (ABE) can be "split" and reconstituted using a split intein. Polynucleotides encoding the N- and C-terminal fragments of ABE fused to intein-N and intein-C of a split intein pair, respectively, were delivered to cells on separate vectors along with a single guide RNA. The encoded ABE fragments were spliced ​​together to reconstitute a functional nucleobase editor fusion protein that is particularly useful for targeted editing of nucleic acid sequences.

[0185] [Intein] Intein ( in tervening pro tein ) are self-processing domains found in a wide variety of organisms that carry out the process known as protein splicing. Protein splicing is a multi-step biochemical reaction that consists of both the cleavage and formation of peptide bonds. The endogenous substrates for protein splicing are proteins found in organisms that contain inteins, but inteins can also be used to chemically engineer virtually any polypeptide backbone.

[0186] In protein splicing, an intein excises itself from a precursor polypeptide by cleaving two peptide bonds, thereby linking adjacent extein (external protein) sequences through the formation of new peptide bonds. This rearrangement occurs post-translationally (although it can also occur co-translationally). Intein-mediated protein splicing occurs spontaneously and requires only the folding of the intein domain.

[0187] Approximately 5% of inteins are split inteins, which are transcribed and translated as two separate polypeptides, the N-intein and the C-intein, each of which is fused to an extein. During translation, the intein fragments spontaneously and non-covalently assemble into a canonical intein structure and undergo protein splicing in trans. The mechanism of protein splicing involves a series of acyl transfer reactions that result in the cleavage of two peptide bonds at the intein-extein junction and the formation of a new peptide bond between the N-extein and the C-extein. This process is initiated by the activation of the peptide bond connecting the N-extein to the N-terminus of the intein. Virtually all inteins have a cysteine ​​or serine at their N-terminus that attacks the carbonyl carbon of the C-terminal residue of the N-extein. This N to O / S acyl transfer is facilitated by conserved threonine and histidine (termed the TXXH motif) and the commonly found aspartic acid, resulting in the formation of a linear (thio)ester intermediate. This intermediate is then trans-(thio)esterified by nucleophilic attack of the first residue (+1) of the C-extein, which can be a cysteine, serine, or threonine. The resulting branched (thio)ester intermediate is resolved by a unique transformation, the cyclization of the highly conserved C-terminal asparagine of the intein. This process is facilitated by histidine (as found in the highly conserved HNF motif) and the penultimate histidine, and can also involve aspartic acid. This succinimide-forming reaction excises the intein from the reaction complex, leaving the extein attached via a non-peptide bond. This structure rapidly rearranges in an intein-independent manner to a stable peptide bond.

[0188] [Adenosine deaminase] In some embodiments, the fusion protein of the present invention comprises an adenosine deaminase domain. In some embodiments, the adenosine deaminase provided herein can deaminate adenine. In some embodiments, the adenosine deaminase provided herein can deaminate adenine in deoxyadenosine residues of DNA. The adenosine deaminase can be derived from any suitable organism (e.g., E. coli). In some embodiments, the adenosine deaminase is a naturally occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). One of skill in the art can identify the corresponding residues in any homologous protein, for example, by sequence alignment and determining homologous residues. Thus, one of skill in the art can generate a mutation in any naturally occurring adenosine deaminase (e.g., one that has homology to ecTadA) that corresponds to any of the mutations described herein (e.g., any of the mutations identified in ecTadA). In some embodiments, the adenosine deaminase is derived from a prokaryote. In some embodiments, the adenosine deaminase is derived from a bacterium. In some embodiments, the adenosine deaminase is derived from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is derived from E. coli.

[0189] In one embodiment, the fusion protein of the invention comprises wild-type TadA linked to TadA7.10, which is linked to Cas9 nickase. In certain embodiments, the fusion protein comprises a single TadA7.10 domain (e.g., provided as a monomer). In other embodiments, the ABE7.10 editor comprises TadA7.10 and TadA (wt) capable of forming a heterodimer. The relevant sequences are as follows:

[0190] TadA(wt): SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD

[0191] TadA7.10: SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0192] In some embodiments, TadA (eg, one with double-stranded substrate activity) or TadA7.10 is provided as a homodimer or a monomer.

[0193] In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the amino acid sequences described in any of the adenosine deaminases provided herein. It should be understood that the adenosine deaminases provided herein can comprise one or more mutations (e.g., any of the mutations provided herein). The present disclosure provides that the deaminase domain with a particular percent identity additionally comprises any of the mutations described herein or a combination thereof. In some embodiments, the adenosine deaminase 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 a reference sequence or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence having at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues compared to any of the amino acid sequences known in the art or described herein.

[0194] In some embodiments, the adenosine deaminase comprises a D108X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in wild-type adenosine deaminase.In some embodiments, the adenosine deaminase comprises a D108G, D108N, D108V, D108A, or D108Y mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.However, it should be understood that additional deaminases can be aligned in the same way to identify the homologous amino acid residues that can be mutated as provided herein.

[0195] In some embodiments, the adenosine deaminase comprises an A106X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A106V mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0196] In some embodiments, the adenosine deaminase comprises an E155X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E155D, E155G, or E155V mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0197] In some embodiments, the adenosine deaminase comprises a D147X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D147Y mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0198] It should be understood that any of the mutations provided herein (e.g., based on the ecTadA amino acid sequence of the TadA reference sequence) can be introduced into other adenosine deaminases, such as Staphylococcus aureus TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases). It will be clear to one of skill in the art how homologous the mutated residues in ecTadA are. Thus, any of the mutations identified in ecTadA can be made in other adenosine deaminases with homologous amino acid residues. It should also be understood that any of the mutations provided herein can be made in ecTadA or another adenosine deaminase, individually or in any combination. For example, an adenosine deaminase can include D108N, A106V, E155V, and / or D147Y mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the following mutations in the TadA reference sequence (groups of mutations are separated by ";"), or a corresponding mutation in another adenosine deaminase: D108N and A106V; D108N and E155V; D108N and D147Y; A106V and E155V; A106V and D147Y; E155V and D147Y; D 108N, A106V, and E55V; D108N, A106V, and D147Y; D108N, E55V, and D147Y; A106V, E55V, and D147Y; and D108N, A106V, E55V, and D147Y; however, it should be understood that any combination of the corresponding mutations provided herein can be made in an adenosine deaminase (e.g., ecTadA).

[0199] In some embodiments, the adenosine deaminase comprises one or more of the H8X, T17X, ​​L18X, W23X, L34X, W45X, R51X, A56X, E59X, E85X, M94X, I95X, V102X, F104X, A106X, R107X, D108X, K110X, M118X, N127X, A138X, F149X, M151X, R153X, Q154X, I156X, and / or K157X mutations in the TadA reference sequence, or one or more corresponding mutations in other adenosine deaminases, where the occurrence of X indicates any amino acid other than the corresponding amino acid in a wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the H8Y, T17S, L18E, W23L, L34S, W45L, R51H, A56E, or A56S, E59G, E85K, or E85G, M94L, 1951, V102A, F104L, A106V, R107C, or R107H, or R107P, D108G, or D108N, or D108A, D108Y, K110I, M118K, N127S, A138V, F149Y, M151V, R153C, Q154L, I156D, and / or K157R mutations relative to the TadA reference sequence, or one or more corresponding mutations in other adenosine deaminases.

[0200] In certain aspects, the adenosine deaminase comprises one or more of H8X, D108X, and / or N127X mutations in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, where X indicates the presence of any amino acid. In some embodiments, the adenosine deaminase comprises one or more of H8Y, D108N, and / or N127S mutations in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase.

[0201] In some embodiments, the adenosine deaminase comprises one or more of the H8X, R26X, M61X, L68X, M70X, A106X, D108X, A109X, N127X, D147X, R152X, Q154X, E155X, K161X, Q163X, and / or T166X mutations in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the H8Y, R26W, M61I, L68Q, M70V, A106T, D108N, A109T, N127S, D147Y, R152C, Q154H or Q154R, E155G or E155V or E155D, K161Q, Q163H, and / or T166P mutations in the TadA reference sequence, or one or more corresponding mutations in other adenosine deaminases.

[0202] In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8X, D108X, N127X, D147X, R152X, and Q154X in the TadA reference sequence, or the corresponding mutation(s) in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8X, M61X, M70X, D108X, N127X, Q154X, E155X, and Q163X in the TadA reference sequence, or a corresponding mutation(s) in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8X, D108X, N127X, E155X, and T166X in the TadA reference sequence, or a corresponding mutation(s) in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8X, A106X, D108X, a mutation(s) in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8X, R126X, L68X, D108X, N127X, D147X, and E155X in the TadA reference sequence, or a corresponding mutation(s) in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.In certain embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8X, D108X, A109X, N127X, and E155X in the TadA reference sequence, or the corresponding mutation(s) in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.

[0203] In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8Y, D108N, N127S, D147Y, R152C, and Q154H in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8Y, M61I, M70V, D108N, N127S, Q154R, E155G, and Q163H in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8Y, D108N, N127S, E155V, and T166P in the TadA reference sequence, or a corresponding mutation(s) in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8Y, A106T, D108N, N127S, E155D, and K161Q in the TadA reference sequence, or a corresponding mutation(s) in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8Y, R126W, L68Q, D108N, N127S, D147Y, and E155V in the TadA reference sequence, or corresponding mutations in other adenosine deaminases. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8Y, D108N, A109T, N127S, and E155G in the TadA reference sequence, or corresponding mutation(s) in another adenosine deaminase.

[0204] In some embodiments, the adenosine deaminase comprises one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises D108N, D108G, or D108V mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises A106V and D108N mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises R107C and D108N mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises H8Y, D108N, N127S, D147Y, and Q154H mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises H8Y, R24W, D108N, N127S, D147Y, and E155V mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises D108N, D147Y, and E155V mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises H8Y, D108N, and S127S mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises A106V, D108N, D147Y, and E155V mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase.

[0205] In some embodiments, the adenosine deaminase comprises one or more of the S2X, H8X, I49X, L84X, H123X, N127X, I156X and / or K160X mutations in the tadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the S2A, H8Y, I49F, L84F, H123Y, N127S, I156F and / or K160S mutations in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase.

[0206] In some embodiments, the adenosine deaminase comprises an L84X mutant adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an L84F mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0207] In some embodiments, the adenosine deaminase comprises a H123X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a H123Y mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0208] In some embodiments, the adenosine deaminase comprises an I157X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an I157F mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0209] In certain embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations in the TadA reference sequence selected from the group consisting of L84X, A106X, D108X, H123X, D147X, E155X, and I156X, or the corresponding mutation(s) in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of S2X, I49X, A106X, D108X, D147X, and E155X in the tadA reference sequence, or a corresponding mutation(s) in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8X, A106X, D108X, N127X, and K160X in the TadA reference sequence, or a corresponding mutation(s) in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.

[0210] In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations in the TadA reference sequence selected from the group consisting of L84F, A106V, D108N, H123Y, D147Y, E155V, and I156F, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations in the TadA reference sequence selected from the group consisting of S2A, I49F, A106V, D108N, D147Y, and E155V.

[0211] In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8Y, A106T, D108N, N127S, and K160S in the TadA reference sequence, or corresponding mutations or mutations in another adenosine deaminase.

[0212] In some embodiments, the adenosine deaminase comprises one or more of E25X, R26X, R107X, A142X, and / or A143X mutations in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, where the occurrence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In certain embodiments, the adenosine deaminase comprises one or more of the E25M, E25D, E25A, E25R, E25V, E25S, E25Y, R26G, R26N, R26Q, R26C, R26L, R26K, R107P, R07K, R107A, R107N, R107W, R107H, R107S, A142N, A142D, A142G, A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q and / or A143R mutations in the TadA reference sequence, or one or more corresponding mutations in other adenosine deaminases. In some embodiments, the adenosine deaminase comprises one or more of the mutations described herein corresponding to the TadA reference sequence, or one or more of the corresponding mutations in another adenosine deaminase.

[0213] In some embodiments, the adenosine deaminase comprises an E25X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E25M, E25D, E25A, E25R, E25V, E25S, or E25Y mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0214] In some embodiments, the adenosine deaminase comprises a R26X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a R26G, R26N, R26Q, R26C, R26L, or R26K mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0215] In some embodiments, the adenosine deaminase comprises a R107X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a R107P, R07K, R107A, R107N, R107W, R107H, or R107S mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0216] In some embodiments, the adenosine deaminase comprises an A142X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A142N, A142D, A142G mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0217] In some embodiments, the adenosine deaminase comprises an A143X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q and / or A143R mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0218] In some embodiments, the adenosine deaminase comprises one or more of the H36X, N37X, P48X, I49X, R51X, M70X, N72X, D77X, E134X, S146X, Q154X, K157X, and / or K161X mutations in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, where the occurrence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the H36L, N37T, N37S, P48T, P48L, I49V, R51H, R51L, M70L, N72S, D77G, E134G, S146R, S146C, Q154H, K157N, and / or K161T mutations in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase.

