Nucleobase editors with reduced off-target deamination and methods of using same to modify nucleobase target sequences

Abstract

The present invention features nucleobase editors and multi-effector nucleobase editors with improved editing profiles that minimize off-target deamination, compositions comprising such editors, and methods of using the same to generate modifications in target nucleobase sequences.

Description

[Technical Field]

[0001] Cross Reference This application is a continuation of U.S. Provisional Application Nos. 62 / 799,702, filed January 31, 2019, 62 / 835,456, filed April 17, 2019, and 62 / 941,569, filed November 27, 2019. and international PCT applications claiming the benefit of US Provisional Patent Application No. 2004 / 0119994, the contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Targeted editing of nucleic acid sequences, such as targeted cleavage or modification of genomic DNA, is a highly promising approach for studying gene function and may provide novel treatments for human genetic diseases. Currently available base editors include cytidine base editors (e.g., BE4) that convert targeted C·G base pairs to T·A, and adenine base editors (e.g., ABE7.10) that convert A·T to G·C. Summary of the Invention [Problem to be solved by the invention]

[0003] There is a need in the art for improved base editors that can induce modifications in target sequences with greater specificity and efficiency. [Means for solving the problem]

[0004] As described below, the present invention features nucleobase editors and multi-effector nucleobase editors with improved editing profiles that minimize off-target deamination, compositions comprising such editors, and methods of using the same to produce modifications in target nucleobase sequences.

[0005] In one aspect, provided herein is a cytidine base editor comprising: (i) a polynucleotide-programmable DNA-binding domain; and (ii) a cytidine deaminase, wherein the cytidine base editor has an increased ratio of cis to trans activity (in cis:in trans) compared to a standard cytidine base editor.

[0006] In some embodiments, the canonical cytidine base editor comprises (i) a polynucleotide-programmable DNA-binding domain and (ii) an APOBEC cytidine deaminase. In some embodiments, the APOBEC cytidine deaminase of the canonical cytidine base editor is rat APOBEC-1 cytidine deaminase (rAPOBEC-1). In some embodiments, the polynucleotide-programmable DNA-binding domain of the canonical cytidine base editor is a Cas9 nickase. In some embodiments, the canonical cytidine base editor comprises a uracil glycosylase inhibitor (UGI) domain. In some embodiments, the canonical cytidine base editor is BE3 or BE4. In some embodiments, the increased cis-to-trans activity ratio is at least 2-, 2.5-, 5-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 60-fold or more. In some embodiments, the cis-activity is at least 50%, 60%, 70%, 80%, 90%, 95%, 100%, 105%, 110%, 115%, 120% or more relative to the standard cytidine base editor.

[0007] In some embodiments, the transactivation is at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, or more times less than the standard cytidine base editor.

[0008] In some embodiments, the cytidine deaminase is selected from the group consisting of APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, activation-induced (cytidine) deaminase (AID), hAPOBEC1, rAPOBEC1, ppAPOBEC1, AmAPOBEC1 (BEM 3.31), ocAPOBEC1, SSAPOBEC2 (BEM3.39), hAPOBEC3A, maAPOBEC1, mdAPOBEC1, cytidine deaminase 1 (CDA1), hA3A, RrA3F (BEM3.14), PmCDA1, AID (activation-induced cytidine deaminase; AICDA), hAID, and FENRY. In one embodiment, the cytidine deaminase is APOBEC1. In one embodiment, the cytidine deaminase is selected from the group consisting of: (a) APOBEC-1 from Mesocricetus auratus (MaAPOBEC-1), Pongo pygmaeus (PpAPOBEC-1), Oryctolagus cuniculus (OcAPOBEC-1), Monodelphis domestica (MdAPOBEC-1), or Alligator mississippiensis (AmAPOBEC-1); (b) APOBEC-2 from Pongo pygmaeus (PpAPOBEC-2), Bos taurus (BtAPOBEC-2), or Sus scrofa (SsAPOBEC-2); (c) APOBEC-4 from Macaca fascicularis (MfAPOBEC-4); (d) APOBEC-5 from Canis lupus familiaris (ClAID) or Bos (e) yeast cytosine deaminase (yCD) from Saccharomyces cerevisiae; (f) APOBEC-3F (RrA3F) from Rhinopithecus roxellana; or (g) a cytidine deaminase having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of (a) to (f).

[0009] In some embodiments, the cytidine deaminase is APOBEC-1 from Mesocricetus auratus (MaAPOBEC-1), Pongo pygmaeus (PpAPOBEC-1), Oryctolagus cuniculus (OcAPOBEC-1), or Monodelphis domestica (MdAPOBEC-1), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the cytidine deaminase is rAPOBEC1. In some embodiments, the cytidine deaminase is hAPOBEC3A. In some embodiments, the cytidine deaminase is ppAPOBEC1. In one embodiment, the cytidine deaminase is APOBEC-2 from Pongo pygmaeus (PpAPOBEC-2), Bos taurus (BtAPOBEC-2), or Sus scrofa (SsAPOBEC-2), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, the cytidine deaminase is APOBEC-4 from Macaca fascicularis (MfAPOBEC-4), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, the cytidine deaminase is AID from Canis lupus familiaris (ClAID) or Bos Taurus (BtAID), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0010] In one embodiment, the cytidine deaminase is yeast cytosine deaminase (yCD) from Saccharomyces cerevisiae or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, the cytidine deaminase is APOBEC-3F (RrA3F) from Rhinopithecus roxellana or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, the cytidine deaminase is any of the cytidine deaminases provided in Table 13, or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, the cytidine deaminase is APOBEC-3F from Rhinopithecus roxellana (RrA3F), APOBEC-1 from Alligator mississippiensis (AmAPOBEC-1), APOBEC-2 from Sus scrofa (SsAPOBEC-2), or APOBEC-1 from Pongo pygmaeus (PpAPOBEC-1), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0011] In one embodiment, the cytidine deaminase comprises one or more modifications at or corresponding to positions R15X, R16X, H21X, R30X, R33X, K34X, R52X, K60X, R118X, H121X, H122X, R126X, R128X, R169X, R198X, T36X, H53X, V62X, L88X, W90X, Y120X, or R132X numbered in SEQ ID NO: 1, where X is any amino acid.

[0012] In one embodiment, the cytidine deaminase comprises one or more modifications selected from the group consisting of R15A, R16A, H21A, R30A, R33A, K34A, R52A, K60A, R118A, H121A, H122A, H122L, R126A, R128A, R169A, R198A, T36A, H53A, V62A, L88A, W90F, W90A, Y120F, Y120A, H121R, H122R, R126E, W90Y, and R132E according to the numbering in SEQ ID NO: 1, or one or more modifications corresponding thereto. In one embodiment, the cytidine deaminase comprises a combination of modifications selected from the group consisting of K34A+R33A, K34A+H122A, K34A+Y120F, K34A+R52A, K34A+H122A, K34A+H121A, W90A+R126E, W90Y+R126E, H121R+H122R, R126+R132E, W90Y+R132E, and W90Y+R126E+R132E, as numbered in SEQ ID NO: 1, or a corresponding combination of modifications. In one embodiment, the cytidine deaminase comprises a modification at position Y120F, as numbered in SEQ ID NO: 1, and one or more modifications selected from the group consisting of R33A, W90F, K34A, R52A, H122A, and H121A, as numbered in SEQ ID NO: 1, or one or more modifications corresponding thereto. In one embodiment, the cytidine deaminase comprises an alteration at position Y130X or R28X, or an alteration corresponding thereto, in the numbering of SEQ ID NO: 1, where X is any amino acid.

[0013] The cytidine deaminase comprises an alteration at position Y130A or R28A in the numbering of SEQ ID NO: 1, or an alteration corresponding thereto. In one embodiment, the cytidine deaminase comprises an alteration at positions Y130A and R28A in the numbering of SEQ ID NO: 1, or an alteration corresponding thereto. In one embodiment, the cytidine deaminase comprises one or more alterations at positions H122X, K34X, R33X, W90X, or R128X in the numbering of SEQ ID NO: 1, or one or more alterations corresponding thereto, where X is any amino acid. In one embodiment, the cytidine deaminase comprises one or more alterations selected from the group consisting of H122A, K34A, R33A, W90F, W90A, and R128A in the numbering of SEQ ID NO: 1, or one or more alterations corresponding thereto. In one embodiment, the cytidine deaminase comprises an alteration selected from the group consisting of R33A+K34A, W90F+K34A, R33A+K34A+W90F, and R33A+K34A+H122A+W90F, as numbered in SEQ ID NO: 1, or a corresponding combination of alterations.

[0014] In one embodiment, the cytidine deaminase has the following amino acid sequence: MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVAR LFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR and an amino acid sequence having at least 80% identity with

[0015] In one embodiment, the cytidine deaminase has the following amino acid sequence: MKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEIIKQYLPVPWKKGVFRNQVDPETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTIYTARLYYFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREILQ and an amino acid sequence having at least 80% identity with

[0016] In one embodiment, the cytidine deaminase has the following amino acid sequence: MADSSEKMRGQYISRDTFEKNYKPIDGTKEAHLLCEIKWGKYGKPWLHWCQNQRMNIHAEDYFMNNIFKAKKHPVHCYVTWYLSWSPCADCASKIVKFLEERPYLKLTIYVAQLYYHTEEENRKGLRLLRSKKVIIRVMDISDYNYCWKVFVSNQNGNEDYWPLQFDPWVKENYSRLLDIFWESKCRSPNPW and an amino acid sequence having at least 80% identity with

[0017] In one embodiment, the cytidine deaminase has the following amino acid sequence: MDPQRLRQWPGPGPASRGGYGQRPRIRNPEEWFHELSPRTFSFHFRNLRFASGRNRSYICCQVEGKNCFFQGIFQNQVPPDPPCHAELCFLSWFQSWGLSPDEHYYVTWFISWSPCCECAAKVAQFLEENRNVSLSLSAA RLYYFWKSESREGLRRLSDLGAQVGIMSFQDFQHCWNNFVHNLGMPFQPWKKLHKNYQRLVTELKQILREEPATYGSPQAQGKVRIGSTAAGLRHSHSHTRSEAHLRPNHSSRQHRILNPPREARARTCVLVDASWICYR and an amino acid sequence having at least 80% identity with

[0018] In some embodiments, the cytidine deaminase comprises an H122A modification. In some embodiments, the cytidine base editor of any one of the above aspects further comprises at least one adenosine deaminase or a catalytically active fragment thereof. In some embodiments, the adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase is a modified adenosine deaminase that does not occur in nature. In some embodiments, the cytidine base editor comprises two adenosine deaminases, which may be the same or different. In some embodiments, the two adenosine deaminases are capable of forming a heterodimer or a homodimer. In some embodiments, the adenosine deaminase domain is wild-type TadA and TadA7.10.

[0019] In one embodiment, the adenosine deaminase comprises a C-terminal deletion beginning at a residue selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 157. In one embodiment, the adenosine deaminase lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to full-length adenosine deaminase. In one embodiment, the adenosine deaminase lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to full-length adenosine deaminase. In some embodiments, the at least one nucleobase editor domain further comprises an abasic nucleobase editor. In some embodiments, the cytidine base editor of any one of the above aspects further comprises one or more nuclear localization signals (NLS). In some embodiments, the cytidine base editor comprises an N-terminal NLS and / or a C-terminal NLS. In some embodiments, the NLS is a bipartite NLS.

[0020] In some embodiments, the polynucleotide-programmable DNA-binding domain is Cas9. In some embodiments, the polynucleotide-programmable DNA-binding domain is Staphylococcus aureus Cas9 (SaCas9), Streptococcus pyogenes Cas9 (SpCas9), or a variant thereof. In some embodiments, the polynucleotide-programmable DNA-binding domain comprises nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9. In some embodiments, the polynucleotide-programmable DNA-binding domain comprises a catalytic domain capable of cleaving the reverse complement of a nucleic acid sequence. In some embodiments, the polynucleotide-programmable DNA-binding domain does not comprise a catalytic domain capable of cleaving a nucleic acid sequence. In some embodiments, the Cas9 is dCas9. In some embodiments, the Cas9 is Cas9 nickase (nCas9). In one embodiment, the nCas9 comprises the amino acid substitution D10A or a corresponding amino acid substitution.

[0021] In some embodiments, the cytidine base editor of any one of the above aspects further comprises one or more uracil DNA glycosylase inhibitors (UGIs). In some embodiments, the one or more UGIs are derived from Bacillus subtilis bacteriophage PBS1 and inhibit human UDG activity. In some embodiments, the cytidine base editor comprises two uracil DNA glycosylase inhibitors (UGIs). The cytidine base editor of any one of the above aspects further comprises one or more linkers.

[0022] Provided herein is a cell comprising the cytidine base editor of any one of the above aspects. In some embodiments, the cell is a bacterial cell, a plant cell, an insect cell, or a mammalian cell.

[0023] Provided herein is a molecular complex comprising the cytidine base editor of any one of the above embodiments and one or more of a guide RNA sequence, a tracrRNA sequence, or a target DNA sequence.

[0024] Provided herein is a method of editing a nucleobase of a nucleic acid sequence, the method comprising contacting the nucleic acid sequence with a cytidine base editor of any one of the above aspects, thereby converting a first nucleobase of the DNA sequence to a second nucleobase.

[0025] In some embodiments, the method further comprises contacting the nucleic acid sequence with a guide polynucleotide to effect the conversion, hi some embodiments, the first nucleobase is cytosine and the second nucleobase is thymidine.