[0219] In some embodiments, the adenosine deaminase comprises an H36X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H36L mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0220] In some embodiments, the adenosine deaminase comprises an N37X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an N37T or N37S mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0221] In some embodiments, the adenosine deaminase comprises a P48X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a P48T or P48L mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0222] In some embodiments, the adenosine deaminase comprises an R51X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R51H or R51L mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0223] In some embodiments, the adenosine deaminase comprises an S146X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an S146R or S146C mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0224] In some embodiments, the adenosine deaminase comprises a K157X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a K157N mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0225] In some embodiments, the adenosine deaminase comprises a P48X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a P48S, P48T, or P48A mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0226] In some embodiments, the adenosine deaminase comprises an A142X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A142N mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0227] In some embodiments, the adenosine deaminase comprises a W23X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a W23R or W23L mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0228] In some embodiments, the adenosine deaminase comprises a R152X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a R152P or R52H mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0229] In one embodiment, the adenosine deaminase can include the mutations H36L, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N. In some embodiments, the adenosine deaminase includes the following combinations of mutations relative to the tadA reference sequence, where each mutation in the combination is separated by an "_" and each combination of mutations is within parentheses: (A106V_D108N), (R107C_D108N), (H8Y_D108N_S 127S_D 147Y_Q154H), (H8Y_R24W_D108N_N127S_D147Y_E155V), (D108N_D147Y_E155V), (H8Y_D108N_S 127S), (H8Y_D108N_N127S_D147Y_Q154H), (A106V D108N D147Y E155V) (D108Q D147Y E155V) (D108M_D147Y_E155V), (D108L_D147Y_E155V), (D108K_D147Y_E155V), (D108I_D147Y_E155V), (D108F_D147Y_E155V), (A106V_D108N_D147Y), (A106V_D108M_D147Y_E155V), (E59A_A106V_D108N_D147Y_E155V), (E59A cat dead_A106V_D108N_D147Y_E155V), (L84F_A106V_D108N_H123Y_D147Y_E155V_I156Y), (L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (D103A_D014N), (G22P_D 103 A_D 104N), (G22P_D 103 A_D 104N_S 138 A) , (D 103 A_D 104N_S 138A), (R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), (E25G_R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I15 6F), (E25D_R26G_L84F_A106V_R107K_D108N_H123Y_A142N_A143G_D147Y_E155V_I15 6F), (R26Q_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), (E25M_R26G_L84F_A106V_R107P_D108N_H123Y_A142N_A143D_D147Y_E155V_I15 6F), (R26C_L84F_A106V_R107H_D108N_H123Y_A142N_D147Y_E155V_I156F), (L84F_A106V_D108N_H123Y_A142N_A143L_D147Y_E155V_I156F), (R26G_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), (E25A_R26G_L84F_A106V_R107N_D108N_H123Y_A142N_A143E_D147Y_E155V_I15 6F), (R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), (A106V_D108N_A142N_D147Y_E155V), (R26G_A106V_D108N_A142N_D147Y_E155V), (E25D_R26G_A106V_R107K_D108N_A142N_A143G_D147Y_E155V), (R26G_A106V_D108N_R107H_A142N_A143D_D147Y_E155V), (E25D_R26G_A106V_D108N_A142N_D147Y_E155V), (A106V_R107K_D108N_A142N_D147Y_E155V), (A106V_D108N_A142N_A143G_D147Y_E155V), (A106V_D108N_A142N_A143L_D147Y_E155V), (H36L_R51L_L84F_A106V_D108N_H123Y_S 146C_D147Y_E155V_I156F _K157N), (N37T_P48T_M70L_L84F_A106V_D108N_H123Y_D147Y_I49V_E155V_I156F), (N37S_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K161T), (H36L_L84F_A106V_D108N_H123Y_D147Y_Q154H_E155V_I156F), (N72S_L84F_A106V_D108N_H123Y_S 146R_D147Y_E155V_I156F), (H36L_P48L_L84F_A106V_D108N_H123Y_E134G_D147Y_E155V_I156F), 57N), (H36L_L84F_A106V_D108N_H123Y_S 146C_D147Y_E155V_I156F), (L84F_A106V_D108N_H123Y_S 146R_D147Y_E155V_I156F_K161T), (N37S_R51H_D77G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (R51L_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K157N), (D24G_Q71R_L84F_H96L_A106V_D108N_H123Y_D147Y_E155V_I156F_K160E), (H36L_G67V_L84F_A106V_D108N_H123Y_S 146T_D147Y_E155V_I156F), (Q71L_L84F_A106V_D108N_H123Y_L137M_A143E_D147Y_E155V_I156F), (E25G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L), (L84F_A91T_F104I_A106V_D108N_H123Y_D147Y_E155V_I156F), (N72D_L84F_A106V_D108N_H123Y_G125A_D147Y_E155V_I156F), (P48S_L84F_S97C_A106V_D108N_H123Y_D147Y_E155V_I156F), (W23G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (D24G_P48L_Q71R_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L), (L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), (H36L_R51L_L84F_A106V_D108N_H123Y_A142N_S 146C_D147Y_E155V_I156F _K157N), (N37S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F_K161T), (L84F_A106V_D108N_D147Y_E155V_I156F), (R51L_L84F_A106V_D108N_H123Y_S 146C_D147Y_E155V_I156F_K157N_K161T), (L84F_A106V_D108N_H123Y_S 146C_D147Y_E155V_I156F_K161T), (L84F_A106V_D108N_H123Y_S 146C_D147Y_E155V_I156F_K157N_K160E_K161T), (L84F_A106V_D108N_H123Y_S 146C_D147Y_E155V_I156F_K157N_K160E), (R74Q L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (R74A_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (R74Q_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (L84F_R98Q_A106V_D108N_H123Y_D147Y_E155V_I156F), (L84F_A106V_D108N_H123Y_R129Q_D147Y_E155V_I156F), (P48S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), (P48S_A142N), (P48T_I49V_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F_L157N), (P48T_I49V_A142N), (H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S 146C_D147Y_E155V_I156F _K157N), (H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S 146C_A142N_D147Y_E155V_I156F (H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_S 146C_D147Y_E155V_I156F _K157N), (H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_A142N_S 146C_D147Y_E155V_ I156F _K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S 146C_D147Y_E155V_I156F _K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S 146C_D147Y_E155V_I156F _K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S 146C_A142N_D147Y_E155V_I156F _K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S 146C_D147Y_E155V_I156F _K157N), (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S 146C_D147Y_E155V_I156F _K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S 146R_D147Y_E155V_I156F _K161T), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S 146C_D147Y_R152H_E155V_I156F _K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S 146C_D147Y_R152P_E155V_I156F _K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S 146C_D147Y_R152P_E155V _I156F _K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S 146C_D147Y_E155 V_I156F _K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S 146C_D147Y_R152P _E155V_I156F _K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S 146R_D147Y_E155V_I156F _K161T), (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S 146C_D147Y_R152P_E155V _I156F _K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S 146C_D147Y_R152P_E155 V_I156F _K157N).

[0230] [Cytidine deaminase] In one embodiment, the fusion protein of the present invention comprises a cytidine deaminase. In an embodiment, the cytidine deaminase provided herein can deaminate cytosine or 5-methylcytosine to uracil or thymine. In some embodiments, the cytidine deaminase provided herein can deaminate cytosine in DNA. The cytidine deaminase can be derived from any suitable organism. In some embodiments, the cytidine deaminase is one in which the naturally occurring cytidine deaminase comprises one or more mutations corresponding to any of the mutations provided herein. Those skilled in the art can identify the corresponding residues in any homologous protein, for example, by sequence alignment and determining homologous residues. Thus, those skilled in the art can generate mutations corresponding to any of the mutations described herein in any naturally occurring cytidine deaminase. In an embodiment, the cytidine deaminase is derived from a prokaryotic organism. In an embodiment, the cytidine deaminase is derived from a bacterium. In some embodiments, the cytidine deaminase is from a mammal (e.g., a human).

[0231] In some embodiments, the cytidine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the cytidine deaminase amino acid sequences described herein.It should be understood that the cytidine deaminase provided herein can comprise one or more mutations (e.g., any of the mutations provided herein).The present disclosure provides any deaminase domain with a specific percent identity, with any of the mutations described herein or combinations thereof. In some embodiments, the cytidine deaminase 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 a reference sequence or any cytidine deaminase provided herein. In some embodiments, the cytidine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues compared to any of the amino acid sequences known in the art or described herein.

[0232] The fusion protein of the present invention comprises a nucleic acid editing domain. In some embodiments, the nucleic acid editing domain can catalyze a base change from C to U. In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytidine deaminase or an adenosine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBECl deaminase. In some embodiments, the deaminase is an APOBEC 2 deaminase. In some embodiments, the deaminase is an APOBEC3 deaminase. In some embodiments, the deaminase is an APOBEC3A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase. In some embodiments, the deaminase is an APOBEC3E deaminase. In some embodiments, the deaminase is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a vertebrate deaminase. In some embodiments, the deaminase is an invertebrate deaminase. In some embodiments, the deaminase is a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse deaminase. In some embodiments, the deaminase is a human deaminase. In some embodiments, the deaminase is a rat deaminase, such as rAPOBEC1. In some embodiments, the deaminase is Petromyzon marinus cytidine deaminase 1 (pmCDAl). In some embodiments, the deaminase is human APOBEC3G. In some embodiments, the deaminase is a fragment of human APOBEC3G.In some embodiments, the deaminase is a human APOBEC3G variant comprising a D316R D317R mutation. In some embodiments, the deaminase is a fragment of human APOBEC3G and comprises a mutation corresponding to the D316R D317R mutation. In some embodiments, the nucleic acid editing domain is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or at least 99.5% identical to the deaminase domain of any deaminase described herein.

[0233] In certain embodiments, the fusion proteins provided herein include one or more features that improve the base editing activity of the fusion protein. For example, the fusion proteins provided herein may include a Cas9 domain with reduced nuclease activity. In some embodiments, the fusion proteins provided herein may have a Cas9 domain that does not have nuclease activity (dCas9), or a Cas9 domain that cleaves one strand of a double-stranded DNA molecule, called Cas9 nickase (nCas9).

[0234] [Other nucleobase editors] The invention provides nucleobase editor fusion proteins in which the cytidine deaminase or adenosine deaminase domain in the fusion protein of the invention is replaced with substantially any nucleobase editor known in the art.

[0235] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting exemplary Cas9 domains are provided herein. The Cas9 domain can be a nuclease-active Cas9 domain, a nuclease-inactive Cas9 domain, or a Cas9 nickase. In some embodiments, the Cas9 domain is a nuclease-active domain. For example, the Cas9 domain can be a Cas9 domain that cleaves both strands of a double-stranded nucleic acid (e.g., both strands of a double-stranded DNA molecule). In some embodiments, the Cas9 domain comprises any one of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences described herein.In some embodiments, the Cas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical consecutive amino acid residues compared to any one of the amino acid sequences described herein.

[0236] In some embodiments, the Cas9 domain is a nuclease-inactive Cas9 domain (dCas9). For example, the dCas9 domain can bind to a double-stranded nucleic acid molecule (e.g., via a gRNA molecule) without cleaving either strand of the double-stranded nucleic acid molecule. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10X mutation and a H840X mutation of the amino acid sequence described herein, or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid change. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10A mutation and a H840A mutation of the amino acid sequence described herein, or a corresponding mutation in any of the amino acid sequences described herein. As an example, the nuclease-inactive Cas9 domain comprises the following amino acid sequence described in the cloning vector pPlatTET-gRNA2 (Accession No. BAV54124): (See 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.)

[0237] Further suitable nuclease-inactive dCas9 domains will be apparent to those skilled in the art based on this disclosure and knowledge in the art, and are within the scope of this disclosure.Such further 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). In some embodiments, the dCas9 domain comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to any of the dCas9 domains provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any of the amino acid sequences described herein.In some embodiments, the Cas9 domain comprises an amino acid sequence that has at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any of the amino acid sequences described herein.

[0238] In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase can be a Cas9 protein that can cleave only one strand of a double-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, the Cas9 nickase cleaves the target strand of a double-stranded nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is base-paired (complementary) to the gRNA (e.g., sgRNA) that is bound to the Cas9. In some embodiments, the Cas9 nickase cleaves the non-target, non-base-edited strand of a double-stranded nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is not base-paired to the gRNA (e.g., sgRNA) that is bound to the Cas9. In some embodiments, the Cas9 nickase cleaves the non-target, non-base-edited strand of a double-stranded nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is not base-paired to the gRNA (e.g., sgRNA) that is bound to the Cas9. In some embodiments, the Cas9 nickase cleaves the H840A mutation and has an aspartic acid residue at position 10, or a corresponding mutation. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the Cas9 nickases provided herein. Additional suitable Cas9 nickases will be apparent to one of skill in the art based on this disclosure and knowledge in the art, and are within the scope of this disclosure.

[0239] [Cas9 domain with reduced PAM exclusivity] In one particular embodiment, the invention features a nucleobase editor that includes a Cas9 domain split into two fragments, each of the two fragments having a terminal intein, i.e., the N-terminal fragment is fused at its C-terminus to one member of the intein system and the C-terminal fragment has a member of the intein system at its N-terminus.

[0240] Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a canonical NGG PAM sequence to bind to a particular nucleic acid region, where the "N" in "NGG" is adenosine (A), thymidine (T) or cytosine (C), and G is guanosine. This may limit the ability to edit a desired base in a genome. In some embodiments, the base editing fusion proteins provided herein may need to be placed in a precise location, e.g., in a region containing a target base, upstream of the PAM. See, e.g., Komor, AC, et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016), the entire contents of which are incorporated herein by reference. Thus, in some embodiments, any of the fusion proteins provided herein may contain a Cas9 domain capable of binding to a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-standard PAM sequences have been described in the art and will be apparent to those skilled in the art. For example, Cas9 domains that bind to non-standard PAM sequences are described in Kleinstiver, BP, et al., "Engineered CRISPR-Cas9 nucleases with altered PAM specificities" Nature 523, 481-485 (2015); and Kleinstiver, BP, et al., "Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition" Nature Biotechnology 33, 1293-1298 (2015), the entire contents of each of which are incorporated herein by reference. Table 1 below lists some PAM variants.

[0241] Table 1. Cas9 proteins and corresponding PAM sequences [Table 1]

[0242] In some embodiments, the PAM is NGC. In some embodiments, the NGC PAM is recognized by Cas9 variant. In some embodiments, the NGC PAM variant comprises one or more amino acid substitutions selected from D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E and T1337R (collectively referred to as "MQKFRAER").

[0243] In some embodiments, the Cas9 domain is a Cas9 domain from Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domain is a nuclease-active SaCas9, a nuclease-inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, the SaCas9 comprises a N579A mutation, or a corresponding mutation in any of the amino acid sequences provided herein.

[0244] In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence with a non-standard PAM, and in some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence with an NNGRRT PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of E781X, N967X, and R1014X mutations, or corresponding mutations in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of E781K, N967K, and R1014H mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SaCas9 domain comprises E781K, N967K, or R1014H mutations, or corresponding mutations in any of the amino acid sequences provided herein.

[0245] Exemplary SaCas9 Sequences KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQ KKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLPKKVDLSQQK EIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEE NSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNLDVKVKSINGGFTSFLRRKWKFKKERNKGY KHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKL KKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVI KKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG Residue N579, underlined and bolded above, can be mutated (e.g., to A579) to generate a SaCas9 nickase.

[0246] Exemplary SaCas9n Sequences KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQ KKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRKLVPKKVDLSQQK EIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEE ASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNLDVKVKSINGGFTSFLRRKWKFKKERNKGY KHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKL KKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVI KKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG Residue A579, which can be mutated from N579 to generate SaCas9 nickase, is shown underlined and in bold.

[0247] Exemplary SaKKH Cas9 KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQ KKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLPKKVDLSQQK EIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEE A SKKGNRTPFQYLSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEITEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNR K LINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFY KNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPP H IIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG. Residue A579 can be mutated from N579 to generate SaCas9 nickase and is shown in bold and underlined. Residues K781, K967, and H1014 can be mutated from E781, N967, and R1014 to generate SaKKH Cas9 and are shown in italics and underlined.

[0248] In some embodiments, the Cas9 domain is a Cas9 domain from Streptococcus pyogenes (SpCas9). In some embodiments, the SpCas9 domain is a nuclease-active SpCas9, a nuclease-inactive SpCas9 (SpCas9d), or a SpCas9 nickase (SpCas9n). In some embodiments, the SpCas9 comprises a D9X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid other than D. In some embodiments, the SpCas9 comprises a D9A mutation, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence with a non-canonical PAM. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence with a NGG, NGA, or NGCG PAM sequence. In some embodiments, the SpCas9 domain comprises one or more of D1134X, R1334X, and T1336X mutations, or corresponding mutations in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1134E, R1334Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises D1134E, R1334Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises one or more of D1134X, R1334X, and T1336X mutations, or corresponding mutations in any of the amino acid sequences provided herein, where X is any amino acid.In some embodiments, the SpCas9 domain comprises one or more of D1134V, R1334Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises D1134V, R1334Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises one or more of D1134X, G1217X, R1334X, and T1336X mutations, or corresponding mutations in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1134V, G1217R, R1334Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises a D1134V, G1217R, R1334Q, and T1336R mutation, or the corresponding mutations in any of the amino acid sequences provided herein.

[0249] In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a Cas9 polypeptide described herein. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises the amino acid sequence of any Cas9 polypeptide described herein. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein consists of the amino acid sequence of any Cas9 polypeptide described herein.

[0250] Exemplary SpCas9

[0251] Exemplary SpCas9n

[0252] Exemplary SpEQR Cas9 E SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQSITGLYETRIDLSQLGGD Residues E1134, Q1334, and R1336 above can be mutated from D1134, R1334, and T1336 to generate SpEQR Cas9 and are shown underlined and in bold.

[0253] Exemplary SpVQR Cas9 V SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQSITGLYETRIDLSQLGGD Residues V1134, Q1334, and R1336 above can be mutated from D1134, R1334, and T1336 to yield SpVQR Cas9 and are shown underlined and in bold.

[0254] Exemplary SpVRER Cas9 V SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASA R ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK E Y R STKEVLDATLIHQSITGLYETRIDLSQLGGD Residues V1134, R1217, Q1334, and R1336 above can be mutated from D1134, G1217, R1334, and T1336 to generate SpVRER Cas9, and are shown underlined and in bold.

[0255] In certain embodiments, a fusion protein of the invention comprises a dCas9 domain that binds to a canonical PAM sequence and an nCas9 domain that binds to a non-canonical PAM sequence (e.g., a non-canonical PAM identified in Table 1). In another embodiment, a fusion protein of the invention comprises an nCas9 domain that binds to a canonical PAM sequence and a dCas9 domain that binds to a non-canonical PAM sequence (e.g., a non-canonical PAM identified in Table 1).

[0256] [High-fidelity Cas9 domain] Some aspects of the present disclosure provide a high-fidelity Cas9 domain. In some embodiments, the high-fidelity Cas9 domain is an artificial Cas9 domain that comprises one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA compared to the corresponding wild-type Cas9 domain. Without wishing to be bound by a particular theory, a high-fidelity Cas9 domain that reduces the electrostatic interaction with the sugar-phosphate backbone of DNA may have fewer off-target effects. In some embodiments, the Cas9 domain (e.g., a wild-type Cas9 domain) comprises one or more mutations that reduce the binding between the Cas9 domain and the sugar-phosphate backbone of DNA. In some embodiments, the Cas9 domain comprises one or more mutations that reduce the bond between the Cas9 domain and the sugar-phosphate backbone of the DNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70%.

[0257] In some embodiments, any of the Cas9 fusion proteins provided herein comprise one or more of N497X, R661X, Q695X, and / or Q926X mutations, or corresponding mutations in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, any of the Cas9 fusion proteins provided herein comprise one or more of N497A, R661A, Q695A, and / or Q926A mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the Cas9 domain comprises a D10A mutation, or corresponding mutations in any of the amino acid sequences provided herein. Cas9 domains with high fidelity are known in the art and will be apparent to one of skill in the art. For example, high-fidelity Cas9 domains are described in Kleinstiver, BP, et al. "High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects." Nature 529, 490-495 (2016); and Slaymaker, IM, et al. "Rationally engineered Cas9 nucleases with improved specificity." Science 351, 84-88 (2015), the entire contents of each of which are incorporated herein by reference.

[0258] High-fidelity Cas9 domain mutations for Cas9 are shown in bold and underlined. DKKYSIGL AIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLNLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKIKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT A FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRIEMIEERLKTYAHLFDDKVMKQLKRRRYTGWG A LSRKLINGIRDKQSGKTILDFLKSDGFANRNFM A LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR AITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKY FFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0259] The Cas9 nuclease has two functional endonuclease domains, RuvC and HNH. Upon binding to target DNA, Cas9 undergoes a conformational change that positions the nuclease domain to cleave the opposite strand of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) in the target DNA (approximately 3-4 nucleotides upstream of the PAM sequence). The resulting DSB can be repaired by one of two general repair pathways: (1) the efficient but error-prone non-homologous end joining (NHEJ) pathway; or (2) the less efficient but high-fidelity homology-guided repair (HDR) pathway.

[0260] The "efficiency" of non-homologous end joining (NHEJ) and / or homology directed repair (HDR) can be calculated by any convenient method. For example, in some cases, the efficiency can be expressed as a percentage of successful HDR. For example, a test nuclease assay can be used to generate cleavage products, and the ratio of products to substrate can be used to calculate the percentage. For example, a test nuclease enzyme can be used that directly cleaves the DNA containing the newly incorporated restriction sequence as a result of successful HDR. More substrates cleaved indicates a higher rate of HDR (more efficient HDR). As an illustrative example, the rate (percentage) of HDR can be calculated using the following formula [(cleavage products) / (substrate+cleavage products)] (e.g., (b+c) / (a+b+c) where "a" is the band intensity of the DNA substrate and "b" and "c" are the cleavage products).

[0261] In some cases, efficiency can be expressed as the success rate of NHEJ. For example, a T7 endonuclease I assay can be used to generate cleavage products and the ratio of products to substrates can be used to calculate the percentage of NHEJ. T7 endonuclease I cleaves mismatched heteroduplex DNA resulting from hybridization of wild-type and mutant DNA strands (NHEJ produces small random insertions or deletions (indels) at the site of the initial cleavage). More cleavage indicates a higher rate of NHEJ (higher efficiency of NHEJ). As an illustrative example, the rate (percentage) of NHEJ can be calculated using the formula (1-(1-(b+c) / (a+b+c)) 1 / 2 ) × 100, where "a" is the band intensity of the DNA substrate and "b" and "c" are the cleavage products (Ran et. al., 2013 Sep. 12; 154(6):1380-9; and Ran et al., Nat Protoc. 2013 Nov.; 8(11): 2281-2308).

[0262] The NHEJ repair pathway is the most active repair mechanism, frequently resulting in small nucleotide insertions or deletions (indels) at DSB sites. The random nature of NHEJ-mediated DSB repair has important practical implications, as cell populations expressing Cas9 and gRNA or guide polynucleotides can result in an array of diverse mutations. In most cases, NHEJ generates small indels in the target DNA, resulting in amino acid deletions, insertions, or frameshift mutations that lead to premature stop codons within the open reading frame (ORF) of the target gene. The ideal end result is a loss-of-function mutation within the target gene.

[0263] While NHEJ-mediated DSB repair often disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to generate specific nucleotide changes ranging from single nucleotide changes to larger insertions such as the addition of fluorophores or tags.

[0264] To utilize HDR for gene editing, a DNA repair template containing the desired sequence can be delivered to a cell type of interest along with gRNA and Cas9 or Cas9 nickase. The repair template can contain additional homologous sequences immediately upstream and downstream (called left and right homologous arms) of the desired edit as well as its target. The length of each homologous arm can depend on the magnitude of the change to be introduced, with larger insertions requiring longer homologous arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. The efficiency of HDR is generally low (less than 10% corrected alleles), even in cells expressing Cas9, gRNA, and an exogenous repair template. Because HDR occurs between the S and G2 phases of the cell cycle, the efficiency of HDR can be increased by synchronizing the cells. Chemical or genetic inhibition of genes involved in NHEJ can also increase HDR frequency.

[0265] In some embodiments, Cas9 is a modified Cas9. A given gRNA target sequence may have additional sites with partial homology throughout the genome. These sites are called off-targets and need to be considered when designing gRNA, but in addition to optimizing gRNA design, Cas9 can also be modified to increase CRISPR specificity. Cas9 generates 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 generates DNA nicks instead of DSBs. Nickase can also be combined with HDR-mediated gene editing for specific gene editing.

[0266] In some cases, the Cas9 is a variant Cas9 protein. The variant Cas9 polypeptide has an amino acid sequence that differs by one amino acid (e.g., has a deletion, insertion, substitution, fusion) compared to the amino acid sequence of the wild-type Cas9 protein. In some cases, 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 cases, the variant Cas9 polypeptide has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 protein. In some cases, the variant Cas9 protein does not have substantial nuclease activity. When the subject Cas9 protein is a variant Cas9 protein that does not have substantial nuclease activity, it can be referred to as "dCas9".

[0267] In some cases, 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 a wild-type Cas9 protein (e.g., a wild-type Cas9 protein).

[0268] In some cases, variant Cas9 protein can cleave the complementary strand of guide target sequence, but has a reduced ability to cleave the non-complementary strand of double-stranded guide target sequence. For example, variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of RuvC domain. As a non-limiting example, in some embodiments, variant Cas9 protein has D10A (aspartic acid to alanine at amino acid position 10), and thus can cleave the complementary strand of double-stranded guide target sequence, but has a reduced ability to cleave the non-complementary strand of double-stranded guide target sequence (and thus, when this variant Cas9 protein cleaves double-stranded target nucleic acid, it generates a single-strand break (SSB) instead of a double-strand break (DSB)) (see, for example, Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21).

[0269] In some cases, the variant Cas9 protein can cleave the non-complementary strand of the double-stranded guide target sequence, but has a reduced ability to cleave the complementary strand of the guide target sequence. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif). As a non-limiting example, in some embodiments, the variant Cas9 protein has a H840A (histidine to alanine at amino acid position 840) mutation, and thus can cleave the non-complementary strand of the guide target sequence, but has a reduced ability to cleave the complementary strand of the guide target sequence (so that when the variant Cas9 protein cleaves the double-stranded guide target sequence, an SSB is generated instead of a DSB). Such a Cas9 protein has a reduced ability to cleave the guide target sequence (e.g., a single-stranded guide target sequence), but retains the ability to bind to the guide target sequence (e.g., a single-stranded guide target sequence).

[0270] In some cases, the variant Cas9 protein has a reduced ability to cleave both complementary and non-complementary strands of double-stranded target DNA.As a non-limiting example, in some cases, the variant Cas9 protein has both D10A and H840A mutations, so that the polypeptide has a reduced ability to cleave both complementary and non-complementary strands of double-stranded target DNA.Such Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0271] As another non-limiting example, in some cases, a variant Cas9 protein has a W476A and a W1126A mutation such that the polypeptide has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0272] As another non-limiting example, in some cases, variant Cas9 proteins have P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, resulting in a polypeptide with reduced ability to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0273] As another non-limiting example, in some cases, variant Cas9 proteins have H840A, W476A, and W1126A mutations, such that the polypeptide has a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retains the ability to bind to target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some cases, variant Cas9 proteins have H840A, D10A, W476A, and W1126A mutations, such that the polypeptide has a reduced ability to cleave target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). In some embodiments, variant Cas9 has a restored catalytic His residue at position 840 of the Cas9 HNH domain (A840H).

[0274] As another non-limiting example, in some cases, the variant Cas9 protein has H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, such that the polypeptide has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind to target DNA (e.g., single-stranded target DNA).As another non-limiting example, in some cases, the variant Cas9 protein has D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, such that the polypeptide has a reduced ability to cleave target DNA.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). When the variant Cas9 protein has W476A and W1126A mutations, or when the variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, the variant Cas9 protein does not bind to the PAM sequence efficiently. Therefore, in such cases, when such variant Cas9 protein is used in the method of binding, the method does not require a PAM sequence. In other words, in some cases, when such variant Cas9 protein is used in the method of binding, the method may include a guide RNA, but the method can be performed in the absence of a PAM sequence (therefore, the specificity of binding is provided by the target segment of the guide RNA). To achieve the above effect, other residues can be mutated (i.e., inactivate one or the other nuclease part). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 can be altered (i.e., substituted). Mutations other than alanine substitutions are also suitable.

[0275] In some embodiments, a variant Cas9 protein with reduced catalytic activity (e.g., where 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) can bind to the target DNA in a site-specific manner, as long as it retains the ability to interact with the guide RNA (since it is guided to the target DNA sequence by the guide RNA).

[0276] 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.