[0026] In one embodiment, a fusion protein is provided comprising a polynucleotide-programmable DNA binding domain and at least one nucleobase editor domain comprising a cytidine deaminase, wherein the cytidine deaminase is selected from the group consisting of: (i) APOBEC-1 from Mesocricetus auratus (MaAPOBEC-1), Pongo pygmaeus (PpAPOBEC-1), Oryctolagus cuniculus (OcAPOBEC-1), Monodelphis domestica (MdAPOBEC-1), or Alligator mississippiensis (AmAPOBEC-1); (ii) APOBEC-2 from Pongo pygmaeus (PpAPOBEC-2), Bos taurus (BtAPOBEC-2), or Sus scrofa (SsAPOBEC-2); (iii) APOBEC-4 from Macaca fascicularis (MfAPOBEC-4); (iv) APOBEC-5 from Canis lupus (CsAPOBEC-5); Provided herein are fusion proteins that are AID from Lupus familiaris (ClAID) or Bos Taurus (BtAID); (v) yeast cytosine deaminase from Saccharomyces cerevisiae (yCD); (vi) APOBEC-3F from Rhinopithecus roxellana (RrA3F); or (vii) a cytidine deaminase having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of (i)-(viii).

[0027]

[0010] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and at least one nucleobase editor domain comprising a cytidine deaminase, wherein the cytidine deaminase is APOBEC-1 from Mesocricetus auratus (MaAPOBEC-1), Pongo pygmaeus (PpAPOBEC-1), Oryctolagus cuniculus (OcAPOBEC-1), or Monodelphis domestica (MdAPOBEC-1), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0028]

[0010] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and at least one nucleobase editor domain comprising a cytidine deaminase, wherein the cytidine deaminase is APOBEC-2 from Pongo pygmaeus (PpAPOBEC-2), Bos taurus (BtAPOBEC-2), or Sus scrofa (SsAPOBEC-2), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0029]

[0010] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and at least one nucleobase editor domain comprising a cytidine deaminase, wherein said cytidine deaminase is APOBEC-4 from Macaca fascicularis (MfAPOBEC-4), or a cytidine deaminase having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0030]

[0010] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA binding domain and at least one nucleobase editor domain comprising a cytidine deaminase, wherein said cytidine deaminase is AID from Canis lupus familiaris (ClAID), or from Bos Taurus (BtAID), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0031]

[0010] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and at least one nucleobase editor domain comprising a cytidine deaminase, wherein said cytidine deaminase is yeast cytosine deaminase (yCD) from Saccharomyces cerevisiae, or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0032]

[0010] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and at least one nucleobase editor domain comprising a cytidine deaminase, wherein said cytidine deaminase is APOBEC-3F from Rhinopithecus roxellana (RrA3F), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0033]

[0013] In one embodiment, provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and at least one nucleobase editor domain comprising a cytidine deaminase, wherein said cytidine deaminase is any one of the cytidine deaminases provided in Table 13, or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0034]

[0010] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and at least one nucleobase editor domain comprising a cytidine deaminase, wherein said cytidine deaminase is APOBEC-3F from Rhinopithecus roxellana (RrA3F), APOBEC-1 from Alligator mississippiensis (AmAPOBEC-1), APOBEC-2 from Sus scrofa (SsAPOBEC-2), APOBEC-1 from Pongo pygmaeus (PpAPOBEC-1), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0035] In one embodiment, the cytidine deaminase comprises one or more modifications at or corresponding to positions R15X, R16X, H21X, R30X, R33X, K34X, R52X, K60X, R118X, H121X, H122X, R126X, R128X, R169X, R198X, T36X, H53X, V62X, L88X, W90X, Y120X, or R132X numbered in SEQ ID NO: 1, where X is any amino acid. In one embodiment, the cytidine deaminase comprises one or more modifications selected from the group consisting of R15A, R16A, H21A, R30A, R33A, K34A, R52A, K60A, R118A, H121A, H122A, H122L, R126A, R128A, R169A, R198A, T36A, H53A, V62A, L88A, W90F, W90A, Y120F, Y120A, H121R, H122R, R126E, W90Y, and R132E according to the numbering in SEQ ID NO: 1, or one or more modifications corresponding thereto. In one embodiment, the cytidine deaminase comprises a combination of modifications selected from the group consisting of K34A+R33A, K34A+H122A, K34A+Y120F, K34A+R52A, K34A+H122A, K34A+H121A, W90A+R126E, W90Y+R126E, H121R+H122R, R126+R132E, W90Y+R132E, and W90Y+R126E+R132E, or a corresponding combination of modifications, according to the numbering in SEQ ID NO: 1. In one embodiment, the cytidine deaminase comprises Y120F and one or more modifications selected from the group consisting of R33A, W90F, K34A, R52A, H122A, and H121A, or a corresponding combination of one or more modifications.

[0036] In some embodiments, the cytidine deaminase comprises one or more modifications at, or one or more corresponding to, positions Y130X or R28X in the numbering of SEQ ID NO: 1, where X is any amino acid. In some embodiments, the cytidine deaminase comprises one or more modifications selected from, or one or more corresponding to, the group consisting of Y130A and R28A in the numbering of SEQ ID NO: 1. In some embodiments, the cytidine deaminase comprises the modifications Y130A and R28A in the numbering of SEQ ID NO: 1, or one or more corresponding modifications. In some embodiments, the cytidine deaminase comprises one or more modifications at, or one or more corresponding to, positions H122X, K34X, R33X, W90X, or R128X in the numbering of SEQ ID NO: 1, where X is any amino acid. In one embodiment, the cytidine deaminase comprises one or more modifications selected from the group consisting of H122A, K34A, R33A, W90F, W90A, and R128A, as numbered in SEQ ID NO: 1, or one or more modifications corresponding thereto.

[0037] In one embodiment, the cytidine deaminase comprises a combination of one or more modifications selected from the group consisting of R33A+K34A, W90F+K34A, R33A+K34A+W90F, and R33A+K34A+H122A+W90F, or a corresponding modification, according to the numbering in SEQ ID NO: 1. In one embodiment, the cytidine deaminase comprises an H122A modification, or a corresponding modification, according to the numbering in SEQ ID NO: 1. In one embodiment, the cytidine deaminase is rAPOBEC1 and comprises one or more modifications selected from the group consisting of R15A, R16A, H21A, R30A, R33A, K34A, R52A, K60A, R118A, H121A, H122A, H122L, R126A, R128A, R169A, R198A, T36A, H53A, V62A, L88A, W90F, W90A, Y120F, Y120A, H121R, H122R, R126E, W90Y, and R132E as numbered in SEQ ID NO: 1, or one or more modifications corresponding thereto. In one embodiment, the cytidine deaminase comprises modifications selected from the group consisting of K34A+R33A, K34A+H122A, K34A+Y120F, K34A+R52A, K34A+H122A, K34A+H121A, W90A+R126E, W90Y+R126E, H121R+H122R, R126+R132E, W90Y+R132E, and W90Y+R126E+R132E, as numbered in SEQ ID NO: 1, or a corresponding combination of one or more modifications thereof.

[0038] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and at least one nucleobase editor domain comprising a cytidine deaminase selected from the group consisting of an APOBEC2 family member, an APOBEC3 family member, an APOBEC4 family member, a cytidine deaminase 1 family member (CDA1), an A3A family member, an RrA3F family member, a PmCDA1 family member, and a FENRY family member.

[0039] In one embodiment, the APOBEC3 family member is selected from the group consisting of APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3G, and APOBEC3H. In one embodiment, the APOBEC2 family member is SsAPOBEC2.

[0040] Provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and at least one nucleobase editor domain comprising an APOBEC1 selected from the group consisting of ppAPOBEC1, AmAPOBEC1 (BEM3.31), ocAPOBEC1, SsAPOBEC2 (BEM3.39), hAPOBEC3A, maAPOBEC1, and mdAPOBEC1.

[0041] In one embodiment, the cytidine deaminase comprises one or more modifications at, or corresponding to, positions R15X, R16X, H21X, R30X, R33X, K34X, R52X, K60X, R118X, H121X, H122X, R126X, R128X, R169X, R198X, T36X, H53X, V62X, L88X, W90X, Y120X, or R132X numbered in SEQ ID NO: 1, wherein X is any amino acid. In one embodiment, the one or more modifications are selected from the group consisting of R15A, R16A, H21A, R30A, R33A, K34A, R52A, K60A, R118A, H121A, H122A, H122L, R126A, R128A, R169A, R198A, T36A, H53A, V62A, L88A, W90F, W90A, Y120F, Y120A, H121R, H122R, R126E, W90Y, and R132E as numbered in SEQ ID NO: 1, or one or more modifications corresponding thereto. In one embodiment, the cytidine deaminase comprises modifications selected from the group consisting of K34A+R33A, K34A+H122A, K34A+Y120F, K34A+R52A, K34A+H122A, K34A+H121A, W90A+R126E, W90Y+R126E, H121R+H122R, R126+R132E, W90Y+R132E, and W90Y+R126E+R132E, as numbered in SEQ ID NO: 1, or a corresponding combination of one or more modifications thereof. In one embodiment, the cytidine deaminase comprises Y120F and one or more modifications selected from the group consisting of R33A, W90F, K34A, R52A, H122A, and H121A, or a combination of one or more modifications corresponding thereto, as numbered in SEQ ID NO: 1.

[0042]

[0013] In one embodiment, provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and at least one nucleobase editor domain comprising a cytidine deaminase, wherein said cytidine deaminase comprises one or more modifications at, or one or more corresponding modifications to, positions R15X, R16X, H21X, R30X, R33X, K34X, R52X, K60X, R118X, H121X, H122X, R126X, R128X, R169X, R198X, T36X, H53X, V62X, L88X, W90X, Y120X or R132X in the numbering in SEQ ID NO: 1, wherein X is any amino acid.

[0043] In one embodiment, the cytidine deaminase comprises one or more modifications selected from the group consisting of R15A, R16A, H21A, R30A, R33A, K34A, R52A, K60A, R118A, H121A, H122A, H122L, R126A, R128A, R169A, R198A, T36A, H53A, V62A, L88A, W90F, W90A, Y120F, Y120A, H121R, H122R, R126E, W90Y, and R132E according to the numbering in SEQ ID NO: 1, or one or more modifications corresponding thereto. In one embodiment, the cytidine deaminase comprises modifications selected from the group consisting of K34A+R33A, K34A+H122A, K34A+Y120F, K34A+R52A, K34A+H122A, K34A+H121A, W90A+R126E, W90Y+R126E, H121R+H122R, R126+R132E, W90Y+R132E, and W90Y+R126E+R132E, as numbered in SEQ ID NO: 1, or a combination of one or more modifications corresponding thereto. In one embodiment, the cytidine deaminase comprises a modification at position Y120F in the numbering of SEQ ID NO: 1 and one or more modifications selected from the group consisting of R33A, W90F, K34A, R52A, H122A, and H121A, or one or more modifications corresponding thereto.

[0044] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and at least one nucleobase editor domain comprising a cytidine deaminase, wherein said cytidine deaminase comprises one or more modifications at, or one or more modifications corresponding to, positions Y130X and R28X in the numbering in SEQ ID NO: 1, wherein X is any amino acid.

[0045] In one embodiment, the cytidine deaminase comprises one or more modifications selected from the group consisting of Y130A and R28A, or one or more modifications corresponding thereto, in the numbering of SEQ ID NO: 1. In one embodiment, the cytidine deaminase comprises the modifications Y130A and R28A.

[0046]

[0013] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and at least one nucleobase editor domain comprising a cytidine deaminase, wherein said cytidine deaminase comprises one or more modifications at, or one or more modifications corresponding to, positions H122X, K34X, R33X, W90X, or R128X in the numbering in SEQ ID NO: 1, where X is any amino acid.

[0047] In one embodiment, the cytidine deaminase comprises one or more modifications selected from the group consisting of H122A, K34A, R33A, W90F, W90A, and R128A, or one or more modifications corresponding thereto, according to the numbering in SEQ ID NO: 1. In one embodiment, the cytidine deaminase comprises one or more modifications selected from the group consisting of R33A+K34A, W90F+K34A, R33A+K34A+W90F, and R33A+K34A+H122A+W90F, according to the numbering in SEQ ID NO: 1. In some embodiments, the cytidine deaminase is selected from the group consisting of APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, activation-induced (cytidine) deaminase (AID), hAPOBEC1, rAPOBEC1, ppAPOBEC1, AmAPOBEC1 (BEM3.31), ocAPOBEC1, ssAPOBEC2 (BEM3.39), hAPOBEC3A, maAPOBEC1, mdAPOBEC1, cytidine deaminase 1 (CDA1), hA3A, RrA3F (BEM3.14), PmCDA1, AID (activation-induced cytidine deaminase; AICDA), hAID, and FENRY. In one embodiment, the cytidine deaminase is APOBEC1. In one embodiment, the cytidine deaminase is rAPOBEC1. In one embodiment, the cytidine deaminase is hAPOBEC3A. In one embodiment, the cytidine deaminase is ppAPOBEC1.