[0277] Alternatives to S. pyogenes Cas9 could include RNA-guided endonucleases from the Cpf1 family that show cleavage activity in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of a class II CRISPR / Cas system. This adaptive 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 Cas9 nuclease, the result of Cpf1-mediated DNA cleavage is a double-stranded break with a short 3' overhang. The staggered cleavage pattern of Cpf1 can open up the possibility of directional gene transfer, similar to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Similar to the Cas9 variants and orthologues mentioned above, Cpf1 can also expand the number of sites that CRISPR can target to AT-rich regions or AT-rich genomes that lack the NGG PAM sites preferred by SpCas9. The Cpf1 locus contains an α / β mixed domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. In addition, Cpf1 does not have the HNH endonuclease region, and the N-terminus of Cpf1 does not have the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain organization showed that Cpf1 is functionally unique and classified as a class 2, type V CRISPR system. The Cpf1 locus encoded Cas1, Cas2 and Cas4 proteins that were more similar to type I and III systems than to type II systems. Functional Cpf1 does not require trans-activating CRISPR RNA (tracrRNA); therefore, it only requires CRISPR (crRNA).This is beneficial for genome editing because Cpf1 is not only smaller than Cas9 but also has a smaller sgRNA molecule (approximately half the number of nucleotides as Cas9). In contrast to the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex cleaves the target DNA or RNA by identification of a protospacer adjacent to the motif 5'-YTN-3'. After identification of the PAM, Cpf1 introduces a sticky-end-like DNA double-strand break with a 4- or 5-nucleotide overhang.

[0278] [Protospacer adjacent motif] The term "protospacer adjacent motif (PAM)" or PAM-like motif refers to a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. In some embodiments, the PAM can be a 5'PAM (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3'PAM (i.e., located downstream of the 5' end of the protospacer).

[0279] The PAM sequence is essential for target binding, but its exact sequence depends on the type of Cas protein.

[0280] Base editors provided herein can include a domain derived from a CRISPR protein that can bind to a nucleotide sequence that includes a canonical or non-canonical protospacer adjacent motif (PAM) sequence. A PAM site is a nucleotide sequence that is adjacent to a target polynucleotide sequence. Some aspects of the disclosure provide base editors that include all or part of a CRISPR protein with different PAM specificities. For example, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), typically require a canonical NGG PAM sequence to bind to a specific nucleic acid region, where "N" in "NGG" is adenine (A), thymine (T), guanine (G), or cytosine (C), and G is guanine. The PAM can be CRISPR protein specific and can vary between different base editors that include domains derived from different CRISPR proteins. The PAM can be 5' or 3' of the target sequence. The PAM can be upstream or downstream of the target sequence. A PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. Often, PAMs are 2-6 nucleotides in length. Several PAM variants are listed in Table 1.

[0281] In some embodiments, the SpCas9 has specificity for the PAM nucleic acid sequence 5'-NGC-3' or 5'-NGC-3'. In various embodiments of the above aspects, the SpCas9 is Cas9 or a Cas9 variant listed in Table 1. In various embodiments of the above aspects, the modified SpCas9 is spCas9-MQKFRAER. In some embodiments, the variant Cas protein can be spCas9, spCas9-VRQR, spCas9-VRER, xCas9 (sp), saCas9, saCas9-KKH, SpCas9-MQKFRAER, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL. In one particular embodiment, a modified SpCas9 is used that contains the amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and has specificity for the modified PAM 5'-NGC-3'.

[0282] In some embodiments, the PAM is NGT. In some embodiments, the NGT PAM is a variant. In some embodiments, the NGT PAM variant is generated through targeted mutation at one or more of residues 1335, 1337, 1135, 1136, 1218, and / or 1219. In some embodiments, the NGT PAM variant is generated through targeted mutation at one or more of residues 1219, 1335, 1337, 1218. In some embodiments, the NGT PAM variant is generated through targeted mutation at one or more of residues 1135, 1136, 1218, 1219, and 1335. In some embodiments, the NGT PAM variant is selected from the set of targeted mutations provided in Tables 2 and 3 below.

[0283] Table 2: NGT PAM variant mutations at residues 1219, 1335, 1337, and 1218 [Table 2]

[0284] Table 3: NGT PAM variant mutations at residues 1135, 1136, 1218, 1219, and 1335 [Table 3]

[0285] In some embodiments, the NGT PAM variant is selected from variants 5, 7, 28, 31, or 36 of Tables 2 and 3. In some embodiments, the variant has improved NGT PAM recognition.

[0286] In some embodiments, the NGT PAM variant has mutations at residues 1219, 1335, 1337, and / or 1218. In some embodiments, the NGT PAM variant is selected with mutations to improve recognition from the variants provided in Table 4 below.

[0287] Table 4: NGT PAM variant mutations at residues 1219, 1335, 1337, and 1218 [Table 4]

[0288] In some embodiments, the NGT PAM is selected from the variants provided in Table 5 below.

[0289] Table 5: NGT PAM variants [Table 5]

[0290] In some embodiments, the Cas9 domain is a Cas9 domain from Streptococcus pyogenes (SpCas9). In some embodiments, the SpCas9 domain is a nuclease-active SpCas9, a nuclease-inactive SpCas9 (SpCas9d), or a SpCas9 nickase (SpCas9n). In some embodiments, the SpCas9 comprises a D9X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid other than D. In some embodiments, the SpCas9 comprises a D9A mutation, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence with a non-canonical PAM. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence with a NGG, NGA, or NGCG PAM sequence.

[0291] In some embodiments, the SpCas9 domain comprises one or more of D1135X, R1335X, and T1336X mutations, or corresponding mutations in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1135E, R1335Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises D1135E, R1335Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises one or more of D1135X, R1335X, and T1336X mutations, or corresponding mutations in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1135V, R1335Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises D1135V, R1335Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises one or more of D1135X, G1217X, R1335X, and T1336X mutations, or corresponding mutations in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1135V, G1217R, R1335Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises a D1135V, G1217R, R1335Q, and T1336R mutation, or the corresponding mutations in any of the amino acid sequences provided herein.

[0292] In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a Cas9 polypeptide described herein. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises the amino acid sequence of any Cas9 polypeptide described herein. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein consists of the amino acid sequence of any Cas9 polypeptide described herein.

[0293] In some examples, the PAM recognized by the CRISPR protein-derived domain of a base editor disclosed herein can be provided to a cell on an oligonucleotide that is separate from the insert (e.g., an AAV insert) that encodes the base editor. In such embodiments, providing the PAM on a separate oligonucleotide allows for cleavage of a target sequence that would otherwise be unable to be cleaved due to the absence of an adjacent PAM on the same polynucleotide as the target sequence.

[0294] In one embodiment, S. pyogenes Cas9 (SpCas9) can be used as a CRISPR endonuclease for genome engineering, although others can be used. In some embodiments, different endonucleases can be used to target specific genomic targets. In some embodiments, synthetic SpCas9-derived variants with non-NGG PAM sequences can be used. Additionally, other Cas9 orthologs from various species have been identified, and these "non-SpCas9" can bind to various PAM sequences that can also be useful in the present disclosure. For example, the relatively large size of SpCas9 (about 4 kilobases (kb) coding sequence) can result in SpCas9 cDNA plasmids that cannot be efficiently expressed in cells. Conversely, the coding sequence of Staphylococcus aureus Cas9 (SaCas9) is about 1 kilobase shorter than SpCas9, and can therefore be efficiently expressed in cells. Similar to SpCas9, SaCas9 endonuclease is capable of modifying target genes in mammalian cells in vitro and in mice in vivo. In some embodiments, Cas proteins can target different PAM sequences. In some embodiments, target genes can be adjacent to Cas9 PAMs, e.g., 5'-NGG. In other embodiments, other Cas9 orthologs can have different PAM requirements. For example, PAMs such as those of S. thermophilus (5'-NNAGAA for CRISPR1, 5'-NGGNG for CRISPR3) and those of Neisseria meningiditis (5'-NNNNGATT) can also be found adjacent to target genes.

[0295] In some embodiments, for S. pyogenes systems, the target gene sequence can precede (i.e., 5' to) the 5'-NGG PAM, and a 20 nt guide RNA sequence can base-pair with the opposite strand to mediate Cas9 cleavage adjacent to the PAM. In some embodiments, the adjacent cleavage can be (about) 3 base pairs upstream of the PAM. In some embodiments, the adjacent cleavage can be (about) 10 base pairs upstream of the PAM. In some embodiments, the adjacent cleavage can be (about) 0-20 base pairs upstream of the PAM. For example, the adjacent cleavage can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs next to the PAM upstream. The adjacent cleavage can also be 1-30 base pairs downstream of the PAM. The following are sequences of exemplary SpCas9 proteins that can bind to PAM sequences:

[0296] The amino acid sequence of an exemplary PAM-bound SpCas9 is as follows:

[0297] The amino acid sequence of an exemplary PAM-bound SpCas9n is as follows:

[0298] The amino acid sequence of an exemplary PAM-bound SpEQR Cas9 is as follows: E SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQSITGLYETRIDLSQLGGD In this sequence, residues E1135, Q1335, and R1337, which can be mutated from D1135, R1335, and T1337 to generate SpEQR Cas9, are underlined and in bold.

[0299] The amino acid sequence of an exemplary PAM-linked SpVQR Cas9 is as follows: V SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQSITGLYETRIDLSQLGGD In this sequence, residues V1135, Q1335, and R1336, which can be mutated from D1135, R1335, and T1336 to generate SpVQR Cas9, are underlined and shown in bold.

[0300] The amino acid sequence of an exemplary PAM-linked SpVRER Cas9 is as follows: V SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASA R ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK E Y R STKEVLDATLIHQSITGLYETRIDLSQLGGD

[0301] In some embodiments, the Cas9 domain is a recombinant Cas9 domain. In some embodiments, the recombinant Cas9 domain is a SpyMacCas9 domain. In some embodiments, the SpyMacCas9 domain is a nuclease-active SpyMacCas9, a nuclease-inactive SpyMacCas9 (SpyMacCas9d), or a SpyMacCas9 nickase (SpyMacCas9n). In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence with a non-canonical PAM. In some embodiments, the SpyMacCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence with a NAA PAM sequence.

[0302] Example SpyMacCas9

[0303] In some cases, the variant Cas9 protein has H840A, P475A, W476A, N477A, ​​D1125A, W1126A, D1218A mutations, resulting in a reduced ability to cleave target DNA or RNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some cases, the variant Cas9 protein has D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1218A mutations, resulting in a reduced ability of the polypeptide to cleave target DNA (e.g., single-stranded target DNA). Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). When the variant Cas9 protein has W476A and W1126A mutations, or when the variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A, and D1218A mutations, the variant Cas9 protein does not bind to the PAM sequence efficiently. Therefore, in such cases, when such variant Cas9 protein is used in the method of binding, the method does not require a PAM sequence. In other words, in some cases, when such variant Cas9 protein is used in the method of binding, the method may include a guide RNA, but the method can be performed in the absence of a PAM sequence (therefore, the specificity of binding is provided by the target segment of the guide RNA). To achieve the above effect, other residues can be mutated (i.e., inactivate one or the other nuclease part). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 can be altered (i.e., substituted). Mutations other than alanine substitutions are also suitable.

[0304] In some embodiments, the base editor CRISPR protein-derived domain can include all or a portion of a Cas9 protein with a canonical PAM sequence (NGG). In other embodiments, the base editor Cas9-derived domain can use a non-canonical PAM sequence. Such sequences are described in the art and will be apparent to one of skill in the art. For example, Cas9 domains that bind to non-canonical PAM sequences are described in Kleinstiver, BP, et al., "Engineered CRISPR-Cas9 nucleases with altered PAM specificities" Nature, 523, 481-485 (2015); and Kleinstiver, BP, et al., "Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition" Nature Biotechnology, 33, 1293-1298 (2015), the entire contents of each of which are incorporated herein by reference.

[0305] [Fusion protein containing a nuclear localization sequence (NLS)] In some embodiments, the fusion proteins provided herein further comprise one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, e.g., nuclear localization sequences (NLSs). In one embodiment, a bipartite NLS is used. In some embodiments, the NLS comprises an amino acid sequence that facilitates import of the protein comprising the NLS into the cell nucleus (e.g., by nuclear transport). In some embodiments, any of the fusion proteins provided herein further comprise a nuclear localization sequence (NLS). In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the Cas9 domain. In some embodiments, the NLS is fused to the C-terminus of the nCas9 domain or dCas9 domain. In some embodiments, the NLS is fused to the N-terminus of the deaminase. In some embodiments, the NLS is fused to the C-terminus of the deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises the amino acid sequence of any one of the NLS sequences provided or referenced herein. Additional nuclear localization sequences are known in the art and will be apparent to those skilled in the art. For example, NLS sequences are described in Plank et al., PCT / EP2000 / 011690, the contents of which are incorporated herein by reference for disclosure of exemplary nuclear localization sequences. In some embodiments, the NLS comprises the amino acid sequence PKKKRKVEGADKRTADGSEFESPKKKRKV, KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRKPKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.In some embodiments, the NLS is in a linker or is flanked by a linker, such as a linker described herein. In some embodiments, the N-terminal or C-terminal NLS is a bipartite NLS. A bipartite NLS contains two basic amino acid clusters separated by a relatively short spacer sequence (hence the term bipartite, different from monopartite NLS). The nucleoplasmin NLS KR[PAATKKAGQA]KKKK is a prototype of a ubiquitous bipartite signal, with two clusters of basic amino acids separated by a spacer of about 10 amino acids. An exemplary bipartite NLS sequence is PKKKRKVEGADKRTADGSEFES PKKKRKV.

[0306] In some embodiments, the fusion proteins of the invention do not contain a linker sequence. In certain embodiments, there is a linker sequence between one or more domains or proteins.

[0307] It should be understood that the fusion protein of the present disclosure may include one or more additional features. For example, in some embodiments, the fusion protein may include an inhibitor, a cytoplasmic localization sequence, an export sequence such as a nuclear export sequence, or other localization sequence, as well as a sequence tag that is useful for solubilizing, purifying, or detecting the fusion protein. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier tag (BCCP) tag, myc-tag, calmodulin tag, FLAG-tag, hemagglutinin (HA)-tag, polyhistidine tag (also called histidine tag or His-tag), maltose binding protein (MBP)-tag, nus-tag, glutathione-S-transferase (GST)-tag, green fluorescent protein (GFP)-tag, thioredoxin tag, S-tag, soft tag (e.g. Softag1, Softag3), strepto tag, biotin ligase tag, FLASH tag, V5 tag, and SBP-tag. Further suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein comprises one or more His tags.

[0308] [Linker] In certain embodiments, a linker may be used to link any of the peptides or peptide domains of the invention. The linker may be as simple as a covalent bond, or it may be a polymeric linker that is multiatomic in length. The linker may be a peptide linker or a non-peptide linker. In certain embodiments, the linker may be a UV-cleavable linker. In some embodiments, the linker may be a polynucleotide linker, such as an RNA linker. In certain embodiments, the linker is a polypeptide or is based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, a disulfide bond, a carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched, aliphatic or heteroaliphatic linker. In certain embodiments, the linker is a polymer (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain aspects, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises an amino acid. In certain aspects, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include a functionalized moiety to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker.Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0309] In certain embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is a bond (e.g., a covalent bond), an organic molecule, a group, a polymer, or a chemical moiety. In certain embodiments, the linker is about 3 to about 104 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 61, 62, 63, 64, 65, 70, 75, 80, 85, 90, 95, or 100) amino acids in length.

[0310] [Cas9 complex with guide RNA] Some aspects of the disclosure provide complexes comprising any of the fusion proteins provided herein, wherein a guide RNA may be utilized to achieve an optimal length for activity of the nucleobase editor (e.g., (GGGS) n , (GGGGS) n and (G) n From a very flexible linker of the form (EAAAK) n , (SGGS) n , to more rigid linkers in the form of SGSETPGTSESATPES (see, e.g., Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82, the entire contents of which are incorporated herein by reference), and (XP) nIn some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker is (GGS) n motif, where n is 1, 3, or 7. In some embodiments, the Cas9 domain of the fusion proteins provided herein is fused via a linker comprising the amino acid sequence SGSETPGTSESATPES.

[0311] In some embodiments, the guide nucleic acid (e.g., guide RNA) is 15-100 nucleotides in length and comprises a sequence of at least 10 consecutive nucleotides that are complementary to the target sequence. In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive nucleotides that are complementary to the target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is a sequence in a bacterial, yeast, fungal, insect, plant, or animal genome. In some embodiments, the target sequence is a sequence in a human genome. In some embodiments, the 3' end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is immediately adjacent to a non-canonical PAM sequence (e.g., a sequence listed in Table 1). In some embodiments, the guide nucleic acid (e.g., guide RNA) is complementary to a sequence associated with a disease or disorder.

[0312] Some aspects of the disclosure provide methods of using the fusion proteins or complexes provided herein. For example, some aspects of the disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins provided herein and at least one guide RNA, where the guide RNA is about 15-100 nucleotides in length and comprises a sequence of at least 10 contiguous nucleotides that are complementary to the target sequence. In some embodiments, the 3' end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence. In some embodiments, the 3' end of the target sequence is immediately adjacent to an NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, NGTN, NGTN, or 5' (TTTV) sequence.

[0313] In some embodiments, the fusion proteins of the present invention are used to induce mutations in a target of interest. These mutations can affect the function of the target. For example, using a nucleobase editor to target a regulatory region can change the function of the regulatory region and reduce the expression of a downstream protein.

[0314] It will be understood that the numbering of a particular position or residue in each sequence depends on the particular protein and numbering scheme used. For example, the numbering of a precursor of a mature protein may differ from that of the mature protein itself, and sequence differences between species may affect the numbering. Those skilled in the art can identify any homologous protein and each residue in the respective encoding nucleic acid by methods well known to those skilled in the art, such as sequence alignment and determining homologous residues.

[0315] It will be apparent to one of skill in the art that in order to target any of the fusion proteins disclosed herein to a target site, e.g., a site containing a mutation to be edited, it is typically necessary to co-express the fusion protein with a guide RNA. As described in more detail elsewhere herein, the guide RNA typically comprises a tracrRNA framework that allows Cas9 binding and a guide sequence that confers sequence specificity to the Cas9:nucleic acid editing enzyme / domain fusion protein. Alternatively, the guide RNA and the tracrRNA can be provided separately as two nucleic acid molecules. In some embodiments, the guide RNA comprises a structure in which the guide sequence comprises a sequence that is complementary to the target sequence. The guide sequence is typically 20 nucleotides in length. The sequence of a suitable guide RNA for targeting a Cas9:nucleic acid editing enzyme / domain fusion protein to a specific genomic target site will be apparent to one of skill in the art based on this disclosure. Such a suitable guide RNA sequence typically comprises a guide sequence that is complementary to a nucleic acid sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting any of the provided fusion proteins to a specific target sequence are provided herein.

[0316] [Method of using a fusion protein containing cytidine deaminase, adenosine deaminase and Cas9 domain] Some aspects of the disclosure provide methods of using the fusion proteins or complexes provided herein. For example, some aspects of the disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins provided herein and at least one guide RNA, where the guide RNA is about 15-100 nucleotides in length and comprises a sequence of at least 10 contiguous nucleotides that are complementary to the target sequence. In some embodiments, the 3' end of the target sequence is immediately adjacent to the canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is not immediately adjacent to the canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence. In some embodiments, the 3' end of the target sequence is immediately adjacent to an NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, NGTN, NGTN, or 5' (TTTV) sequence.

[0317] In some embodiments, the fusion protein of the present invention is used to mutate a target of interest. In particular, the multi-effector nucleobase editor described herein can create multiple mutations in a target sequence. These mutations can affect the function of the target. For example, using a multi-effector nucleobase editor to target a regulatory region can change the function of the regulatory region and reduce the expression of downstream proteins.

[0318] It will be understood that the numbering of a particular position or residue in each sequence depends on the particular protein and numbering scheme used. For example, the numbering of a precursor of a mature protein may differ from that of the mature protein itself, and sequence differences between species may affect the numbering. Those skilled in the art can identify any homologous protein and each residue in the respective encoding nucleic acid by methods well known to those skilled in the art, such as sequence alignment and determining homologous residues.

[0319] It will be apparent to one of skill in the art that in order to target any of the fusion proteins comprising a Cas9 domain and a cytidine deaminase or an adenosine deaminase disclosed herein to a target site, e.g., a site containing a mutation to be edited, it is typically necessary to co-express the fusion protein with a guide RNA, e.g., an sgRNA. As described in more detail elsewhere herein, the guide RNA typically comprises a tracrRNA framework that allows Cas9 binding and a guide sequence that confers sequence specificity to the Cas9:nucleic acid editing enzyme / domain fusion protein. Alternatively, the guide RNA and the tracrRNA can be provided separately as two nucleic acid molecules. In some embodiments, the guide RNA comprises a structure in which the guide sequence comprises a sequence that is complementary to the target sequence. The guide sequence is typically 20 nucleotides in length. The sequence of a suitable guide RNA for targeting a Cas9:nucleic acid editing enzyme / domain fusion protein to a specific genomic target site will be apparent to one of skill in the art based on this disclosure. Such a suitable guide RNA sequence typically comprises a guide sequence that is complementary to a nucleic acid sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Provided herein are several exemplary guide RNA sequences suitable for targeting any of the provided fusion proteins to a specific target sequence.

[0320] [Efficiency of base editors] The fusion proteins of the present invention improve base editor efficiency by modifying specific nucleotide bases without generating a significant proportion of indels. As used herein, "indel" refers to an insertion or deletion of a nucleotide base in a nucleic acid. Such an insertion or deletion can cause a frameshift mutation in the coding region of a gene. In some embodiments, it is desirable to generate a base editor that efficiently modifies (e.g., mutates) specific nucleotides in a nucleic acid without generating a large number of insertions or deletions (i.e., indels) in the nucleic acid. In certain embodiments, any of the base editors provided herein can generate a greater proportion of intended modifications (e.g., mutations) compared to indels. In some embodiments, the base editors provided herein can generate a ratio of intended mutations to indels that is greater than 1:1. In some embodiments, base editors provided herein are capable of producing a ratio of intended mutations to indels of at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1, or more. The number of intended mutations and indels can be determined using any suitable method.

[0321] In some embodiments, the base editors provided herein can limit the formation of indels in a region of a nucleic acid. In some embodiments, the region is at the nucleotide targeted by the base editor or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the nucleotide targeted by the base editor. In some embodiments, any of the base editors provided herein can limit the formation of indels in a region of a nucleic acid to less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, or less than 20%. The number of indels formed in a nucleic acid region can depend on the amount of time that the nucleic acid (e.g., a nucleic acid in the genome of a cell) is exposed to the base editor. In some embodiments, the number or percentage of indels is determined at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days after exposing a nucleic acid (e.g., a nucleic acid in the genome of a cell) to a base editor.

[0322] Some aspects of the present disclosure are based on the recognition that any of the base editors provided herein can efficiently generate an intended mutation in a nucleic acid (e.g., a nucleic acid in a genome of a subject) without generating a significant number of unintended mutations. In some embodiments, the intended mutation is a mutation generated by a specific base editor coupled to a gRNA specifically designed to generate the intended mutation. In some embodiments, the intended mutation is a mutation that generates a stop codon, e.g., a premature stop codon in the coding region of a gene. In some embodiments, the intended mutation is a mutation that removes a stop codon. In some embodiments, the intended mutation is a mutation that changes the splicing of a gene. In some embodiments, the intended mutation is a mutation that changes a regulatory sequence of a gene (e.g., a gene promoter or a gene repressor). In some embodiments, any of the base editors provided herein can provide a ratio of intended mutations to unintended mutations (e.g., intended mutations:unintended mutations) of greater than 1:1. In some embodiments, any of the base editors provided herein can provide a ratio of intended mutations to unintended mutations of at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least 500:1, or at least 1000:1. It should be understood that the features of the base editors described in the "Efficiency of Base Editors" section herein can be applied to any of the fusion proteins, or methods of using the fusion proteins, provided herein.

[0323] [Method of editing nucleic acid] Some aspects of the present disclosure provide a method for editing a nucleic acid. In some embodiments, the method is a method for editing a nucleobase of a nucleic acid (e.g., a base pair of a double-stranded DNA sequence). In some embodiments, the method includes: a) contacting a target region of a nucleic acid (e.g., a double-stranded DNA sequence) with a complex comprising a base editor and a guide nucleic acid (e.g., a gRNA); b) inducing strand separation of the target region; c) converting a first nucleobase of the target nucleobase pair in a single strand of the target region to a second nucleobase; and d) using nCas9 to cut no more than one strand of the target region, where a third nucleobase complementary to the first nucleobase is replaced by a fourth nucleobase complementary to the second nucleobase. In some embodiments, the method results in less than 20% indel formation in the nucleic acid. It should be understood that in some embodiments, step b is omitted. In some embodiments, the method results in less than 19%, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% indel formation. In some embodiments, the method further comprises replacing the second nucleobase with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited base pair (e.g., G·C to A·T). In some embodiments, at least 5% of the intended base pairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended base pairs are edited.

[0324] In some embodiments, the ratio of intended product to unintended product at the target nucleotide is at least 2:1, at least 5:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, at least 100:1, or at least 200:1, or more. In some embodiments, the ratio of intended mutation to indel formation is greater than 1:1, greater than 10:1, greater than 50:1, greater than 100:1, greater than 500:1, or greater than 1000:1, or more. In some embodiments, the cleaved single strand (nicked strand) is hybridized to the guide nucleic acid. In some embodiments, the cleaved single strand is the opposite strand to the strand containing the first nucleobase. In some embodiments, the base editor comprises a dCas9 domain. In some embodiments, the base editor protects or binds the unedited strand. In some embodiments, the intended editing base pair is upstream of the PAM site. In some embodiments, the intended editing base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended editing base pair is downstream of the PAM site. In some embodiments, the intended editing base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In certain embodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In one embodiment, the linker is 32 amino acids in length. In another embodiment, a "long linker" is at least about 60 amino acids in length.In other embodiments, the linker is about 3-100 amino acids in length. In some embodiments, the target region comprises a target window, and the target window comprises the target nucleic acid base pair. In certain embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotide in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended editing base pair is within the target window. In some embodiments, the target window comprises the intended editing base pair. In some embodiments, the method is performed using any of the base editors provided herein.

[0325] In some embodiments, the present disclosure provides a method for editing nucleotides. In some embodiments, the present disclosure provides a method for editing nucleobase pairs of a double-stranded DNA sequence. In some embodiments, the method includes: a) contacting a target region of a double-stranded DNA sequence with a complex comprising a base editor and a guide nucleic acid (e.g., gRNA), wherein the target region comprises a target nucleobase pair; b) inducing strand separation of the target region; c) converting a first nucleobase of the target nucleobase pair in a single strand of the target region to a second nucleobase; and d) cleaving no more than one strand of the target region, wherein a third nucleobase complementary to the first nucleobase is replaced by a fourth nucleobase complementary to the second nucleobase, and the second nucleobase is replaced by a fifth nucleobase complementary to the fourth nucleobase, thereby generating an intended edited base pair, wherein the efficiency of generating the intended base pair is at least 5%. It should be understood that in some embodiments, step b is omitted. In some embodiments, at least 5% of the intended base pairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended base pairs are edited. In some embodiments, the method causes less than 19%, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% indel formation. In some embodiments, the ratio of intended product to unintended product at the target nucleotide is at least 2:1, at least 5:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, at least 100:1, or at least 200:1, or more. In some embodiments, the ratio of intended mutations to indel formation is greater than 1:1, greater than 10:1, greater than 50:1, greater than 100:1, greater than 500:1, or greater than 1000:1, or more.In some embodiments, the cut single strand is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is the opposite strand to the strand containing the first nucleobase. In some embodiments, the intended editing base pair is upstream of the PAM site. In some embodiments, the intended editing base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended editing base pair is downstream of the PAM site. In some embodiments, the intended editing base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the target region comprises a target window, and the target window comprises a target nucleic acid base pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotide in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended editing base pair occurs within the target window. In some embodiments, the target window comprises the intended editing base pair. In some embodiments, the nucleobase editor is any of the base editors provided herein.