[0048] In one embodiment, there is provided a fusion protein comprising a polynucleotide programmable DNA binding domain and a cytidine deaminase, wherein said cytidine deaminase has the following amino acid sequence: MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVAR LFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR Provided herein are fusion proteins comprising an amino acid sequence having at least 80% identity to

[0049] In one embodiment, there is provided a fusion protein comprising a polynucleotide programmable DNA binding domain and a cytidine deaminase, wherein said cytidine deaminase has the following amino acid sequence: MKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEIIKQYLPVPWKKGVFRNQVDPETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTIYTARLYYFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREILQ Provided herein are fusion proteins comprising an amino acid sequence having at least 80% identity to

[0050] In one embodiment, there is provided a fusion protein comprising a polynucleotide programmable DNA binding domain and a cytidine deaminase, wherein said cytidine deaminase has the following amino acid sequence: MADSSEKMRGQYISRDTFEKNYKPIDGTKEAHLLCEIKWGKYGKPWLHWCQNQRMNIHAEDYFMNNIFKAKKHPVHCYVTWYLSWSPCADCASKIVKFLEERPYLKLTIYVAQLYYHTEEENRKGLRLLRSKKVIIRVMDISDYNYCWKVFVSNQNGNEDYWPLQFDPWVKENYSRLLDIFWESKCRSPNPW Provided herein are fusion proteins comprising an amino acid sequence having at least 80% identity to

[0051] In one embodiment, there is provided a fusion protein comprising a polynucleotide programmable DNA binding domain and a cytidine deaminase, wherein said cytidine deaminase has the following amino acid sequence: MDPQRLRQWPGPGPASRGGYGQRPRIRNPEEWFHELSPRTFSFHFRNLRFASGRNRSYICCQVEGKNCFFQGIFQNQVPPDPPCHAELCFLSWFQSWGLSPDEHYYVTWFISWSPCCECAAKVAQFLEENRNVSLSLSAA RLYYFWKSESREGLRRLSDLGAQVGIMSFQDFQHCWNNFVHNLGMPFQPWKKLHKNYQRLVTELKQILREEPATYGSPQAQGKVRIGSTAAGLRHSHSHTRSEAHLRPNHSSRQHRILNPPREARARTCVLVDASWICYR Provided herein are fusion proteins comprising an amino acid sequence having at least 80% identity to

[0052] In one embodiment, the cytidine deaminase comprises an H122A mutation.

[0053] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and a cytidine deaminase, wherein said cytidine deaminase is APOBEC1 deaminase and comprises an H122A modification.

[0054]

[0010] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and a cytidine deaminase, wherein said cytidine deaminase is rAPOBEC1 and comprises one or more modifications selected from the group consisting of R15A, R16A, H21A, R30A, R33A, K34A, R52A, K60A, R118A, H121A, H122A, H122L, R126A, R128A, R169A, R198A, T36A, H53A, V62A, L88A, W90F, W90A, Y120F, Y120A, H121R, H122R, R126E, W90Y, and R132E. In one embodiment, the cytidine deaminase comprises a combination of modifications selected from the group consisting of K34A+R33A, K34A+H122A, K34A+Y120F, K34A+R52A, K34A+H122A, K34A+H121A, W90A+R126E, W90Y+R126E, H121R+H122R, R126+R132E, W90Y+R132E, and W90Y+R126E+R132E.

[0055] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and at least one nucleobase editor domain comprising an APOBEC1 selected from the group consisting of ppAPOBEC1, AmAPOBEC1 (BEM3.31), ocAPOBEC1, SsAPOBEC2 (BEM3.39), hAPOBEC3A, maAPOBEC1, and mdAPOBEC1.

[0056] In certain embodiments, the APOBEC1 comprises one or more modifications at or corresponding to positions R15X, R16X, H21X, R30X, R33X, K34X, R52X, K60X, R118X, H121X, H122X, R126X, R128X, R169X, R198X, T36X, H53X, V62X, L88X, W90X, Y120X, or R132X numbered in SEQ ID NO: 1, where X is any amino acid.

[0057] In one embodiment, the one or more modifications are selected from the group consisting of R15A, R16A, H21A, R30A, R33A, K34A, R52A, K60A, R118A, H121A, H122A, H122L, R126A, R128A, R169A, R198A, T36A, H53A, V62A, L88A, W90F, W90A, Y120F, Y120A, H121R, H122R, R126E, W90Y, and R132E as numbered in SEQ ID NO: 1, or one or more modifications corresponding thereto. In one embodiment, the APOBEC1 comprises a combination of one or more modifications selected from the group consisting of K34A+R33A, K34A+H122A, K34A+Y120F, K34A+R52A, K34A+H122A, K34A+H121A, W90A+R126E, W90Y+R126E, H121R+H122R, R126+R132E, W90Y+R132E, and W90Y+R126E+R132E, according to the numbering in SEQ ID NO: 1. In one embodiment, the APOBEC1 comprises a modification at Y120F and one or more modifications selected from the group consisting of R33A, W90F, K34A, R52A, H122A, and H121A, according to the numbering in SEQ ID NO: 1, or one or more modifications corresponding thereto.

[0058] In one embodiment, the fusion protein of any one of the above aspects further comprises at least one adenosine deaminase or a catalytically active fragment thereof. In one embodiment, the adenosine deaminase is TadA deaminase. In one embodiment, the TadA deaminase is a modified adenosine deaminase that does not occur in nature. In one embodiment, the fusion protein comprises two adenosine deaminases, which may be the same or different. In one embodiment, the two adenosine deaminases can form a heterodimer or a homodimer. In one embodiment, the two adenosine deaminase domains are wild-type TadA and TadA7.10.

[0059] In one embodiment, the adenosine deaminase comprises a C-terminal deletion beginning at a residue selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 157. In one embodiment, the adenosine deaminase lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues compared to full-length adenosine deaminase. In one embodiment, the adenosine deaminase lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues compared to full-length adenosine deaminase. In some embodiments, the at least one nucleobase editor domain further comprises an abasic nucleobase editor.

[0060] In some embodiments, the fusion protein of any one of the above aspects further comprises one or more nuclear localization signals (NLS). In some embodiments, the fusion protein comprises an N-terminal NLS and / or a C-terminal NLS. In some embodiments, the NLS is a bipartite NLS.

[0061] In some embodiments, the polynucleotide-programmable DNA-binding domain is Cas9. In some embodiments, the polynucleotide-programmable DNA-binding domain is Staphylococcus aureus Cas9 (SaCas9), Streptococcus pyogenes Cas9 (SpCas9), or a variant thereof. In some embodiments, the polynucleotide-programmable DNA-binding domain comprises nuclease-inactive Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9. In some embodiments, the polynucleotide-programmable DNA-binding domain comprises a catalytic domain capable of cleaving the reverse complement of a nucleic acid sequence. In some embodiments, the polynucleotide-programmable DNA-binding domain does not comprise a catalytic domain capable of cleaving a nucleic acid sequence.

[0062] In some embodiments, the Cas9 is dCas9. In some embodiments, the Cas9 is Cas9 nickase (nCas9). In some embodiments, the nCas9 comprises the amino acid substitution D10A or a corresponding amino acid substitution. In some embodiments, the Cas9 further comprises one or more uracil DNA glycosylase inhibitors (UGIs). In some embodiments, the one or more UGIs are derived from Bacillus subtilis bacteriophage PBS1 and inhibit human UDG activity. In some embodiments, the fusion protein comprises two uracil DNA glycosylase inhibitors (UGIs). In some embodiments, the fusion protein further comprises one or more linkers. In some embodiments, the fusion protein deaminates a nucleobase in a target nucleotide sequence, and the deamination has an increased cis to trans activity (in cis:in trans) ratio compared to a standard cytidine base editor.

[0063] In one embodiment, the canonical cytidine base editor comprises (i) a polynucleotide-programmable DNA-binding domain and (ii) an APOBEC cytidine deaminase.

[0064] In some embodiments, the APOBEC cytidine deaminase of the canonical cytidine base editor is rat APOBEC-1 cytidine deaminase (rAPOBEC-1). In some embodiments, the polynucleotide-programmable DNA-binding domain of the canonical cytidine base editor is Cas9 nickase. In some embodiments, the canonical cytidine base editor comprises a uracil glycosylase inhibitor (UGI) domain. In some embodiments, the canonical cytidine base editor is BE3 or BE4. In some embodiments, the increased cis-to-trans activity ratio is at least 2-, 2.5-, 5-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 60-fold or more. In some embodiments, the cytidine base editor has at least 50%, 60%, 70%, 80%, 90%, 95%, 100%, 105%, 110%, 115%, 120% or more cis activity relative to the standard cytidine base editor, hi some embodiments, the cytidine base editor has at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 or more times less in trans activity relative to the standard cytidine base editor.

[0065] In one aspect, provided herein is a polynucleotide molecule encoding the fusion protein of any one of the above aspects. In one embodiment, the polynucleotide is codon-optimized.

[0066] In one aspect, provided herein is an expression vector comprising the polynucleotide molecule. In one embodiment, the expression vector is a mammalian expression vector. In one embodiment, the vector is a viral vector selected from the group consisting of adeno-associated virus (AAV), retroviral vector, adenoviral vector, lentiviral vector, Sendai virus vector, and herpes virus vector. In one embodiment, the vector comprises a promoter.

[0067] In one aspect, provided herein is a cell comprising the polynucleotide or the vector, hi some embodiments, the cell is a bacterial cell, a plant cell, an insect cell, a human cell, or a mammalian cell.

[0068] In one aspect, provided herein is a molecular complex comprising the fusion protein of any one of the above aspects and one or more of a guide RNA sequence, a tracrRNA sequence, or a target DNA sequence.

[0069] In one aspect, provided herein is a kit comprising the fusion protein, the polynucleotide, the vector, or the molecular complex of any one of the above aspects.

[0070]

[0010] In one aspect, provided herein is a method of editing a nucleobase of a nucleic acid sequence, comprising contacting the nucleic acid sequence with a base editor comprising the fusion protein of any one of the above aspects to convert a first nucleobase of a DNA sequence to a second nucleobase. In one embodiment, the first nucleobase is cytosine and the second nucleobase is thymidine.

[0071] In one aspect, provided herein is a method for editing nucleobases of a nucleic acid sequence, comprising contacting the nucleic acid sequence with a base editor comprising the fusion protein of any one of the above aspects to convert a first nucleobase of a DNA sequence to a second nucleobase. In some embodiments, the first nucleobase is cytosine and the second nucleobase is thymidine, or the first nucleobase is adenine and the second nucleobase is guanine. In some embodiments, the method further comprises converting a third nucleobase to a fourth nucleobase. In some embodiments, the third nucleobase is guanine and the fourth nucleobase is adenine, or the third nucleobase is thymine and the fourth nucleobase is cytosine.

[0072]

[0009] In one aspect, provided herein is a method for optimized base editing, comprising contacting a target nucleobase in a target nucleotide sequence with a cytidine base editor comprising (i) a polynucleotide-programmable DNA-binding domain and (ii) a cytidine deaminase, wherein the cytidine base editor deaminates the target nucleobase with lower unintended deamination in the target nucleobase sequence compared to a canonical cytidine base editor comprising rAPOBEC1. In some embodiments, the cytidine base editor deaminates the target nucleobase with higher efficiency compared to the canonical cytidine base editor. In some embodiments, the canonical cytidine base editor further comprises a uracil glycosylase inhibitor (UGI) domain. In some embodiments, the canonical cytidine base editor is BE3 or BE4. In some embodiments, the cytidine base editor produces at least 20%, 30%, 50%, 70%, or 90% less undesired deamination than the canonical cytidine base editor as measured by a cis / trans deamination assay. In some embodiments, the cytidine base editor has at least 50%, 60%, 70%, 80%, 90%, 95%, 100%, 105%, 110%, 115%, 120% or more cis activity than the canonical cytidine base editor. In some embodiments, the cytidine base editor has at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold or more less in trans activity than the canonical cytidine base editor.In one embodiment, the cytidine deaminase is selected from the group consisting of: (a) APOBEC-1 from Mesocricetus auratus (MaAPOBEC-1), Pongo pygmaeus (PpAPOBEC-1), Oryctolagus cuniculus (OcAPOBEC-1), Monodelphis domestica (MdAPOBEC-1), or Alligator mississippiensis (AmAPOBEC-1); (b) APOBEC-2 from Pongo pygmaeus (PpAPOBEC-2), Bos taurus (BtAPOBEC-2), or Sus scrofa (SsAPOBEC-2); (c) APOBEC-4 from Macaca fascicularis (MfAPOBEC-4); (d) APOBEC-5 from Canis lupus familiaris (ClAID) or Bos (e) yeast cytosine deaminase (yCD) from Saccharomyces cerevisiae; (f) APOBEC-3F (RrA3F) from Rhinopithecus roxellana; or (g) a cytidine deaminase having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of (a) to (f).

[0073] In one embodiment, the cytidine deaminase is AID from Canis lupus familiaris (ClAID) or Bos taurus (BtAID), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, the cytidine deaminase is APOBEC-3F from Rhinopithecus roxellana (RrA3F), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0074] In one embodiment, the cytidine deaminase comprises an alteration selected from the group consisting of R15X, R16X, H21X, R30X, R33X, K34X, R52X, K60X, R118X, H121X, H122X, R126X, R128X, R169X, R198X, T36X, H53X, V62X, L88X, W90X, Y120X, and R132X according to the numbering in SEQ ID NO: 1, or a corresponding alteration, wherein X is any amino acid. In one embodiment, the cytidine deaminase comprises an alteration selected from the group consisting of R15A, R16A, H21A, R30A, R33A, K34A, R52A, K60A, R118A, H121A, H122A, H122L, R126A, R128A, R169A, R198A, T36A, H53A, V62A, L88A, W90F, W90A, Y120F, Y120A, H121R, H122R, R126E, W90Y, and R132E as numbered in SEQ ID NO: 1, or an alteration corresponding thereto. In one embodiment, the cytidine deaminase comprises a combination of modifications selected from the group consisting of K34A+R33A, K34A+H122A, K34A+Y120F, K34A+R52A, K34A+H122A, K34A+H121A, W90A+R126E, W90Y+R126E, H121R+H122R, R126+R132E, W90Y+R132E, and W90Y+R126E+R132E, as numbered in SEQ ID NO: 1, or a corresponding combination of modifications.