[0326] [Expression of fusion proteins in host cells] The fusion proteins of the invention can be expressed in virtually any host cell of interest, including, but not limited to, bacterial, yeast, fungal, insect, plant, and animal cells, using routine methods known to those of skill in the art. The fusion proteins are produced by operably linking one or more polynucleotides encoding one or more domains having nucleobase modifying activity (e.g., adenosine deaminase or cytidine deaminase) to a polynucleotide encoding napDNAbp to prepare a polynucleotide encoding the fusion protein of the invention.

[0327] The DNA encoding the protein domains described herein can be obtained by any method known in the art, such as by chemically synthesizing a DNA strand, by PCR, or by Gibson assembly. The advantage of constructing a full-length DNA by a combination of chemical synthesis or PCR or Gibson assembly is that the codons can be optimized to ensure that the fusion protein is expressed at a high level in the host cell. Optimized codons can be selected using the Genetic Code Usage Database (http: / / www.kazusa.or.jp / codon / index.html) disclosed on the website of the Kazusa DNA Research Institute. Once obtained, the polynucleotide encoding the fusion protein is inserted into an appropriate expression vector.

[0328] Suitable expression vectors include Escherichia coli-derived plasmids (e.g., pBR322, pBR325, pUC12, pUC13); Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5, pC194); yeast-derived plasmids (e.g., pSH19, pSH15); plasmids suitable for expression in insect cells (e.g., pFast-Bac); plasmids suitable for expression in mammalian cells (e.g., pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); and bacteriophages such as lambda phage. Other vectors that may be used include insect viral vectors such as baculovirus (e.g., BmNPV, AcNPV); viral vectors suitable for expression in mammalian cells, such as retroviruses, vaccinia virus, adenovirus, etc.

[0329] The polynucleotide encoding the fusion protein is typically expressed under the control of a suitable promoter useful for expression in the desired host cell. For example, when the host is an animal cell, the SR alpha promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, etc. are used. In one embodiment, the promoter is a CMV promoter or an SR alpha promoter. When the host cell is E. coli, any of the promoters can be used, such as the trp promoter, the lac promoter, the recA promoter, the lambda P.sub.L promoter, the lpp promoter, the T7 promoter, etc. When the host is Bacillus, any of the promoters can be used, such as the SPO1 promoter, the SPO2 promoter, the penP promoter, etc. When the host is yeast, any of the promoters can be used, such as the Gal1 / 10 promoter, the PHO5 promoter, the PGK promoter, the GAP promoter, the ADH promoter, etc. When the host is an insect cell, the polyhedrin promoter, P10 promoter, etc. may be used. When the host is a plant cell, any of the promoters such as the CaMV35S promoter, CaMV19S promoter, NOS promoter, etc. may be used.

[0330] If desired, the expression vector also contains one or more of an enhancer, a splicing signal, a terminator, a poly A addition signal, a selection marker (eg, a drug resistance gene, an auxotrophy complementing gene, etc.), or an origin of replication.

[0331] RNA encoding the protein domains described herein can be prepared, for example, by transcribing mRNA in an in vitro transcription system.

[0332] The fusion protein of the present invention can be expressed by introducing an expression vector encoding the fusion protein into a host cell and culturing the host cell. Host cells useful in the present invention include bacterial cells, yeast, insect cells, mammalian cells, etc.

[0333] Escherichia genus includes Escherichia coli K12.cndot.DH1 [Proc. Natl. Acad. USA, 60, 160 (1968)], Escherichia coli JM103 [Nucleic Acids Research, 9, 309 (1981)], Escherichia coli JA221 [Journal of Molecular Biology, 120, 517 (1978)], Escherichia coli HB101 [Journal of Molecular Biology, 41, 459 (1969)], Escherichia coli C600 [Genetics, 39, 440 (1954)], etc.

[0334] The genus Bacillus includes Bacillus subtilis M1114 [Gene, 24, 255 (1983)], Bacillus subtilis 207-21 [Journal of Biochemistry, 95, 87 (1984)], and the like.

[0335] Yeast useful for expressing the fusion protein of the present invention include Saccharomyces cerevisiae AH22, AH22R.sup.-, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris KM71, etc.

[0336] The fusion protein is expressed in insect cells using a viral vector such as AcNPV. As the insect host cell, any of the cell lines such as an established strain derived from the armyworm larvae (Spodoptera frugiperda cells; SF cells), MG1 cells derived from the midgut of Trichoplusiani, High Five, cells derived from Trichoplusiani eggs, cells derived from Mamestra brassicae, and cells derived from Estigmena acrea are used. When the virus is BmNPV, cells derived from a strain derived from Bombyx mori (Bombyx mori N cells; BmN cells) are used. As the Sf cell, for example, Sf9 cells (ATCC CRL1711), Sf 21 cells [all of the above, In Vivo, 13, 213-217 (1977)], etc. are used.

[0337] In insects, fusion proteins are expressed in larvae of Bombyx mori, Drosophila, crickets, etc. [Nature, 315,592 (1985)].

[0338] Mammalian cell lines may be used to express the fusion protein. Examples of such cell lines include monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary (CHO) cells, dhfr gene-deficient CHO cells, mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, and human FL cells. Human and other mammalian iPS cells, pluripotent stem cells such as ES cells, and primary cultured cells prepared from various tissues may be used. Zebrafish embryos, Xenopus oocytes, and the like may also be used.

[0339] Plant cells can be maintained in culture using methods well known to those skilled in the art. Plant cell cultures involve suspension cultures of cells, calli, protoplasts, leaf fragments, root fragments, etc., prepared from a variety of plants (e.g., rice, wheat, corn, tomato, cucumber, eggplant, carnation, Eustoma russellianum, tobacco, Arabidopsis thaliana).

[0340] All of the above host cells may be haploid (haploid) or polyploid (eg, diploid, triploid, tetraploid, etc.).

[0341] An expression vector encoding the fusion protein of the invention is introduced into a host cell using any transfection method (e.g., lysozyme, PEG, CaCl 2 (using co-precipitation, electroporation, microinjection, particle gun, lipofection, Agrobacterium, etc.). The transfection method is selected based on the host cell to be transfected. Escherichia coli can be transformed according to the methods described, for example, in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), etc. Methods for transduction in the genus Bacillus are described, for example, in Molecular & General Genetics, 168, 111 (1979).

[0342] Yeast cells can be transformed using the methods described, for example, in Methods in Enzymology, 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978), and the like.

[0343] Insect cells are transfected using the method described, for example, in Bio / Technology, 6, 47-55 (1988).

[0344] Mammalian cells are transfected using the methods described, for example, in Cell Engineering additional volume 8, New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha), and Virology, 52, 456 (1973).

[0345] Cells containing the expression vector of the present invention are cultured according to known methods which vary depending on the host.

[0346] For example, when culturing Escherichia coli or Bacillus cells, a liquid medium is used. The medium preferably contains a carbon source, a nitrogen source, inorganic substances, and other components necessary for the growth of the transformant. Examples of carbon sources include glucose, dextrin, soluble starch, sucrose, etc. Examples of nitrogen sources include inorganic or organic substances such as ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean cake, potato extract, etc., and inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, etc. The medium may also contain yeast extract, vitamins, growth promoting factors, etc. The pH of the medium is preferably about 5 to about 8.

[0347] As a medium for culturing Escherichia coli, for example, M9 medium containing glucose and casamino acids is used [Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972]. Escherichia coli is usually cultured at about 15 to about 43° C. Aeration and stirring may be performed as necessary.

[0348] The genus Bacillus is generally cultured at about 30 to about 40° C. Aeration and stirring are performed as necessary.

[0349] Examples of media suitable for culturing yeast include Burkholder's minimal medium [Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)] and SD medium containing 0.5% casamino acids [Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. The pH of the medium is preferably about 5 to about 8. The culture is generally carried out at about 20°C to about 35°C. Aeration and stirring may be performed as necessary.

[0350] As a medium for culturing insect cells or insects, Grace's insect medium (Nature, 195, 788 (1962)) containing an additive of inactivated 10% bovine serum is used. The pH of the medium is preferably about 6.2 to about 6.4. The cells are cultured at about 27°C. Aeration and stirring may be performed as necessary.

[0351] Mammalian cells are cultured in, for example, minimum essential medium (MEM) containing about 5 to about 20% fetal bovine serum (Science, 122, 501 (1952)), Dulbecco's modified Eagle's medium (DMEM) (Virology, 8, 396 (1959)), RPMI 1640 medium (The Journal of the American Medical Association, 199, 519 (1967)), 199 medium (Proceeding of the Society for the Biological Medicine, 73, 1 (1950)), etc. The pH of the medium is preferably about 6 to about 8. The culture is performed at about 30°C to about 40°C. Aeration and stirring may be performed as necessary.

[0352] As a medium for culturing plant cells, for example, MS medium, LS medium, B5 medium, etc. are used. The pH of the medium is preferably about 5 to about 8. The culture is generally carried out at about 20° C. to about 30° C. Aeration and stirring may be performed as necessary.

[0353] Expression of the fusion protein can be regulated using an inducible promoter (e.g., a metallothionein promoter (induced by heavy metal ions), a heat shock protein promoter (induced by heat shock), a Tet-ON / Tet-OFF system promoter (induced by the addition or removal of tetracycline or its derivatives), a steroid-responsive promoter (induced by a steroid hormone or its derivatives), etc.), and an inducer is added to (or removed from) the medium at an appropriate stage to induce expression of the fusion protein.

[0354] Prokaryotic cells such as E. coli can utilize inducible promoters, including but not limited to the lac promoter (induced by IPTG), the cspA promoter (induced by cold shock), and the araBAD promoter (induced by arabinose).

[0355] [Nucleic acid-based delivery of nucleobase editors] Nucleic acids encoding nucleobase editors according to the disclosure can be administered to a subject or delivered to cells (e.g., bacterial, yeast, fungal, insect, plant, and animal cells) by methods known in the art or as described herein. For example, nucleobase editors can be delivered, for example, by vectors (e.g., viral or non-viral vectors), non-vector-based methods (e.g., the use of naked DNA or DNA complexes), or combinations thereof.

[0356] Nucleic acids encoding nucleobase editors can be delivered directly to cells (e.g., bacterial, yeast, fungal, insect, plant, and animal cells) as naked DNA or RNA, for example by transfection or electroporation, or can be conjugated to a molecule that facilitates uptake by the target cell (e.g., N-acetylgalactosamine). Nucleic acid vectors such as vectors can also be used.

[0357] The nucleic acid vector can include one or more sequences encoding the domains of the fusion proteins described herein. The vector can also include a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization) associated with (e.g., inserted or fused to) the protein-encoding sequence. As an example, the nucleic acid vector can include a Cas9 coding sequence that includes one or more nuclear localization sequences (e.g., nuclear localization sequences from SV40) and one or more deaminases.

[0358] The nucleic acid vector can also include any suitable number of regulatory / control elements, such as promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, or internal ribosome entry sites (IRES), which are well known in the art.

[0359] The nucleic acid vector according to the present disclosure includes recombinant viral vectors. Exemplary viral vectors are described herein above. Other viral vectors known in the art can also be used. In addition, viral particles can be used to deliver genome editing system components in the form of nucleic acids and / or peptides. For example, "empty" viral particles can be assembled to contain any suitable cargo. Viral vectors and viral particles can also be engineered to incorporate targeting ligands to change target tissue specificity.

[0360] In addition to viral vectors, non-viral vectors can also be used to deliver the nucleic acid encoding the genome editing system according to the present disclosure. One important category of non-viral nucleic acid vectors is that of nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art, and any suitable nanoparticle design can be used to deliver genome editing system components or the nucleic acid encoding such components. For example, organic (e.g., lipid and / or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of the present disclosure. Exemplary lipids for use in nanoparticle formulations and / or gene transfer are shown in Table 6 (below).

[0361] [Table 6]

[0362] Table 7 lists exemplary polymers for use in gene transfer and / or nanoparticle formulations. [Table 7]

[0363] Table 8 summarizes delivery methods for polynucleotides encoding the fusion proteins described herein. [Table 8]

[0364] In some embodiments, the present disclosure relates to viral delivery of fusion proteins, for example, using viral vectors. Exemplary viral vectors include retroviral vectors (e.g., Maloney murine leukemia virus, MML-V), adenoviral vectors (e.g., AD 100), lentiviral vectors (HIV and FIV-based vectors), herpes viruses (e.g., HSV-2), and adeno-associated viral vectors.

[0365] [Adeno-associated virus vector] AAV is a small, single-stranded DNA-dependent virus belonging to the parvovirus family. The 4.7 kb wild-type (wt) AAV genome consists of two genes encoding four replication and three capsid proteins, flanked by 145 bp inverted terminal repeats (ITRs). Virions consist of three capsid proteins, Vp1, Vp2, and Vp3, which are produced in a 1:1:10 ratio from the same open reading frame, but from differential splicing (Vp1) and alternative translation initiation sites (Vp2 and Vp3, respectively). Vp3 is the most abundant subunit in virions and is involved in receptor recognition at the cell surface, which determines viral tropism. A phospholipase domain has been identified in the unique N-terminus of Vp1 that functions in viral infectivity.

[0366] Recombinant AAV (rAAV), like wt AAV, utilizes cis-acting 145 bp ITRs flanking the vector transgene cassette, providing up to 4.5 kb for packaging of foreign DNA. After infection, rAAV can express the fusion protein of the invention and persist as an episome in the form of a circular head-to-tail concatemer without integrating into the host genome. Although there have been many successful examples of rAAV using this system, in vitro and in vivo, limited packaging capacity limits the use of AAV-mediated gene delivery when the length of the gene coding sequence is equal to or greater than the length of the wt AAV genome.

[0367] The small packaging capacity of AAV vectors makes it difficult to deliver a large number of genes and / or use large physiological regulatory elements that exceed this size. These challenges can be addressed, for example, by splitting the protein to be delivered into two or more fragments, where the N-terminal fragment is fused to a split intein-N and the C-terminal fragment is fused to a split intein-C. These fragments are then packaged into two or more AAV vectors. In one embodiment, the fragments or portions of the nucleobase editor protein that are grafted onto the AAV capsid protein are linked together using an intein. As used herein, "intein" refers to a self-splicing protein intron (e.g., a peptide) that ligates adjacent N-terminal and C-terminal exteins (e.g., fragments to be linked). The use of certain inteins to link heterologous protein fragments is described, for example, in Wood et al., J. Biol. Chem. 289(21); 14512-9 (2014). For example, the inteins IntN and IntC, when fused to separate protein fragments, recognize each other and splice themselves out while simultaneously ligating adjacent N- and C-terminal exteins of the fused protein fragments, thereby reconstituting a full-length protein from the two protein fragments. Other suitable inteins will be apparent to those skilled in the art.

[0368] The fragments of the fusion proteins of the present invention can vary in length. In some embodiments, the protein fragments range from 2 amino acids to about 1000 amino acids in length. In some embodiments, the protein fragments range from about 5 amino acids to about 500 amino acids in length. In some embodiments, the protein fragments range from about 20 amino acids to about 200 amino acids in length. In some embodiments, the protein fragments range from about 10 amino acids to about 100 amino acids in length. Other lengths of suitable protein fragments will be apparent to those of skill in the art.

[0369] In some embodiments, a portion or fragment of a nuclease (e.g., a fragment of a deaminase such as cytidine deaminase, adenosine deaminase, or a fragment of Cas9) is fused to an intein. The nuclease can be fused to the N-terminus or C-terminus of the intein. In some embodiments, a portion or fragment of a fusion protein is fused to an intein and fused to an AAV capsid protein. The intein, nuclease, and capsid protein can be fused together in any configuration (e.g., nuclease-intein-capsid, intein-nuclease-capsid, capsid-intein-nuclease, etc.). In some embodiments, the N-terminus of the intein is fused to the C-terminus of the fusion protein, and the C-terminus of the intein is fused to the N-terminus of the AAV capsid protein.

[0370] In one embodiment, a dual AAV vector is generated by splitting a large transgene expression cassette into two separate halves (5' and 3' ends, or head and tail), and each half of the cassette is packaged into a single AAV vector (<5 kb). Then, following co-infection of the same cell with both dual AAV vectors, reconstitution of the full-length transgene expression cassette is achieved by (1) homologous recombination (HR) between the 5' and 3' genomes (dual AAV overlapping vector); (2) tail-to-head concatemerization of the 5' and 3' genomes (dual AAV trans-splicing vector) via ITRs; or (3) a combination of the two mechanisms (dual AAV hybrid vector). The use of dual AAV vectors in vivo results in the expression of full-length proteins. The use of dual AAV vector platform represents an efficient and viable gene transfer strategy for transgenes >4.7 kb in size.

[0371] [Screening of nucleobase editors] The suitability of the nucleobase editor fusion proteins containing the "split" and reconstituted Cas9 can be evaluated in various screening approaches as described herein. Each fragment of the fusion protein to be tested is delivered to a single cell of interest (e.g., bacterial, yeast, fungal, insect, plant, or animal cell) together with a small amount of vector encoding a reporter (e.g., GFP). These cells can be immortalized in human cell lines, such as 293T, K562, or U20S. Alternatively, primary human cells can be used. Such cells can be related to the final cellular target.

[0372] Delivery can be performed using a viral vector. In one embodiment, transfection can be performed using lipid transfection (such as Lipofectamine or Fugene) or by electroporation. After transfection, GFP expression can be measured by either fluorescent microscopy or flow cytometry to confirm consistent high levels of transfection. These preliminary transfections can include different nucleobase editors to determine the editor combination that gives the highest activity.

[0373] The activity of the nucleobase editors is assessed as described herein, i.e., by sequencing the genome of the cells to detect changes in the target sequence. For Sanger sequencing, purified PCR amplicons are cloned into a plasmid backbone, transformed, miniprepped, and sequenced with a single primer. Sequencing may also be performed using next-generation sequencing techniques. When using next-generation sequencing, the amplicons may be 300-500 bp with the intended cleavage site asymmetrically positioned. Following PCR, next-generation sequencing adapters and barcodes (e.g., Illumina multiplex adapters and indexes) may be added to the ends of the amplicons (e.g., for use in high-throughput sequencing, e.g., on an Illumina MiSeq).

[0374] Fusion proteins that induce the greatest levels of target-specific modification in initial tests can be selected for further evaluation.

[0375] In certain embodiments, nucleobase editors are used to target polynucleotides of interest. In one embodiment, nucleobase editors are used to target regulatory sequences, including but not limited to splice sites, enhancers, and transcriptional regulatory elements. The effect of the modification on the expression of genes controlled by the regulatory elements is then assayed using any method known in the art.

[0376] In other embodiments, nucleobase editors of the invention are used to target polynucleotides encoding complementarity determining regions (CDRs), thereby resulting in modifications in the expressed CDRs. The effect of these modifications on CDR function is then assayed, for example, by measuring specific binding of the CDR to an antigen.

[0377] In yet other embodiments, nucleobase editors of the invention are used to target polynucleotides of interest within the genome of an organism (e.g., bacteria, yeast, fungi, insects, plants, and animals). In one embodiment, nucleobase editors of the invention are delivered to a cell along with a library of guide RNAs that are used to tile various sequences within the genome of the cell, thereby systematically modifying sequences throughout the genome.

[0378] [Applications of nucleobase editors] Nucleobase editors can be used to target polynucleotides of interest and create changes that modify protein expression. In one embodiment, nucleobase editors are used to modify non-coding or regulatory sequences, including but not limited to splice sites, enhancers, and transcriptional regulatory elements. Any method known in the art is then used to assay the effect of the modification on the expression of the gene controlled by the regulatory element. In certain embodiments, nucleobase editors can substantially change regulatory sequences, thereby eliminating their ability to regulate gene expression. Advantageously, this can be done without creating double-strand breaks in genomic target sequences, in contrast to other RNA programmable nucleases.

[0379] Nucleic acid base editors can be used to target polynucleotides of interest and create changes that modify protein activity.For example, in the context of mutagenesis, nucleic acid base editors have many advantages over error-prone PCR and other polymerase-based methods.Unlike error-prone PCR, which induces random changes throughout polynucleotides, the nucleic acid base editors of the present invention can be used to target specific amino acids within a defined region of target protein.

[0380] In other embodiments, the nucleobase editors of the invention are used to target a polynucleotide of interest within the genome of an organism. In one embodiment, the organism is a bacterium of the microbiome (e.g., Bacteriodetes, Verrucomicrobia, Firmicutes; Gammaproteobacteria, Alphaproteobacteria, Bacteriodetes, Clostridia, Erysipelotrichia, Bacilli; Enterobacteriales, Bacteriodales, Verrucomicrobiales, Clostridiales, Erysiopelotrichales, Lactobacillales; Enterobacteriaceae, Bacteroidaceae, Erysiopelotrichaceae, Prevotellaceae, Coriobacteriaceae, and Alcaligenaceae; Escherichia, Bacteroides, Alistipes, Akkermansia, Clostridium, Lactobacillus). In another embodiment, the organism is an agriculturally important animal (e.g., cow, sheep, goat, horse, chicken, turkey) or plant (e.g., soybean, wheat, corn, cotton, canola, rice, tobacco, apple, grape, peach, plum, cherry). In one embodiment, the nucleobase editor of the invention is delivered to a cell along with a library of guide RNAs that are used to tile various sequences within the genome of the cell, thereby systematically altering sequences throughout the genome.

[0381] Mutations may be made in any of a variety of proteins to facilitate structure-function analysis or to alter the intrinsic activity of a protein. Mutations may be made, for example, in enzymes (e.g., kinases, phosphatases, carboxylases, phosphodiesterases) or enzyme substrates, receptors or their ligands, and antibodies and their antigens. In one embodiment, the nucleobase editor targets a nucleic acid molecule encoding the active site of an enzyme, the ligand binding site of a receptor, or the complementarity determining region (CDR) of an antibody. In the case of an enzyme, introducing a mutation into the active site may increase, decrease, or eliminate enzyme activity. The effect of the mutation on the enzyme is characterized in an enzyme activity assay, including any of a number of assays known in the art and / or apparent to one of skill in the art. In the case of a receptor, a mutation made in the ligand binding site may increase, decrease, or eliminate the affinity of the receptor for the ligand. The effect of such a mutation is assayed in a receptor / ligand binding assay, including any of a number of assays known in the art and / or apparent to one of skill in the art. In the case of a CDR, a mutation made in the CDR may increase, decrease, or eliminate binding to the antigen. Alternatively, mutations made in the CDRs may change the specificity of the antibody for the antigen, and the effect of these modifications on CDR function is then assayed, for example, by measuring the specific binding of the CDR to its antigen, or in any other type of immunoassay.

[0382] The present disclosure provides methods for the treatment of subjects diagnosed with a disease associated with or caused by a genetic mutation (including gene conversion), a point mutation affecting splicing (e.g., altering a splice donor or acceptor site), or an abnormal or misfolded protein resulting from a point mutation that can be corrected by the base editor system provided herein. For example, in some embodiments, methods are provided that include administering to a subject with such a disease (e.g., a disease caused by gene conversion or other genetic mutation) an effective amount of a nucleoside base editor (e.g., an adenosine deaminase base editor or a cytidine deaminase base editor that includes one or more DNA binding protein domains) that edits a nucleoside base that is directly or indirectly associated with a mutation in the disease-associated gene. In certain embodiments, methods are provided for the treatment of additional diseases or disorders, such as diseases or disorders associated with or caused by a point mutation that can be corrected or ameliorated by deaminase-mediated gene editing. Several such diseases are described herein, and further suitable diseases that can be treated with the strategies and fusion proteins provided herein will be apparent to one of skill in the art based on the present disclosure.

[0383] [Pharmaceutical composition] Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the base editors, fusion proteins, or fusion protein-guide polynucleotide complexes described herein. The term "pharmaceutical composition" as used herein refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutical acceptable carrier. In some embodiments, the pharmaceutical composition comprises an additional agent (e.g., for specific delivery, half-life extension, or other therapeutic compound).

[0384] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, involved in carrying or transporting a compound from one site in the body (e.g., a delivery site) to another site (e.g., an organ, tissue or part of the body). A pharmaceutically acceptable carrier is acceptable in the sense of being compatible with other ingredients of the formulation and not deleterious to the tissues of the subject (e.g., physiological compatibility, sterility, physiological pH, etc.).

[0385] Some non-limiting examples of materials that can serve as pharma-ceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch, potato starch, etc.; (3) cellulose and its derivatives (sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, cellulose acetate, etc.); (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate, talc, etc.; (8) additives such as cocoa butter, suppository waxes, etc.; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, etc.; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) Buffers such as magnesium hydroxide, aluminum hydroxide, etc.; (15) alginic acid; (16) pyrogen-free water; (17) saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffers; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents such as polypeptides and amino acids; (23) serum alcohols such as ethanol; and (23) other non-toxic compatible substances used in the formulation. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives and antioxidants may also be present in the formulation. The terms "excipient", "carrier", "pharmaceutical acceptable carrier", "vehicle" and the like are used interchangeably herein.

[0386] The pharmaceutical composition may include one or more pH buffer compounds to maintain the pH of the formulation at a predetermined level that reflects physiological pH, such as in the range of about 5.0 to about 8.0. The pH buffer compound used in the aqueous liquid formulation may be an amino acid or a mixture of amino acids such as histidine, or a mixture of amino acids such as histidine and glycine. Alternatively, the pH buffer compound is preferably an agent that maintains the pH of the formulation at a predetermined level, for example in the range of about 5.0 to about 8.0, and does not chelate calcium ions. Typical examples of such pH buffer compounds include, but are not limited to, imidazole and acetate ions. The pH buffer compound may be present in any amount suitable for maintaining the pH of the formulation at a predetermined level.

[0387] The pharmaceutical composition may also contain one or more osmotic modifiers, i.e., compounds that adjust the osmotic properties (e.g., isotonicity, osmolality, and / or osmolarity) of the formulation to levels acceptable to the bloodstream and blood cells of the recipient individual. The osmotic modifier may be an agent that does not chelate calcium ions. The osmotic modifier may be any compound known or available to those skilled in the art that adjusts the osmotic properties of the formulation. Those skilled in the art may empirically determine the suitability of a given osmotic modifier for use in the formulations of the present invention. Illustrative examples of suitable types of osmotic modifiers include, but are not limited to, salts such as sodium chloride and sodium acetate; sugars such as sucrose, dextrose, mannitol; amino acids such as glycine; and mixtures of one or more of these agents and / or agent types. The osmotic modifier may be present in any concentration sufficient to adjust the osmotic properties of the formulation.

[0388] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, for example, for gene editing.Suitable routes for administering the pharmaceutical composition described herein include, but are not limited to, topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, intracerebral, and intraventricular administration.

[0389] In some aspects, the pharmaceutical compositions described herein are administered locally to the affected area (e.g., tumor site). In some embodiments, the pharmaceutical compositions described herein are administered to the subject by injection, catheter, suppository, or implant, the implant being a porous, non-porous, or gelatinous material, including, for example, a membrane such as a sialastic membrane, or a fiber.

[0390] In another aspect, the pharmaceutical composition described herein is delivered in a controlled release system.In one embodiment, pumps can be used (see, for example, Langer, 1990, Science 249: 1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al, 1989, N. Engl. J. Med. 321:574).In another embodiment, polymeric materials can be used. (E.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71: 105.) Other controlled release systems are described, for example, in Langer, supra.