[0075] In some embodiments, the cytidine deaminase comprises an alteration at position Y120F in the numbering of SEQ ID NO: 1 and one or more alterations selected from the group consisting of R33A, W90F, K34A, R52A, H122A, and H121A, or one or more alterations corresponding thereto. In some embodiments, the cytidine deaminase comprises an alteration at position Y130X or R28X in the numbering of SEQ ID NO: 1, or an alteration corresponding thereto, where X is any amino acid. In some embodiments, the cytidine deaminase comprises a Y130A mutation or a R28A mutant in the numbering of SEQ ID NO: 1, or an alteration corresponding thereto. In some embodiments, the cytidine deaminase comprises the alterations Y130A and R28A in the numbering of SEQ ID NO: 1, or an alteration corresponding thereto.

[0076] In some embodiments, the cytidine deaminase comprises modifications at positions H122X, K34X, R33X, W90X, and R128X, or modifications corresponding thereto, according to the numbering in SEQ ID NO: 1, where X is any amino acid. In some embodiments, the cytidine deaminase comprises modifications selected from the group consisting of H122A, K34A, R33A, W90F, W90A, and R128A, or modifications corresponding thereto, according to the numbering in SEQ ID NO: 1. In some embodiments, the cytidine deaminase comprises a combination of modifications selected from the group consisting of R33A+K34A, W90F+K34A, R33A+K34A+W90F, and R33A+K34A+H122A+W90F, according to the numbering in SEQ ID NO: 1, or a combination of modifications corresponding thereto.

[0077] In one embodiment, the cytidine deaminase has the following amino acid sequence: MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVAR LFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR and an amino acid sequence having at least 80% identity with

[0078] In one embodiment, the cytidine deaminase has the following amino acid sequence: MKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEIIKQYLPVPWKKGVFRNQVDPETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTIYTARLYYFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREILQ and an amino acid sequence having at least 80% identity with

[0079] In one embodiment, the cytidine deaminase has the following amino acid sequence: MADSSEKMRGQYISRDTFEKNYKPIDGTKEAHLLCEIKWGKYGKPWLHWCQNQRMNIHAEDYFMNNIFKAKKHPVHCYVTWYLSWSPCADCASKIVKFLEERPYLKLTIYVAQLYYHTEEENRKGLRLLRSKKVIIRVMDISDYNYCWKVFVSNQNGNEDYWPLQFDPWVKENYSRLLDIFWESKCRSPNPW and an amino acid sequence having at least 80% identity with

[0080] In one embodiment, the cytidine deaminase has the following amino acid sequence: MDPQRLRQWPGPGPASRGGYGQRPRIRNPEEWFHELSPRTFSFHFRNLRFASGRNRSYICCQVEGKNCFFQGIFQNQVPPDPPCHAELCFLSWFQSWGLSPDEHYYVTWFISWSPCCECAAKVAQFLEENRNVSLSLSAA RLYYFWKSESREGLRRLSDLGAQVGIMSFQDFQHCWNNFVHNLGMPFQPWKKLHKNYQRLVTELKQILREEPATYGSPQAQGKVRIGSTAAGLRHSHSHTRSEAHLRPNHSSRQHRILNPPREARARTCVLVDASWICYR and an amino acid sequence having at least 80% identity with

[0081] In some embodiments, the cytidine deaminase comprises an H122A modification. In some embodiments, the contacting is performed in a cell. In some embodiments, the cell is a human cell or a mammalian cell. In some embodiments, the contacting is performed in vivo or ex vivo.

[0082] In one aspect, provided herein is a cytidine deaminase comprising an amino acid sequence having at least 80% identity to an amino acid sequence selected from the following: MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVAR LFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR; MKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEIIKQYLPVPWKKGVFRNQVDPETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTIYTARLYYFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREILQ; MADSSEKMRGQYISRDTFEKNYKPIDGTKEAHLLCEIKWGKYGKPWLHWCQNQRMNIHAEDYFMNNIFKAKKHPVHCYVTWYLSWSPCADCASKIVKFLEERPYLKLTIYVAQLYYHTEEENRKGLRLLRSKKVIIRVMDISDYNYCWKVFVSNQNGNEDYWPLQFDPWVKENYSRLLDIFWESKCRSPNPW; and MDPQRLRQWPGPGPASRGGYGQRPRIRNPEEWFHELSPRTFSFHFRNLRFASGRNRSYICCQVEGKNCFFQGIFQNQVPPDPPCHAELCFLSWFQSWGLSPDEHYYVTWFISWSPCCECAAKVAQFLEENRNVSLSLSAA RLYYFWKSESREGLRRLSDLGAQVGIMSFQDFQHCWNNFVHNLGMPFQPWKKLHKNYQRLVTELKQILREEPATYGSPQAQGKVRIGSTAAGLRHSHSHTRSEAHLRPNHSSRQHRILNPPREARARTCVLVDASWICYR.

[0083] The description and examples herein show in detail embodiments of the present disclosure. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will recognize that there are numerous variations and modifications that fall within the scope of the present disclosure.

[0084] The practice of some embodiments disclosed herein will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of those in the art. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds.); the series Methods in Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0085] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0086] While various features of the present disclosure may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0087] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and by consideration of the accompanying drawings that are described below.

[0088] definition The following definitions supplement those in the art and are directed to this application and are not to be construed as attributing any related or unrelated matter, such as commonly owned patents or applications. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing of the present disclosure, the preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0089] Unless otherwise defined, all technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art to which this invention belongs.The following references provide those skilled in the art with the general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The HarperCollins Dictionary of Biology (1991).

[0090] In this application, the use of the singular includes the plural unless specifically stated otherwise. It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" and should be understood to be inclusive unless specifically stated otherwise. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is non-limiting.

[0091] As used in the specification and claims, the terms "comprising" (and any of its forms, such as "comprise" and "comprises"), "having" (and any of its forms, such as "have" and "has"), "including" (and any of its forms, such as "include" and "includes"), or "containing" (and any of its forms, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0092] The terms "about" or "approximately" mean within an acceptable error range for a particular value, as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations, according to practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean a value within the same order of magnitude, such as within 5-fold or within 2-fold. When specific values ​​are described in applications and claims, unless otherwise specified, the term "about" meaning within an acceptable error range for that particular value should be presumed.

[0093] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0094] References in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0095] An "abasic base editor" refers to an agent that can excise a nucleobase and insert a DNA nucleobase (A, T, C, or G). An abasic base editor comprises a nucleic acid glycosylase polypeptide or a fragment thereof. In one embodiment, the nucleic acid glycosylase is a mutant human uracil DNA glycosylase or an active fragment thereof that contains Asp at amino acid 204 of the sequence below, or the corresponding position in uracil DNA glycosylase (e.g., substituting Asn at amino acid 204), and has cytosine-DNA glycosylase activity. In one embodiment, the nucleic acid glycosylase is a mutant human uracil DNA glycosylase or an active fragment thereof that contains Ala, Gly, Cys, or Ser at amino acid 147 of the sequence below, or the corresponding position in uracil DNA glycosylase (e.g., substituting Tyr at amino acid 147), and has thymine-DNA glycosylase activity. The sequence of an exemplary human uracil-DNA glycosylase, isoform 1, is as follows: 1 mgvfclgpwg lgrklrtpgk gplqllsrlc gdhlqaipak kapagqeepg tppssplsae 61 qldriqrnka aallrlaarn vpvgfgeswk khlsgefgkp yfiklmgfva eerkhytvyp 121 pphqvftwtq mcdikdvkvv ilgqdp y hgp nqahglcfsv qrpvppppsl eniykelstd 181 iedfvhpghg dlsgwakqgv lll n avltvr ahqanshker gweqftdavv swlnqnsngl 241 vfllwgsyaq kkgsaidrkr hhvlqtahps p l svy r gffg crhfsktnel lqksgkkpid 301 wkel

[0096] The sequence of human uracil-DNA glycosylase, isoform 2 is as follows: 1 migqktlysf fspsparkrh apspepavqg tgvagvpees gdaaaipakk apagqeepgt 61 ppssplsaeq ldriqrnkaa allrlaarnv pvgfgeswkk hlsgefgkpy fiklmgfvae 121 erkhytvypp phqvftwtqm cdikdvkvvi lgqdp y hgpn qahglcfsvq rpvppppsle 181 niykelstdi edfvhpghgd lsgwakqgvl ll n avltvra hqanshkerg weqftdavvs 241 wlnqnsnglv fllwgsyaqk kgsaidrkrh hvlqtahpsp l svy r gffgc rhfsktnell 301 qksgkkpidw kel

[0097] In other embodiments, the abasic editor is any of the abasic editors described in PCT / JP205 / 080958 and US20170321210, which are incorporated herein by reference. In certain embodiments, the abasic editor comprises a mutation at the bolded, underlined position in the above sequence, or at the corresponding amino acid in any other abasic editor or uracil deglycosylase known in the art. In one embodiment, the abasic editor comprises a mutation at Y147, N204, L272, and / or R276 or a corresponding position. In another embodiment, the abasic editor comprises a Y147A or Y147G mutation or a corresponding mutation. In another embodiment, the abasic editor comprises an N204D mutation or a corresponding mutation. In another embodiment, the abasic editor comprises an L272A mutation or a corresponding mutation. In another embodiment, the abasic editor comprises an R276E or R276C mutation or a corresponding mutation.

[0098] "Adenosine deaminase" refers to a polypeptide or fragment thereof that can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine or deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., engineered adenosine deaminases, evolved adenosine deaminases) can be from any organism, such as bacteria.

[0099] In some embodiments, the adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase is a TadA variant. In some embodiments, the TadA variant is TadA*7.10. In some embodiments, the deaminase or deaminase domain is a variant of a natural deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is non-naturally occurring. For example, in some embodiments, the deaminase or deaminase domain has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identity to a naturally occurring deaminase. Deaminase domains are described, for example, in International PCT Applications PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), each of which is incorporated by reference herein in its entirety.Also, Komor, AC, et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage," Nature 533, 420-424 (2016); Gaudelli, NM, et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage," Nature 551, 464-471 (2017); Komor, AC, et al., "Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity," Science Advances 3:eaao4774 (2017), and Rees, HA, et al., "Base editing: precision chemistry on the genome and transcriptome of living cells." Nat Rev Genet. 2018, the entire contents of which are incorporated herein by reference. Dec;19(12):770-788. See also doi: 10.1038 / s41576-018-0059-1.

[0100] In certain embodiments, the adenosine deaminase comprises a modification in the following sequence: MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG LHDPTAHAEI MALRQGGLVM QNYRLIDATL YVTFEPCVMC AGAMIHSRIG RVVFGVRNAK TGAAGSLMDV LHYPGMNHRV EITEGILADE CAALLCYFFR MPRQVFNAQK KAQSSTD (also known as TadA*7.10).

[0101] In certain embodiments, the adenosine deaminase homodimer comprises a TadA*7.10 domain and an adenosine deaminase domain selected from one of the following: Staphylococcus aureus (S. aureus) TadA: MGSHMTNDIYFMTLAIEEAKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN Bacillus subtilis (B. subtilis) TadA: MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE Salmonella typhimurium (S. typhimurium) TadA: MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV Shewanella putrefaciens (S. putrefaciens) TadA: MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE Haemophilus influenzae F3031 (H. influenzae) TadA: MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTΑΗAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK Caulobacter crescentus (C. crescentus) TadA: MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIIAAMRAAAAKLGNYRLTDLTVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI Geobacter sulfurreducens (G. sulfurreducens) TadA: MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP TadA*7.10 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0102] "Administering" refers herein to providing one or more compositions described herein to a patient or subject. By way of example, and not limitation, administration, e.g., injection, of a composition can be by intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more such routes can be used. Parenteral administration can be by, for example, bolus injection or by gradual perfusion over time. In some embodiments, parenteral administration includes intravascular, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, intradermal, intraperitoneal, transtracheal, subcutaneous, subkeratinous, intraarticular, intracapsular, subarachnoid, and intrasternal infusion or injection. Alternatively, or concurrently, administration can be by the oral route.

[0103] By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0104] "Alteration" refers to a change (e.g., an increase or decrease) in the structure, expression level, or activity of a gene or polypeptide, as detected by standard art known methods, such as those described herein. As used herein, alteration includes a change in the sequence of a polynucleotide or polypeptide, or a change in expression level, e.g., a 10% change, a 25% change, a 40% change, a 50% change, or more change in expression level.

[0105] "Ameliorate" means to lessen, inhibit, attenuate, reduce, arrest, or stabilize the occurrence or progression of a disease.