[0391] In some embodiments, the pharmaceutical composition is formulated according to routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human. In some embodiments, the pharmaceutical composition for administration by injection is a solution of sterile isotonic use as a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. In general, the components are supplied separately or together in unit dosage form, e.g., as a dry lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachette, indicating the amount of active agent. When the agent is administered by injection, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. When the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0392] Pharmaceutical compositions for systemic administration may be liquid, such as sterile saline, lactated Ringer's solution, or Hank's solution. Additionally, pharmaceutical compositions may be in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also contemplated. Pharmaceutical compositions may be contained within lipid particles or vesicles, such as liposomes or microcrystals, which are also suitable for parenteral administration. The particles may be of any suitable structure, such as monolamellar or multilamellar, so long as the composition is contained therein. Compounds may be entrapped in "stabilized plasmid lipid particles" (SPLPs) that contain the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), a low amount (5-10 mole %) of cationic lipid, and stabilized by a polyethylene glycol (PEG) coating (Zhang YP et al., Gene Ther. 1999, 6: 1438-47). Positively charged lipids such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methyl sulfate, or "DOTAP", are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known, see, e.g., U.S. Patent Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757, each of which is incorporated herein by reference.

[0393] The pharmaceutical compositions described herein can be administered or packaged, for example, as unit doses. The term "unit dose", when used in relation to the pharmaceutical compositions of the present disclosure, refers to a physically discrete unit suitable as a single dose for a subject, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect, together with a required diluent (e.g., carrier, or vehicle).

[0394] Additionally, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a compound of the invention in lyophilized form, and (b) a second container containing a pharma- ceutically acceptable diluent (e.g., sterile) used for reconstituting or diluting the lyophilized compound of the invention. If desired, such container can bear a notice in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use, or sale for human administration.

[0395] In another aspect, the article of manufacture includes materials useful for treating the above diseases. In some embodiments, the article of manufacture includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials, such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating the diseases described herein and can have a sterile access port. For example, the container can be an intravenous solution bag, or a vial with a stopper that can be pierced by a hypodermic needle. The active agent in the composition is a compound of the present invention. In some embodiments, a label on or associated with the container indicates that the composition is used to treat the selected disease. The article of manufacture can further include a second container that includes a pharma- ceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, or dextrose solution. In addition, it can include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0396] In some embodiments, any of the fusion proteins, gRNAs, and / or complexes described herein are provided as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any of the fusion proteins provided herein. In some embodiments, the pharmaceutical composition comprises any of the complexes provided herein. In some embodiments, the pharmaceutical composition comprises a ribonucleoprotein complex comprising a gRNA and an RNA-guided nuclease (e.g., Cas9) complexed with a cationic lipid. In some embodiments, the pharmaceutical composition comprises a gRNA, a nucleic acid programmable DNA binding protein, a cationic lipid, and a pharma- ceutically acceptable excipient. The pharmaceutical composition can optionally include one or more additional therapeutically active substances.

[0397] In some embodiments, any of the fusion proteins, gRNAs, systems, and / or complexes described herein are provided as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any of the fusion proteins provided herein. In some embodiments, the pharmaceutical composition comprises any of the systems or complexes provided herein. In some embodiments, the pharmaceutical composition comprises a ribonucleoprotein complex comprising an RNA-guided nuclease (e.g., Cas9) or a fragment thereof complexed with a gRNA and a cationic lipid. In some embodiments, the pharmaceutical composition comprises a ribonucleoprotein complex comprising a plurality of programmable DNA binding proteins (e.g., Cas9, zinc finger, TALE, TALE-N proteins or fragments thereof). The programmable DNA binding proteins may comprise nuclease activity, or nickase activity, or may not comprise nuclease activity. In some embodiments, the pharmaceutical composition comprises a gRNA, a nucleic acid programmable DNA binding protein, a cationic lipid, and a pharma- ceutically acceptable excipient. The pharmaceutical compositions described herein can optionally comprise one or more additional therapeutically active substances.

[0398] In some embodiments, the compositions provided herein are administered to a subject, for example a human subject, to perform targeted genomic modification in the subject. In some embodiments, cells are obtained from the subject and contacted with any of the pharmaceutical compositions provided herein. In some embodiments, the cells removed from the subject and contacted with the pharmaceutical composition ex vivo are optionally reintroduced into the subject after the desired genomic modification has been performed or detected in the cell. Methods for delivering pharmaceutical compositions containing nucleases are known and are described, for example, in U.S. Patent Nos. 6,453,242, 6,503,717, 6,534,261, 6,599,692, 6,607,882, 6,689,558, 6,824,978, 6,933,113, 6,979,539, 7,013,219, and 7,163,824, the disclosures of all of which are incorporated herein by reference in their entirety. Although the description of pharmaceutical compositions provided herein is directed primarily to pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any type of animal or organism. Modifications of pharmaceutical compositions suitable for administration to humans to provide compositions suitable for administration to various animals are well understood, and an ordinary skilled veterinary pharmacologist can design and / or perform such modifications, if necessary, with no more than routine experimentation. Subjects to which the pharmaceutical compositions are intended to be administered include, but are not limited to, humans and / or other primates; mammals, livestock, pets, and commercially relevant mammals, such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats; commercially relevant birds, such as chickens, ducks, geese, and / or turkeys.

[0399] The formulation of pharmaceutical composition described herein can be prepared by any method known or hereafter developed in the field of pharmacy.In general, such preparation method includes the step of bringing active ingredient into association with excipient and / or one or more other auxiliary ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into desired single or multiple dosage units.

[0400] Pharmaceutical formulations can further include pharma- ceutically acceptable excipients, which as used herein includes any and all of solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006, incorporated herein by reference in its entirety, discloses various excipients used in formulating pharmaceutical compositions and known techniques for their preparation. See also PCT Application PCT / US2010 / 055131 (filed November 2, 2010, Publication No. WO2011053982 A8), incorporated herein by reference in its entirety, for additional suitable methods, reagents, excipients and solvents for producing pharmaceutical compositions comprising nucleases. Use of any conventional excipient medium is contemplated to be within the scope of the present disclosure unless it is incompatible with the substance or its derivatives, such as by producing undesirable biological effects or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition. In some embodiments, the compositions according to the present invention may be used for the treatment of any of a variety of diseases, disorders, and / or conditions.

[0401] [Kit, Vector, Cells] Various embodiments of the disclosure provide kits comprising a nucleic acid construct comprising a nucleotide sequence encoding a nucleobase editor fusion protein comprising a deaminase, an SpnCas9 domain "split" into two fragment...

Claims

1. (a) a first polynucleotide encoding a fusion protein comprising, in N-terminal to C-terminal order, a deaminase and an N-terminal fragment of Cas9, the N-terminal fragment of Cas9 being a contiguous sequence beginning at the N-terminus of Cas9 and ending at position 302, 309, 312, or 354 of Cas9 as numbered in SEQ ID NO:2, wherein the N-terminal fragment of Cas9 is fused at its C-terminus to a split intein-N; and (b) a second polynucleotide encoding a C-terminal fragment of Cas9, the C-terminal fragment of Cas9 being a contiguous sequence beginning at position S303 of Cas9 if the N-terminal fragment terminates at position 302, at position T310 of Cas9 if the N-terminal fragment terminates at position 309, at position T313 of Cas9 if the N-terminal fragment terminates at position 312, or at position S355 of Cas9 if the N-terminal fragment terminates at position 354, according to the numbering in SEQ ID NO:2, and ending at the C-terminus of Cas9, wherein the C-terminal fragment of Cas9 is fused at its N-terminus to a split intein-C. A composition comprising:

2. (a) a first polynucleotide encoding a fusion protein comprising, in N-terminal to C-terminal order, a deaminase and an N-terminal fragment of Cas9, the N-terminal fragment of Cas9 being a contiguous sequence beginning at the N-terminus of Cas9 and ending at position 302, 309, 312, 354, 465, 471, or 576 of Cas9 as numbered in SEQ ID NO:2, wherein the N-terminal fragment of Cas9 is fused at its C-terminus to a split intein-N; and (b) a second polynucleotide encoding a C-terminal fragment of Cas9, the C-terminal fragment of Cas9 being at position S303 of Cas9 if the N-terminal fragment terminates at position 302, at position T310 of Cas9 if the N-terminal fragment terminates at position 309, at position T313 of Cas9 if the N-terminal fragment terminates at position 312, at position S355 of Cas9 if the N-terminal fragment terminates at position 354, and at position S365 of Cas9 if the N-terminal fragment terminates at position 465, according to the numbering in SEQ ID NO:2; a contiguous sequence beginning at T466 of Cas9 if the N-terminal fragment terminates at position 471, at T472 of Cas9 if the N-terminal fragment terminates at position 471, or at S577 of Cas9 if the N-terminal fragment terminates at position 576, and terminating at the C-terminus of Cas9, wherein the N-terminal residue of the C-terminal fragment of Cas9 is Cys substituted for Ala, Ser, or Thr, and wherein the C-terminal fragment of Cas9 is fused at its N-terminus to the split intein-C. A composition comprising:

3. 3. The composition of claim 1 or 2, further comprising a single guide RNA (sgRNA) or a polynucleotide encoding same.

4. The composition of any one of claims 1 to 3, wherein the first and second polynucleotides are linked or expressed separately.

5. the deaminase is adenosine deaminase, the deaminase is wild-type TadA or TadA7.10; the deaminase is a TadA dimer; or the deaminase is a TadA dimer comprising wild-type TadA and TadA7.10; The composition according to any one of claims 1 to 4.

6. the fusion protein comprises a nuclear localization signal (NLS); the N-terminal fragment of Cas9 or the C-terminal fragment of Cas9 is linked to an NLS; or wherein the NLS is a two-part NLS; The composition according to any one of claims 1 to 5.

7. The composition of any one of claims 1 to 6, wherein the Cas9 has nickase activity or is catalytically inactive.

8. A composition comprising the fusion protein of any one of claims 1 to 7 and a C-terminal fragment of Cas9.

9. The composition of any one of claims 1 to 8, wherein the N-terminal fragment of Cas9 and the deaminase are linked by a linker, which is optionally a peptide linker.

10. A vector comprising the first and second polynucleotides according to any one of claims 1 to 9.

11. the vector comprises a promoter; or the vector comprises a promoter that is a constitutive promoter; or the vector comprises a constitutive promoter which is a CMV or CAG promoter; or The vector is selected from the group consisting of a retroviral vector, an adenoviral vector, a lentiviral vector, a herpes virus vector, and an adeno-associated virus vector; The vector of claim 10.

12. A cell comprising the composition of any one of claims 1 to 9 or the vector of claim 10 or 11, optionally said cell being a mammalian cell.

13. Cells, The first and second polynucleotides according to claim 1 or 2; and a single guide RNA (sgRNA) or a polynucleotide encoding the same. contacting the In vitro or ex vivo methods for delivering base editor systems to cells.

14. The method of claim 13 , wherein the cell is an animal, plant, or bacterial cell.

15. 15. The method of claim 13 or 14, wherein the first polynucleotide, the second polynucleotide, and / or the polynucleotide encoding the sgRNA are contained in one or more vectors that are contacted with the cell.

16. the vector is selected from the group consisting of a retroviral vector, an adenoviral vector, a lentiviral vector, a herpes virus vector, and an adeno-associated virus vector; the C-terminal fragment of Cas9 or the N-terminal fragment of Cas9 comprises Ala / Cys, Ser / Cys, or Thr / Cys mutations at residues corresponding to amino acids S303, T310, T313, S355, T466, T472, or S577 in the numbering of SEQ ID NO:2; the deaminase is adenosine deaminase; the deaminase is an adenosine deaminase that is TadA or a variant thereof; the deaminase is an adenosine deaminase that is wild-type TadA or Tad7.10; the deaminase is a TadA dimer; the deaminase is a TadA dimer comprising wild-type TadA and TadA7.10; the N-terminal fragment of Cas9 and / or the C-terminal fragment of Cas9 comprises an NLS, which is optionally a bipartite NLS; and / or the N-terminal fragment of Cas9 and the C-terminal fragment of Cas9 are linked to form SpCa9, which optionally has nickase activity or is catalytically inactive; 16. The method of claim 15.

17. A polynucleotide encoding a fusion protein, (i) the fusion protein comprises, in N-terminal to C-terminal order, a deaminase and an N-terminal fragment of Cas9, the N-terminal fragment of Cas9 being a contiguous sequence beginning at the N-terminus of Cas9 and ending at position 302, 309, 312, or 354 of Cas9 as numbered in SEQ ID NO:2, and the N-terminal fragment of Cas9 is fused at its C-terminus to split intein-N; or (ii) the fusion protein comprises, in N-terminal to C-terminal order, a deaminase and an N-terminal fragment of Cas9, the N-terminal fragment of Cas9 being a contiguous sequence beginning at the N-terminus of Cas9 and ending at position 465 or 471 of Cas9 as numbered in SEQ ID NO:2, and the N-terminal fragment of Cas9 is fused at its C-terminus to split intein-N; or (iii) the fusion protein comprises, in N-terminal to C-terminal order, a deaminase and an N-terminal fragment of Cas9, the N-terminal fragment of Cas9 being a contiguous sequence beginning at the N-terminus of Cas9 and ending at position 576 of Cas9 as numbered in SEQ ID NO:2, and the N-terminal fragment of Cas9 is fused at its C-terminus to split intein-N; Polynucleotide.

18. the deaminase is adenosine deaminase; the deaminase is an adenosine deaminase that is TadA or a variant thereof; the deaminase is an adenosine deaminase that is wild-type TadA or Tad7.10; The fusion protein comprises two deaminases linked together; the fusion protein comprises both wild-type TadA and TadA7.10; the N-terminal fragment of Cas9 or the C-terminal fragment of Cas9 comprises the amino acid sequence of SpCas9; and / or the N-terminal fragment of Cas9 or the C-terminal fragment of Cas9 comprises one or more amino acid substitutions associated with reduced nuclease activity; 18. The polynucleotide of claim 17.

19. A fusion protein comprising, in N-terminus to C-terminus order, a deaminase and an N-terminal fragment of Cas9, wherein the N-terminal fragment of Cas9 is a contiguous sequence beginning at the N-terminus of Cas9 and ending at position 302, 309, 312, 354, 465, 471, or 576 of Cas9 as numbered in SEQ ID NO:2, wherein the N-terminal fragment of Cas9 is fused at its C-terminus to a split intein-N.

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