[0106] "Analog" refers to a molecule that is not identical but has similar functional or structural characteristics. For example, a polynucleotide or polypeptide analog retains the biological activity of the corresponding naturally occurring polynucleotide or polypeptide while possessing certain modifications that enhance the analog's function compared to the naturally occurring polynucleotide or polypeptide. Such modifications can increase the analog's DNA affinity, efficiency, specificity, protease or nuclease resistance, membrane permeability, and / or half-life, for example, without altering ligand binding. Analogs can include non-naturally occurring polynucleotides or amino acids.

[0107] "Base editor (BE)" or "nucleobase editor (NBE)" refers to an agent that binds to a polynucleotide and has nucleobase-modifying activity. In various embodiments, the base editor comprises a nucleobase-modifying polypeptide (e.g., a deaminase) and a nucleic acid-programmable nucleotide-binding domain together with a guide polynucleotide (e.g., a guide RNA). In various embodiments, the agent is a biomolecular complex that includes a protein domain with base-editing activity, i.e., a domain that can modify bases (e.g., A, T, C, G, U) in a nucleic acid molecule (e.g., DNA). In some embodiments, the polynucleotide-programmable DNA-binding domain is fused or linked to one or more deaminase domains. In one embodiment, the agent is a fusion protein that includes one or more domains with base-editing activity. In another embodiment, the protein domain with base-editing activity is linked to a guide RNA (e.g., via an RNA-binding motif on the guide RNA and an RNA-binding domain fused to a deaminase). In some embodiments, the domain with base-editing activity can deaminate a base in a nucleic acid molecule. In some embodiments, the base editor can deaminate one or more bases in a DNA molecule. In some embodiments, the base editor is capable of deaminating cytosine (C) or adenosine (A) in DNA. In some embodiments, the base editor is capable of deaminating cytosine (C) and adenosine (A) in DNA. In some embodiments, the base editor is capable of deaminating cytosine (C) in DNA. In some embodiments, the base editor is a cytidine base editor (CBE) (e.g., BE4). In some embodiments, the base editor is capable of deaminating adenosine (A) in DNA. In some embodiments, the base editor is a standard base editor comprising a naturally occurring protein domain with base editing activity and / or programmable DNA binding activity.For example, the canonical cytidine base editor can include a cytidine deaminase (e.g., an APOBEC cytidine deaminase or an AID deaminase). In some embodiments, the canonical cytidine deaminase includes an APOBEC1 cytidine deaminase (e.g., rAPOBEC1). In some embodiments, the canonical cytidine base editor further includes additional domains associated with or linked to the cytidine deaminase, for example, one or more UGI domains can be linked to the cytidine deaminase. In some embodiments, the base editor is an adenosine base editor (ABE) and a cytidine base editor (CBE).

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

[0109] In some embodiments, adenosine base editors are generated by cloning adenosine deaminase variants into a scaffold comprising a circularly permuted Cas9 (e.g., spCAS9 or saCAS9) and a bipartite nuclear localization sequence. Circularly permuted Cas9s are known in the art and are described, for example, in Oakes et al., Cell 176, 254-267, 2019. Exemplary circular permutations are described below, where bolded sequences represent Cas9-derived sequences, italicized sequences represent linker sequences, and underlined sequences represent the bipartite nuclear localization sequence.

[0110] TIFF0007600122000001.tif187163

[0111] In some embodiments, the polynucleotide-programmable DNA-binding domain is a CRISPR-associated (e.g., Cas or Cpf1) enzyme. In some embodiments, the base editor is a catalytically inactive Cas9 (dCas9) fused to one or more deaminase domains. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to one or more deaminase domains. In some embodiments, the base editor is fused to a base excision repair (BER) inhibitor. In certain embodiments, the base excision repair inhibitor is a uracil DNA glycosylase inhibitor (UGI). In certain embodiments, the base excision repair inhibitor is an inosine base excision repair inhibitor.

[0112] Details of base editors are described in International PCT Applications PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), each of which is incorporated by reference herein in its entirety. Also, Komor, AC, et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage," Nature 533, 420-424 (2016); Gaudelli, NM, et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage," Nature 551, 464-471 (2017); Komor, AC, et al., "Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity," Science Advances 3:eaao4774 (2017), and Rees, HA, et al., "Base editing: precision chemistry on the genome and transcriptome of living cells." Nat Rev Genet. 2018, the entire contents of which are incorporated herein by reference. Dec;19(12):770-788. See also doi: 10.1038 / s41576-018-0059-1.

[0113] As an example, the adenine base editor (ABE) used in the base editing compositions, systems, and methods described herein has the following nucleic acid sequence (8877 base pairs) (Addgene, Watertown, MA; Gaudelli NM, et al., Nature. 2017 Nov 23;551(7681):464-471. doi: 10.1038 / nature24644; Koblan LW, et al., Nat Biotechnol. 2018 Oct;36(9):843-846. doi: 10.1038 / nbt.4172.). Polynucleotide sequences having at least 95% identity to this ABE nucleic acid sequence are also included.

[0114] ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT GACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGATTTCCAAGTCTCCACCCCATTG ACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCC ATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGT CAGATCCGCTAGAGATCCGCGGCCGCTAATACGACTCACTATAGGGAGAGCCGCCACCATGAAACGGACA GCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTCTCTGAAGTCGAGTTTAGCCACGAGT ATTGGATGAGGCACGCACTGACCCTGGCAAAGCGAGCATGGGATGAAAGAGAAGTCCCCGTGGGCGCCGT GCTGGTGCACAACAATAGAGTGATCGGAGAGGGATGGAACAGGCCAATCGGCCGCCACGACCCTACCGCA CACGCAGAGATCATGGCACTGAGGCAGGGAGGCCTGGTCATGCAGAATTACCGCCTGATCGATGCCACCC TGTATGTGACACTGGAGCCATGCGTGATGTGCGCAGGAGCAATGATCCACAGCAGGATCGGAAGAGTGGT GTTCGGAGCACGGGACGCCAAGACCGGCGCAGCAGGCTCCCTGATGGATGTGCTGCACCACCCCGGCATG AACCACCGGGTGGAGATCACAGAGGGAATCCTGGCAGACGAGTGCGCCGCCCTGCTGAGCGATTTCTTTA GAATGCGGAGACAGGAGATCAAGGCCCAGAAGAAGGCACAGAGCTCCACCGACTCTGGAGGATCTAGCGG AGGATCCTCTGGAAGCGAGACACCAGGCACAAGCGAGTCCGCCACACCAGAGAGCTCCGGCGGCTCCTCC GGAGGATCCTCTGAGGTGGAGTTTTCCCACGAGTACTGGATGAGACATGCCCTGACCCTGGCCAAGAGGG CACGCGATGAGAGGGAGGTGCCTGTGGGAGCCGTGCTGGTGCTGAACAATAGAGTGATCGGCGAGGGCTG GAACAGAGCCATCGGCCTGCACGACCCAACAGCCCATGCCGAAATTATGGCCCTGAGACAGGGCGGCCTG GTCATGCAGAACTACAGACTGATTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTGATGTGCGCCG GCGCCATGATCCACTCTAGGATCGGCCGCGTGGTGTTTGGCGTGAGGAACGCAAAAACCGGCGCCGCAGG CTCCCTGATGGACGTGCTGCACTACCCCGGCATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCA GATGAATGTGCCGCCCTGCTGTGCTATTTCTTTCGGATGCCTAGACAGGTGTTCAATGCTCAGAAGAAGG CCCAGAGCTCCACCGACTCCGGAGGATCTAGCGGAGGCTCCTCTGGCTCTGAGACACCTGGCACAAGCGA GAGCGCAACACCTGAAAGCAGCGGGGGCAGCAGCGGGGGGTCAGACAAGAAGTACAGCATCGGCCTGGCC ATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGG TGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGA AACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGC TATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGT CCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGC CTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGAC CTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACC TGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGA GGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGA CGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGGAGAAAGAATGGCCTGTTCGGAAACCTGATTGCCC TGAGCCTGGGCCTGACCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAG CAAGGACACCTACGACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGGCCGACCTGTTTT CTGGCCGCCAAGAACCTGCTCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCA AGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGC TCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCC GGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGG ACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAA CGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTAC CCATTCCTGAAGGACAACCGGGAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCC CTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAA CTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAG AACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGC TGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGC CATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAG AAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACAT ACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGA AGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCC CACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCC GGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGG CTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAA GCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTA AGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGA ATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACA CCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGA ACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGAC TCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAG AGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTT CGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAG CTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACG ACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCG GAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAAC GCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACA AGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTT CTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGG CCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGC GGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAA AGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAG TACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGT CCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAA TCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAG TACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAA ACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGG CTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATC GAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCT ACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAA TCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAA GAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTC AGCTGGGAGGTGACTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAG GAAAGTCTAACCGGTCATCATCACCATCACCATTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTT CTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGT GGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCT CTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGATACCGTCGACCTCTAGCTAGAGCTTGGCGTA ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA AGCATAAAGTGTAAAGCCTAGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGC CCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGG TTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGA GCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCT CCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGAT ACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTA ACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTA CACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACACTCAGTGGAACGAAAACTC ACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTAC GATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCT CCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGT TGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCC CAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGA TCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTAC TGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGT ATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAA AAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGA GCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATAC TCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATG TATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGA TCGGGAGATCGATCTCCCGATCCCCTAGGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAA GCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAAC AAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGAT GTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCAT TAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCAT TGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC

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

[0116] 1 ATATGCCAAG TACGCCCCCT ATTGACGTCA ATGACGGTAA ATGGCCCGCC TGGCATTATG 61 CCCAGTACAT GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA TTAGTCATCG 121 CTATTACCAT GGTGATGCGG TTTTGGCAGT ACATCAATGG GCGTGGATAG CGGTTTGACT 181 CACGGGGATT TCCAAGTCTC CACCCCATTG ACGTCAATGG GAGTTTGTTT TGGCACCAAA 241 ATCAACGGGA CTTTCCAAAA TGTCGTAACA ACTCCGCCCC ATTGACGCAA ATGGGCGGTA 301 GGCGTGTACG GTGGGAGGTC TATATAAGCA GAGCTGGTTT AGTGAACCGT CAGATCCGCT 361 AGAGATCCGC GGCCGCTAAT ACGACTCACT ATAGGGAGAG CCGCCACCAT GAGCTCAGAG 421 ACTGGCCCAG TGGCTGTGGA CCCCACATTG AGACGGCGGA TCGAGCCCCA TGAGTTTGAG 481 GTATTCTTCG ATCCGAGAGA GCTCCGCAAG GAGACCTGCC TGCTTTACGA AATTAATTGG 541 GGGGGCCGGC ACTCCATTTG GCGACATACA TCACAGAACA CTAACAAGCA CGTCGAAGTC 601 AACTTCATCG AGAAGTTCAC GACAGAAAGA TATTTCTGTC CGAACACAAG GTGCAGCATT 661 ACCTGGTTTC TCAGCTGGAG CCCATGCGGC GAATGTAGTA GGGCCATCAC TGAATTCCTG 721 TCAAGGTATC CCCACGTCAC TCTGTTTATT TACATCGCAA GGCTGTACCA CCACGCTGAC 781 CCCCGCAATC GACAAGGCCT GCGGGATTTG ATCTCTTCAG GTGTGACTAT CCAAATTATG 841 ACTGAGCAGG AGTCAGGATA CTGCTGGAGA AACTTTGTGA ATTATAGCCC GAGTAATGAA 901 GCCCACTGGC CTAGGTATCC CCATCTGTGG GTACGACTGT ACGTTCTTGA ACTGTACTGC 961 ATCATACTGG GCCTGCCTCC TTGTCTCAAC ATTCTGAGAA GGAAGCAGCC ACAGCTGACA 1021 TTCTTTACCA TCGCTCTTCA GTCTTGTCAT TACCAGCGAC TGCCCCCACA CATTCTCTGG 1081 GCCACCGGGT TGAAATCTGG TGGTTCTTCT GGTGGTTCTA GCGGCAGCGA GACTCCCGGG 1141 ACCTCAGAGT CCGCCACACC CGAAAGTTCT GGTGGTTCTT CTGGTGGTTC TGATAAAAAG 1201 TATTCTATTG GTTTAGCCAT CGGCACTAAT TCCGTTGGAT GGGCTGTCAT AACCGATGAA 1261 TACAAAGTAC CTTCAAAGAA ATTTAAGGTG TTGGGGAACA CAGACCGTCA TTCGATTAAA 1321 AAGAATCTTA TCGGTGCCCT CCTATTCGAT AGTGGCGAAA CGGCAGAGGC GACTCGCCTG 1381 AAACGAACCG CTCGGAGAAG GTATACACGT CGCAAGAACC GAATATGTTA CTTACAAGAA 1441 ATTTTTAGCA ATGAGATGGC CAAAGTTGAC GATTCTTTCT TTCACCGTTT GGAAGAGTCC 1501 TTCCTTGTCG AAGAGGACAA GAAACATGAA CGGCACCCCA TCTTTGGAAA CATAGTAGAT 1561 GAGGTGGCAT ATCATGAAAA GTACCCAACG ATTTATCACC TCAGAAAAAA GCTAGTTGAC 1621 TCAACTGATA AAGCGGACCT GAGGTTAATC TACTTGGCTC TTGCCCATAT GATAAAGTTC 1681 CGTGGGCACT TTCTCATTGA GGGTGATCTA AATCCGGACA ACTCGGATGT CGACAAACTG 1741 TTCATCCAGT TAGTACAAAC CTATAATCAG TTGTTTGAAG AGAACCCTAT AAATGCAAGT 1801 GGCGTGGATG CGAAGGCTAT TCTTAGCGCC CGCCTCTCTA AATCCCGACG GCTAGAAAAAC 1861 CTGATCGCAC AATTACCCGG AGAGAAGAAA AATGGGTTGT TCGGTAACCT TATAGCGCTC 1921 TCACTAGGCC TGACACCAAA TTTTAAGTCG AACTTCGACT TAGCTGAAGA TGCCAAATTG 1981 CAGCTTAGTA AGGACACGTA CGATGACGAT CTCGACAATC TACTGGCACA AATTGGAGAT 2041 CAGTATGCGG ACTTATTTTT GGCTGCCAAA AACCTTAGCG ATGCAATCCT CCTATCTGAC 2101 ATACTGAGAG TTAATACTGA GATTACCAAG GCGCCGTTAT CCGCTTCAAT GATCAAAAGG 2161 TACGATGAAC ATCACCAAGA CTTGACACTT CTCAAGGCCC TAGTCCGTCA GCAACTGCCT 2221 GAGAAATATA AGGAAATATT CTTTGATCAG TCGAAAAACG GGTACGCAGG TTATATTGAC 2281 GGCGGAGCGA GTCAAGAGGA ATTCTACAAG TTTATCAAAC CCATATTAGA GAAGATGGAT 2341 GGGACGGAAG AGTTGCTTGT AAAACTCAAT CGGAAGATC TACTGCGAAA GCAGCGGACT 2401 TTCGACAACG GTAGCATTCC ACATCAAATC CACTTAGGCG AATTGCATGC TATACTTAGA 2461 AGGCAGGAGG ATTTTTATCC GTTCCTCAAA GACAATCGTG AAAAGATTGA GAAAATCCTA 2521 ACCTTTCGCA TACCTTACTA TGTGGGACCC CTGGCCCGAG GGAACTCTCG GTTCGCATGG 2581 REPEAT AGTCCREACT AACGATTACT CCATGGAATTT TTREPEAT TGTCREADAA 2641 GGTGCGTCAG CTCAATCGTT CATCGAGAGG ATGACCAACT TTGACAAA TTTACCGAAC 2701 GAAAAGTAT TGCCTAXASSOCIATTACTT CHAPTER TGACTACTACATCHACTC 2761 ACGAAAGTTA AGTATGTCAC TGAGGGCATG CGTAAACCCG CCTTTCTAAG CGGAGAACAG 2821 AAGAAAGCAA TAGTAGATCT GTTATTCAAG ACCAACCGCA AAGTGACAGT TAAGCAATTG 2881 AAAGAGGACT ACTTTAAGAA AATTGAATGC TTCGATTCTG TCGAGATCTC CGGGGTAGAA 2941 GATCGATTTA ATGCGTCACT TGGTACGTAT CATGACCCTCC WINDOW INTERFACE 3001 GACTTCCTGG ATTACK REPEAT ATCTCTGG ATTACKGTT GACTCTTACC 3061 CTCTTTGAAG ATCGGGAAAT GATTGAGGAA AGACTAAAAAA CATACGCTCA CCTGTTCGAC 3121 GATAAGGTTA TGAAACAGTT AAAGAGGCGT CGCTATACGG GCTGGGGACG ATTGTCGCGG 3181 AAACTTATCA ACGGGATAAG AGACAAGCAA AGTGGTAAAA CTATTCTCGA TTTTCTAAAG 3241 AGCGACGGCT TCGCCAATAG GAACTTTATG CAGCTGATCC ATGATGACTC TTTAACCTTC 3301 AAAGAGGATA TACAAAAGGC ACAGGTTTCC GGACAAGGGG ACTCATTGCA CGAACATATT 3361 GCGAATCTTG CTGGTTCGCC AGCCATCAAA AAGGGCATAC TCCAGACAGT CAAAGTAGTG 3421 GATGAGCTAG TTAAGGTCAT GGGACGTCAC AAACCGGAAA ACATTGTAAT CGAGATGGCA 3481 CGCGAAAATC AAACGACTCA GAAGGGGCAA AAAAAACAGTC GAGAGCGGAT GAAGAGAATA 3541 GAAGAGGGTA TTAAAGAACT GGGCAGCCAG ATCTTAAAGG AGCATCCTGT GGAAAATACC 3601 CAATTGCAGA ACGAGAAACT TTACCTCTAT TACCTACAAA ATGGAAGGGA CATGTATGTT 3661 GATCAGGAAC TGGACATAAA CCGTTTATCT GATTACGACG TCGATCACAT TGTACCCCAA 3721 TCCTTTTTGA AGGACGATTC AATCGACAAT AAAGTGCTTA CACGCTCGGA TAAGAACCGA 3781 GGGAAAAGTG ACAATGTTCC AAGCGAGGAA GTCGTAAAGA AAATGAGAA CTATTGGCGG 3841 CAGCTCCTAA ATGCGAAACT GATAACGCAA AGAAAGTTCG ATAACTTAAC TAAAGCTGAG 3901 AGGGGTGGCT TGTCTGAACT TGACAAGGCC GGATTTATTA AACGTCAGCT CGTGGAAACC 3961 CGCCAAATCA CAAAGCATGT TGCACAGATA CTAGATTCCC GAATGAATAC GAAATACGAC 4021 GAGAACGATA AGCTGATTCG GGAAGTCAAA GTAATCACTT TAAAGTCAAA ATTGGTGTCG 4081 GACTTCAGAA AGGATTTTCA ATTCTATAAA GTTAGGGAGA TAATAACTA CCACCATGCG 4141 CACGACGCTT ATCTTAATGC CGTCGTAGGG ACCGCACTCA TTAAGAAATA CCCGAAGCTA 4201 GAAAGTGAGT TTGTGTATGG TGATTACAAA GTTTATGACG TCCGTAAGAT GATCGCGAAA 4261 AGCGAACAGG AGATAGGCAA GGCTACAGCC AAATACTTCT TTTATTCTAA CATTATGAAT 4321 TTCTTTAAGA CGGAAATCAC TCTGGCAAAC GGAGAGATAC GCAAACGACC TTTAATTGAA 4381 ACCAATGGGG AGACAGGTGA AATCGTATGG GATAAGGGCC GGGACTTCGC GACGGTGAGA 4441 AAAGTTTTGT CCATGCCCCA AGTCAACATA GTAAAGAAAA CTGAGGTGCA GACCGGAGGG 4501 TTTTCAAAGG AATCGATTCT TCCAAAAAGG AATAGTGATA AGCTCATCGC TCGTAAAAAG 4561 GACTGGGACC CGAAAAAGTA CGGTGGCTTC GATAGCCCTA CAGTTGCCTA TTCTGTCCTA 4621 GTAGTGGCAA AAGTTGAGAA GGGAAAATCC AAGAAACTGA AGTCAGTCAA AGAATTATTG 4681 GGGATAACGA TTATGGAGCG CTCGTCTTTT GAAAAGAACC CCATCGACTT CCTTGAGGCG 4741 AAAGGTTACA AGGATAAA AAAGGATCTC ATTACK TACTTACA TAGTCTGTTT 4801 GAGTTAGAAA ATGGCCGAAA ACGGATGTTG GCTAGCGCCG GAGAGCTTCA AAAGGGGAAC 4861 GAACTCGCAC TACCGTCTAA ATACGTGAAT TTCCTGTATT TAGCGTCCCA TTACGAGAAG 4921 TTGAAAGGTT CACCTGAAGA TAACGAACAG AAGCAACTTT TTGTTGAGCA GCACAAACAT 4981 TATCTCGACG AAATCATAGA GCAAATTTCG GAATTCAGTA AGAGAGTCAT CCTAGCTGAT 5041 GCCAATCTGG ACAAAGTATT AAGCGCATAC AACAAGCACA GGGATAAACC CATACGTGAG 5101 CAGGCGGAAA ATATTATCCA TTTGTTTACT CTTACCAACC TCGGCGCTCC AGCCGCATTC 5161 AAGTATTTTG ACACAACGAT AGATCGCAAA CGATACACTT CTACCAAGGA GGTGCTAGAC 5221 GCGACACTGA TTCACCAATC CATCACGGGA TTATATGAAA CTCGGATAGA TTTGTCACAG 5281 CTTGGGGGTG ACTCTGGTGG TTCTGGAGGA TCTGGTGGTT CTACTAATCT GTCAGATATT 5341 ATTGAAAAGG AGACCGGTAA GCAACTGGTT ATCCAGGAAT CCATCCTCAT GCTCCCAGAG 5401 GAGGTGGAAG AAGTCATTGG GAACAAGCCG GAAAGCGATA TACTCGTGCA CACCGCCTAC 5461 GACGAGAGCA CCGACGAGAA TGTCATGCTT CTGACTAGCG ACGCCCCTGA ATACAAGCCT 5521 TGGGCTCTGG TCATACAGGA TAGCAACGGT GAGAACAAGA TTAAGATGCT CTCTGGTGGT 5581 TCTGGAGGAT CTGGTGGTTC TACTAATCTG TCAGATATTA TTGAAAAGGA GACCGGTAAG 5641 CAACTGGTTA TCCAGGAATC CATCCTCATG CTCCCAGAGG AGGTGGAAGA AGTCATTGGG 5701 AACAAGCCGG AAAGCGATAT ACTCGTGCAC ACCGCCTACG ACGAGAGCAC CGACGAGAAT 5761 GTCATGCTTC TGACTAGGCGA CGCCCCTGAA TACAAGCCTT GGGCTCTGGT CATACAGGAT 5821 AGCAACGGTG AGAACAAGAT TAAGATGCTC TCTGGTGGTT CTCCCAAGAA GAAGAGGAAA 5881 GTCTAACCGG TCATCATCAC CATCACCATT GAGTTTAAAC CCGCTGATCA GCCTCGACTG 5941 TGCCTTCTAG TTGCCAGCCA TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGG 6001 AAGGTGCCAC TCCCACTGTC CTTTCCTAAT AAAATGAGGA AATTGCATCG CATTGTCTGA 6061 GTAGGTGTCA TTCTATTCTG GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG GAGGATTGGG 6121 AAGACAATAG CAGGCATGCT GGGGATGCGG TGGGCTCTAT GGCTTCTGAG GCGGAAAGAA 6181 CCAGCTGGGG CTCGATACCG TCGACCTCTA GCTAGAGCTT GGCGTAATCA TGGTCATAGC 6241 TGTTTCCTGT GTGAAATTGT TATCCGCTCA CAATTCCACA CAACATACGA GCCGGAAGCA 6301 TAAAGTGTAA AGCCTAGGGT GCCTAATGAG TGAGCTAACT CACATTAATT GCGTTGCGCT 6361 CACTGCCCGC TTTCCAGTCG GGAAACCTGT CGTGCCAGCT GCATTAATGA ATCGGCCAAC 6421 GCGCGGGGAG AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC 6481 TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG GTAATACGGT 6541 TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC CAGCAAAAGG 6601 CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG 6661 AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT 6721 ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA 6781 CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT 6841 GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC 6901 CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA 6961 GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG 7021 TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGAACAG 7081 TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT 7141 GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA 7201 CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC 7261 AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA 7321 CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA 7381 CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT 7441 TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT 7501 TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG GCTCCAGATT 7561 TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT 7621 CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA 7681 ATAGTTTGCG CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC TCGTCGTTTG 7741 GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA TCCCCCATGT 7801 TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG 7861 CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG 7921 TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC 7981 GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA CATAGCAGAA 8041 CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA AGGATCTTAC 8101 CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT 8161 TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG 8221 GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA TATTATTGAA 8281 GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA 8341 AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC GACGGATCGG 8401 GAGATCGATC TCCCGATCCC CTAGGGTCGA CTCTCAGTAC AATCTGCTCT GATGCCGCAT 8461 AGTTAAGCCA GTATCTGCTC CCTGCTTGTG TGTTGGAGGT CGCTGAGTAG TGCGCGAGCA 8521 AAATTTAAGC TACAACAAGG CAAGGCTTGA CCGACAATTG CATGAAGAAT CTGCTTAGGG 8581 TTAGGCGTTT TGCGCTGCTT CGCGATGTAC GGGCCAGATA TACGCGTTGA CATTGATTAT 8641 TGACTAGTTA TTAATAGTAA TCAATTACGG GGTCATTAGT TCATAGCCCA TATATGGAGT 8701 TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC 8761 CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC 8821 GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATC

[0117] In some embodiments, the cytidine base editor is BE4 having a nucleic acid sequence selected from any one of the following:

[0118] Original BE4 nucleic acid sequence:

[0119] BE4 Codon Optimized 1 Nucleic Acid Sequence:

[0120] BE4 Codon Optimized 2 Nucleic Acid Sequence:

[0121] "Base editing activity" refers to acting to chemically change a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is a cytidine deaminase activity, e.g., converting a target C·G to T·A. In another embodiment, the base editing activity is an adenosine or adenine deaminase activity, e.g., converting an A·T to G·C. In another embodiment, the base editing activity is a cytidine deaminase activity (e.g., converting a target C·G to T·A) and an adenosine or adenine deaminase activity (e.g., converting an A·T to G·C).

[0122] The term "base editing system" refers to a system for editing nucleobases of a target nucleotide sequence. In various embodiments, the base editor system comprises: (1) a polynucleotide-programmable nucleotide-binding domain (e.g., Cas9); (2) one or more deaminase domains (e.g., adenosine deaminase and / or cytidine deaminase) for deaminating the nucleobase; and (3) one or more guide polynucleotides (e.g., guide RNA). In some embodiments, the base editor (BE) system comprises: (1) a polynucleotide-programmable nucleotide-binding domain (e.g., Cas9), an adenosine deaminase domain, and a cytidine deaminase domain for deaminating the nucleobase in a target nucleotide sequence; and (2) one or more guide polynucleotides (e.g., guide RNA) in combination with the polynucleotide-programmable nucleotide-binding domain. In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a polynucleotide-programmable DNA-binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor system is BE4. In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE) and a cytidine base editor (CBE). In some embodiments, the base editor is an abasic editor.

[0123] In some embodiments, a base editor system may include two or more base editing components. For example, a base editor system may include one or more deaminases (e.g., adenosine deaminase, cytidine deaminase). In some embodiments, a single guide polynucleotide can be used to target different deaminases to a target nucleic acid sequence. In some embodiments, a single pair of guide polynucleotides can be used to target different deaminases to a target nucleic acid sequence.

[0124] The deaminase domain and polynucleotide-programmable nucleotide-binding component of the base editor system can be bound to each other covalently or non-covalently, or by any combination of such binding and interaction. For example, in some embodiments, one or more deaminase domains can be targeted to a target nucleotide sequence by a polynucleotide-programmable nucleotide-binding domain. In certain embodiments, a polynucleotide-programmable nucleotide-binding domain can be fused or linked to one or more deaminase domains. In some embodiments, a polynucleotide-programmable nucleotide-binding domain can target one or more deaminase domains to a target nucleotide sequence by non-covalently interacting or binding with the deaminase domain. For example, in some embodiments, the deaminase domain can include an additional heterologous moiety or domain that can interact, associate, or form a complex with an additional heterologous moiety or domain that is part of the polynucleotide-programmable nucleotide-binding domain. In some embodiments, the additional heterologous moiety can bind, interact, associate, or form a complex with a polypeptide. In some embodiments, the additional heterologous moiety can bind, interact, associate, or form a complex with a polynucleotide. In some embodiments, an additional heterologous moiety can be attached to a guide polynucleotide. In some embodiments, an additional heterologous moiety can be attached to a polypeptide linker. In some embodiments, an additional heterologous moiety can be attached to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a steryl alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0125] The base editor system can further comprise a guide polynucleotide component. It should be understood that the components of the base editor system can be bound to each other via covalent bonds, non-covalent interactions, or any combination of such bonds and interactions. In certain embodiments, one or more deaminase domains can be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the deaminase domain can include an additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) that can interact, bind, or form a complex with a portion or segment (e.g., a polynucleotide motif) of the guide polynucleotide. In some embodiments, an additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or linked to the deaminase domain. In some embodiments, the additional heterologous moiety can bind, interact, associate, or form a complex with a polypeptide. In some embodiments, the additional heterologous moiety can bind, interact, associate, or form a complex with a polynucleotide. In some embodiments, the additional heterologous moiety can be bound to a guide polynucleotide. In some embodiments, the additional heterologous moiety can be attached to a polypeptide linker. In some embodiments, an additional heterologous moiety can be linked to the polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0126] In certain embodiments, the base editor system can further include a base excision repair (BER) inhibitor component. It should be understood that the components of the base editor system can be bound to each other via covalent bonds, non-covalent interactions, or any combination of these bonds and interactions. The inhibitor of the BER component can include a BER inhibitor. In certain embodiments, the BER inhibitor can be a uracil DNA glycosylase inhibitor (UGI). In certain embodiments, the BER inhibitor can be an inosine BER inhibitor. In certain embodiments, the BER inhibitor can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain. In certain embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to a BER inhibitor. In certain embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to one or more deaminase domains and a BER inhibitor. In some embodiments, the polynucleotide programmable nucleotide binding domain can target a BER inhibitor to a target nucleotide sequence by non-covalently interacting with or associating with the BER inhibitor. For example, in some embodiments, an inhibitor of a BER component may include an additional heterologous moiety or domain that may interact, associate, or complex with an additional heterologous moiety or domain that is part of a polynucleotide programmable nucleotide binding domain.

[0127] In certain embodiments, the BER inhibitor can be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the BER inhibitor can include an additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) that can interact, associate, or complex with a portion or segment (e.g., a polynucleotide motif) of the guide polynucleotide. In some embodiments, the additional heterologous moiety or domain (e.g., a polynucleotide binding domain such as an RNA or DNA binding protein) of the guide polynucleotide can be fused or linked to the BER inhibitor. In some embodiments, the additional heterologous moiety can bind, interact, associate, or complex with the polynucleotide. In some embodiments, the additional heterologous moiety can be linked to the guide polynucleotide. In some embodiments, the additional heterologous moiety can be linked to a polypeptide linker. In some embodiments, the additional heterologous moiety can be linked to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0128] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease containing the Cas9 protein or a fragment thereof (e.g., a protein containing an active, inactive, or partially active DNA cleavage domain of Cas9 and / or a gRNA-binding domain of Cas9). Cas9 nuclease is sometimes referred to as casnl nuclease or CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. CRISPR is an adaptive immune system that provides defense against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In the type II CRISPR system, correct processing of the pre-crRNA requires a transcoding small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. The tracrRNA guides the processing of the pre-crRNA by RNase 3. Cas9 / crRNA / tracrRNA then endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. The target strand not complementary to the crRNA is first endonucleolytically cleaved and then exonucleolytically trimmed 3'-5'. In nature, DNA binding and cleavage typically require both a protein and both RNAs. However, single guide RNAs ("sgRNAs," or simply "gRNAs") can be engineered to incorporate both crRNA and tracrRNA aspects into a single RNA species. See, for example, Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes short motifs (PAM or protospacer-adjacent motifs) in CRISPR repeats to help distinguish self from non-self.The sequence and structure of Cas9 nuclease are well known to those of skill in the art (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes," Ferretti et al., Proc. Natl. Acad. Sci. USA 98:4658-4663(2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III," Deltcheva E. et al., Nature 471:602-607(2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," Jinek M. et al., Science 337:816-821(2012), the entire contents of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, including Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference.

[0129] An exemplary Cas9 is Streptococcus pyogenes Cas9 (spCas9), the amino acid sequence of which is provided below. TIFF0007600122000002.tif173168 (single underline: HNH domain; double underline: RuvC domain)

[0130] Nuclease-inactivated Cas9 proteins may be interchangeably referred to as "dCas9" proteins (meaning nuclease-"dead" Cas9) or catalytically inactive Cas9. Methods for generating Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains are known (see, e.g., Jinek et al., Science. 337:816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152(5): 1173-83, the contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, and the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can suppress the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28;152(5):1173-83(2013)). In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase, referred to as an "nCas9" protein (for "nickase" Cas9). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, the protein comprises one of the following two Cas9 domains: (1) the gRNA-binding domain of Cas9; (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as "Cas9 variants." Cas9 variants share homology with Cas9 or fragments thereof.For example, a Cas9 variant has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to wild-type Cas9. In some embodiments, the Cas9 mutant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) that has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of the corresponding wild-type Cas9.

[0131] In certain embodiments, the fragments are at least 100 amino acids in length, hi certain embodiments, the fragments are at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0132] In one embodiment, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_17053.1, nucleotide and amino acid sequences are as follows):

[0133] TIFF0007600122000003.tif172166 (single underline: HNH domain; double underline: RuvC domain)

[0134] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence: TIFF0007600122000004.tif172165 (single underline: HNH domain; double underline: RuvC domain)

[0135] In some embodiments, wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2 (nucleotide sequence below) and Uniprot Reference Sequence: Q99ZW2 (amino acid sequence below). TIFF0007600122000005.tif168163 (single underline: HNH domain; double underline: RuvC domain)

[0136] In certain embodiments, Cas9 is Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1) or Neisseria meningitidis (NCBI Ref: YP_002342100.1), or Cas9 from any other organism.

[0137] In some embodiments, the dCas9 corresponds to, or comprises part or all of, a Cas9 amino acid sequence with one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises D10A and H840A mutations or corresponding mutations in another Cas9. In some embodiments, the dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A): TIFF0007600122000006.tif171165 (single underline: HNH domain; double underline: RuvC domain)

[0138] 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.

[0139] In other embodiments, dCas9 variants are provided that have mutations other than D10A and H840A, e.g., that result in nuclease-inactivated Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, dCas9 variants or homologs are provided that have at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity. In some embodiments, variants of dCas9 are provided that have amino acid sequences that are shorter or longer by about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids or more.

[0140] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but rather comprise only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0141] It should be understood that additional Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including variants and homologs thereof, are within the scope of this disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is nuclease-dead Cas9 (dCas9). In some embodiments, the Cas9 protein is Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is nuclease-active Cas9.

[0142] Exemplary catalytically inactive Cas9 (dCas9):

[0143] Exemplary catalytic Cas9 nickases (nCas9):

[0144] Exemplary catalytically active Cas9:

[0145] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., nanoarchaea), which constitute the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, Cas9 refers to CasX or CasY, as described, for example, in Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genome-resolved metagenomics, many CRISPR-Cas systems have been identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was discovered as part of an active CRISPR-Cas system in the little-studied nanoarchaea. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, have been discovered, which are among the most compact systems discovered to date. In some embodiments, Cas9 represents CasX or a variant of CasX. In some embodiments, Cas9 represents CasY or a variant of CasY. It should be understood that other RNA-guided DNA binding proteins can also be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0146] In certain embodiments, napDNAbp useful in the methods of the invention comprise circular permutations, which are known and described, for example, in Oakes et al., Cell 176, 254-267, 2019. Below are exemplary circular permutations, where the bolded sequence indicates the sequence derived from Cas9, the italicized sequence represents the linker sequence, and the underlined sequence represents the bipartite nuclear localization sequence. TIFF0007600122000007.tif187163

[0147] Non-limiting examples of polynucleotide-programmable nucleotide binding domains that can be incorporated into base editors include domains from CRISPR proteins, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs).

[0148] 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 Cas12b / C2c1, CasX, and CasY from other bacterial species may also be used in accordance with the present disclosure.

[0149] Cas12b / C2c1 (uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2|C2C1_ALIAG CRISPR-associated endo- nuclease C2c1 OS = Alicyclobacillus acido- terrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) GN=c2c1 PE=1 SV=1

[0150] CasX (uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53) >tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx protein OS = Sulfolobus islandicus (strain HVE10 / 4) GN = SiH_0402 PE=4 SV=1 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE VEPHYLIIAAAGWVLTRLGKVSEGDYVGVNVFTRPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRYIVLANIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAIVNGELIRGEG

[0151] >tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS = Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4 SV=1 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAK VSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0152] Deltaproteobacteria CasX MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQK WYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0153] CasY (ncbi.nlm.nih.gov / protein / APG80656.1) >APG80656.1 CRISPR-associated protein CasY (uncultured Parcubacteria group bacterium]

[0154] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid that shares common properties. A practical method for defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Such analysis allows for the definition of groups of amino acids in which amino acids within a group preferentially exchange with each other and are therefore most similar to each other in their effect on overall protein structure (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include amino acid substitutions such as arginine to lysine, which can maintain a positive charge; aspartic acid to glutamic acid, which can maintain a negative charge; threonine to serine, which maintains a free -OH; and asparagine to glutamine, which can maintain a free NH2.

[0155] The terms "coding sequence" or "protein-coding sequence," as used interchangeably herein, refer to a segment of a polynucleotide that encodes a protein. This region or sequence is bounded at its 5'-proximal end by a start codon and at its 3'-proximal end by a stop codon. A coding sequence is also called an open reading frame.

[0156] "Cytidine deaminase" refers to a polypeptide or fragment thereof that can catalyze a deamination reaction that converts an amino group to a carbonyl group. In one embodiment, the cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine. The cytidine deaminase (e.g., engineered cytidine deaminase, evolved cytidine deaminase) provided herein can be from any organism, such as a bacterium.

[0157] In some embodiments, the base editor cytidine deaminase can comprise all or part of an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. APOBEC is an evolutionarily conserved family of cytidine deaminases. Members of this family are C→U editing enzymes. In some embodiments, the cytidine deaminase is selected from, but not limited to, APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (now referred to as "APOBEC3E"), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, activation-induced (cytidine) deaminase (AID), hAPOBEC1 from Homo sapiens, rAPOBEC1 from Rattus norvegicus, ppAPOBEC1 from Pongo pygmaeus, AmAPOBEC1 (BEM3.31) from Alligator mississippiensis, ocAPOBEC1 from Oryctolagus cuniculus, SsAPOBEC2 (BEM3.39) from Sus scrofa, hAPOBEC3A from Homo sapiens, maAPOBEC1 from Mesocricetus auratus, Monodelphis APOBEC family members include, but are not limited to, mdAPOBEC1 from Homo domestica; cytidine deaminase 1 (CDA1); hA3A, an APOBEC3A from Homo sapiens; RrA3F (BEM3.14), an APOBEC3F from Rhinopithecus roxellana; PmCDA1 from Petromyzon marinus (Petromyzon marinus cytosine deaminase 1, "PmCDA1"); AID (activation-induced cytidine deaminase; AICDA) from mammals (e.g., humans, pigs, cows, horses, monkeys, etc.); hAID from Homo sapiens; and FENRY.

[0158] As used herein, the term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase, which catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytosine deaminase, which catalyzes the hydrolytic deamination of cytosine to uracil. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA). The deaminases provided herein (e.g., engineered deaminases, evolved deaminases) can be derived from any organism, such as bacteria. In some embodiments, the adenosine deaminase is derived from bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Caulobacter crescentus.

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

[0160] "Detectable label" refers to a composition that, when attached to a molecule of interest, renders the latter detectable via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., those commonly used in enzyme-linked immunosorbent assays (ELISAs)), biotin, digoxigenin, or haptens.

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

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

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

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

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

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

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

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

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

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

[0171] Other intein systems can also be used. For example, synthetic inteins based on the dnaE intein, i.e., the intein pair Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C), have been described (e.g., Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5, incorporated herein by reference). Non-limiting examples of intein pairs that can be used according to the present disclosure include Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, and Cne Prp8 intein (e.g., as described in U.S. Patent No. 8,394,604, incorporated herein by reference).

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

[0173] To join the N-terminal portion of split-Cas9 with the C-terminal portion of split-Cas9, an intein-N and an intein-C can be fused to the N-terminal portion of split-Cas9 and the C-terminal portion of split-Cas9, respectively. For example, in some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of split-Cas9, i.e., forming the structure N--[N-terminal portion of split-Cas9]-[intein-N]--C. In some embodiments, intein-C is fused to the N-terminus of the C-terminal portion of split-Cas9, i.e., forming the structure N--[intein-C]--[C-terminal portion of split-Cas9]-C. The mechanism of intein-mediated protein splicing for linking proteins (e.g., split Cas9) to which an intein is fused is known in the art, for example, as described in Shah et al., Chem Sci. 2014; 5(1):446-461, which is incorporated herein by reference. Methods for designing and using inteins are known in the art and are described, for example, by WO2014004336, WO2017132580, US20150344549, and US20180127780, each of which is incorporated herein by reference in its entirety.

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

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

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

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

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

[0179] In some embodiments, a linker connects the gRNA binding domain of an RNA programmable nuclease, including a Cas9 nuclease domain, with the catalytic domain of a nucleic acid editing protein (e.g., cytidine and / or adenosine deaminase). In some embodiments, a linker connects dCas9 and a nucleic acid editing protein. For example, a linker is placed between two groups, molecules, or other moieties, or is flanked by two groups, molecules, or other moieties, and is linked to each other via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5 to 200 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90, 95, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 175, 180, 190, or 200 amino acids in length. Longer or shorter linkers are also contemplated.

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

[0181] In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS. In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

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

[0183] As used herein, the term "mutation" refers to the substitution of a residue in a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or the deletion or insertion of one or more residues in a sequence. Mutations are typically described herein by identifying the original residue, then identifying the position of the residue in the sequence, and identifying the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art and are provided, for example, by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). In some embodiments, the base editors of the present disclosure can efficiently generate "intended mutations," e.g., point mutations, in a nucleic acid (e.g., a nucleic acid in a subject's genome) without generating a significant number of unintended mutations, e.g., unintended point mutations. In some embodiments, the intended mutation is a mutation caused by a particular base editor (e.g., a cytidine base editor and / or an adenosine base editor) attached to a guide polynucleotide (e.g., a gRNA) that is specifically designed to produce the intended mutation.

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

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

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

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

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

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

[0190] The term "nucleobase editing domain" or "nucleobase editing protein," as used herein, refers to a protein or enzyme that can catalyze nucleobase modifications in RNA or DNA, such as the deamination of cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine), and adenine (or adenosine) to hypoxanthine (or inosine), as well as non-templated nucleotide addition and insertion. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., adenine deaminase or adenosine deaminase; or cytidine deaminase or cytosine deaminase). In some embodiments, the nucleobase editing domain is a multiple deaminase domain (e.g., adenine deaminase or adenosine deaminase and cytidine or cytosine deaminase). In some embodiments, the nucleobase editing domain can be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase-editing domain can be a nucleobase-editing domain engineered or evolved from a naturally occurring nucleobase-editing domain. The nucleobase-editing domain can be from any organism, such as a bacterium, human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse.

[0191] As used herein, "obtaining," as in "obtaining a drug," includes synthesizing, purchasing, or otherwise acquiring the drug.

[0192] As used herein, "patient" or "subject" refers to a mammalian subject or individual who has been diagnosed with, is at risk of developing, or is suspected of having or developing a disease or disorder. In some embodiments, the term "patient" refers to a mammalian subject who has a higher than average likelihood of developing a disease or disorder. Exemplary patients may be humans, non-human primates, cats, dogs, pigs, cows, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, guinea pigs), and other mammals that may benefit from the treatments disclosed herein. Exemplary human patients may be male and / or female.

[0193] A "patient in need thereof" or "subject in need thereof" is referred to herein as a patient who has been diagnosed with, is at risk of having, has been predetermined to have, or is suspected of having a disease or disorder.

[0194] The terms "pathogenic mutation," "pathogenic variant," "disease-causing mutation," "disease-causing variant," "deleterious mutation," or "predisposing mutation" refer to a genetic change or mutation that increases an individual's susceptibility or predisposition to a particular disease or disorder. In some embodiments, a pathogenic mutation comprises the substitution of at least one wild-type amino acid in a protein encoded by a gene with at least one pathogenic amino acid.

[0195] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in carrying or transporting a compound from one site in the body (e.g., a delivery site) to another site (e.g., an organ, tissue, or body part). A pharmaceutically acceptable carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the tissues of the subject (e.g., physiological compatibility, sterility, physiological pH, etc.). Terms such as "excipient," "carrier," "pharmaceutically acceptable carrier," "vehicle," and the like are used interchangeably herein.

[0196] The term "pharmaceutical composition" means a composition formulated for pharmaceutical use.

[0197] The terms "protein," "peptide," "polypeptide," and their grammatical equivalents are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The term refers to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide is at least three amino acids in length. A protein, peptide, or polypeptide can refer to an individual protein or a group of proteins. One or more amino acids in a protein, peptide, or polypeptide can be modified by the addition of chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modifications. A protein, peptide, or polypeptide can also be a single molecule or a multimolecular complex. A protein, peptide, or polypeptide can be simply a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide can be naturally occurring, recombinant, synthetic, or any combination thereof. As used herein, the term "fusion protein" refers to a hybrid polypeptide containing protein domains from at least two different proteins. One protein can be located at the amino-terminal (N-terminal) or carboxy-terminal (C-terminal) portion of the fusion protein, thus forming an amino-terminal fusion protein or a carboxy-terminal fusion protein, respectively. The protein can contain different domains, such as a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9, which guides the protein to bind to the target site) and a nucleic acid cleavage domain, or the catalytic domain of a nucleic acid editing protein. In some embodiments, the protein includes a proteinaceous portion, such as an amino acid sequence constituting the nucleic acid binding domain, and an organic compound, such as a compound that can act as a nucleic acid cleavage agent. In some embodiments, the protein is complexed with or associated with a nucleic acid (e.g., RNA or DNA).Any protein provided herein can be produced by any method known in the art.For example, the protein provided herein can be produced through recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers.The method for recombinant protein expression and purification is well known, including that described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), the entire contents of which are incorporated herein by reference.

[0198] The polypeptides and proteins (including functional portions and functional variants thereof) disclosed herein can contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, and indoline-2-carboxylic acid. , 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, aminocyclohexanecarboxylic acid, aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. Polypeptides and proteins can be associated with post-translational modifications of one or more amino acids of the polypeptide construct. Non-limiting examples of post-translational modifications include phosphorylation, acylation, including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation, including methylation and ethylation, ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation, and iodination.

[0199] 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.

[0200] By "decreasing" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0201] "Reference" refers to a standard or control condition. In one embodiment, the reference is a wild-type or healthy cell. In another embodiment, without limitation, the reference is an untreated cell that is not exposed to the test condition or is exposed to a placebo or normal saline, medium, buffer, and / or a control vector that does not carry the polynucleotide of interest.

[0202] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a particular sequence; for example, a segment of a full-length cDNA or gene sequence, or the entire cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is generally at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is generally at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides, or any integer therebetween or thereabout. In some embodiments, the reference sequence is the wild-type sequence of a protein of interest. In other embodiments, the reference sequence is a polynucleotide sequence encoding the wild-type protein.

[0203] The terms "RNA-programmable nuclease" and "RNA-guided nuclease" are used in conjunction with (e.g., bound to or associated with) one or more RNAs that are not the target of cleavage. In certain embodiments, when an RNA-programmable nuclease is complexed with an RNA, it can be referred to as a nuclease:RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA).

[0204] In some embodiments, the RNA-programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 from Streptococcus pyogenes (Casnl) (see, e.g., "Complete genome sequence of an Ml strain of Streptococcus pyogenes," Ferretti JJ et al., Proc. Natl. Acad. Sci. USA 98:4658-4663(2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III," Deltcheva E. et al., Nature 471:602-607(2011)).

[0205] 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.

[0206] The term "single nucleotide polymorphism (SNP)" refers to a single nucleotide variation occurring at a specific position in the genome, where each variation is present to a noticeable degree in a population (e.g., >1%). For example, at a particular base position in the human genome, a C nucleotide can occur in most individuals, but in a minority of individuals, that position is occupied by an A. This means that there is an SNP at this specific position, and the two nucleotide variations, C or A, are alleles at this position. SNPs underlie differences in susceptibility to disease. Disease severity and the body's response to treatment are also manifestations of genetic variation. SNPs can occur in the coding region of a gene, the noncoding region of a gene, or in intergenic regions (regions between genes). In some embodiments, SNPs within a coding sequence do not necessarily change the amino acid sequence of the resulting protein due to the degeneracy of the genetic code. SNPs in coding regions are of two types: synonymous and nonsynonymous SNPs. Synonymous SNPs do not affect the protein sequence, while nonsynonymous SNPs do alter the amino acid sequence of a protein. There are two types of nonsynonymous SNPs: missense and nonsense. SNPs that are not located in protein-coding regions can affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNA. Gene expression affected by this type of SNP is called an eSNP (expressed SNP) and can be upstream or downstream of the gene. Single-nucleotide variants (SNVs) are single-nucleotide variations with unlimited frequency that can occur somatically. Somatic single-nucleotide variations can also be called single-nucleotide modifications.

[0207] By "specifically binds" is meant a nucleic acid molecule, polypeptide, or complex thereof (e.g., a nucleic acid programmable DNA binding domain and a guide nucleic acid), compound, or molecule that recognizes and binds to a polypeptide and / or nucleic acid molecule of the invention, but does not substantially recognize or bind to other molecules in a sample (e.g., a biological sample).

[0208] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to pairing to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof under various stringency conditions. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0209] For example, stringent salt concentrations are typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions will typically include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, detergent (e.g., sodium dodecyl sulfate (SDS)) concentration, and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In one embodiment, hybridization occurs at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization occurs at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In another embodiment, hybridization occurs at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be readily apparent to those of skill in the art.

[0210] In most applications, the washing steps following hybridization also vary in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As described above, washing stringency can be increased by decreasing salt concentration or increasing temperature. For example, stringent salt concentrations for washing steps are preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for washing steps typically include temperatures of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In one embodiment, washing steps are performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washing steps are performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps are performed in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 68° C. Further variations of these conditions will be readily apparent to those of skill in the art.Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0211] "Split" means divided into two or more pieces.

[0212] "Split Cas9 protein" or "split-Cas9" refers to a Cas9 protein that is provided as an N-terminal fragment and a C-terminal fragment encoded by two separate nucleotide sequences. Polypeptides corresponding to the N-terminal and C-terminal portions of the Cas9 protein can be spliced ​​to form a "reconstituted" Cas9 protein. In certain embodiments, the Cas9 protein is split into two fragments within a disordered region of the protein, e.g., as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014, or as described in Jiang et al. (2016) Science 351: 867-871. PDB file: 5F9R (each incorporated herein by reference). In some embodiments, the protein is split into two fragments at any C, T, A, or S within the region between approximately amino acids A292-G364, F445-K483, or E565-T637 of SpCas9, or at the corresponding position in any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other napDNAbp. In certain embodiments, the protein is split into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of splitting a protein into two fragments is referred to as "splitting" the protein.

[0213] In other embodiments, the N-terminal portion of the Cas9 protein comprises amino acids 1-573 or 1-637 of wild-type S. pyogenes Cas9 (SpCas9) (NCBI Reference Sequence: NC_002737.2; Uniprot Reference Sequence: Q99ZW2), and the C-terminal portion of the Cas9 protein comprises amino acids 574-1368 or 638-1368 of wild-type SpCas9.

[0214] The C-terminal portion of a split Cas9 can be joined with the N-terminal portion of a split Cas9 to form a complete Cas9 protein. In some embodiments, the C-terminal portion of the Cas9 protein begins where the N-terminal portion of the Cas9 protein ends. Thus, in some embodiments, the C-terminal portion of the split Cas9 comprises amino acids (551-651)-1368 of spCas9. "(551-651)-1368" means beginning with an amino acid between amino acids 551 and 651 (inclusive) and ending at amino acid 1368.For example, the C-terminal portion of a split-Cas9 consists of amino acids 551-1368, 552-1368, 553-1368, 554-1368, 555-1368, 556-1368, 557-1368, 558-1368, 559-1368, 560-1368, 561-1368, 562-1368, 563-1368, 564-1368, 565-1368, 566-1368, 567-1368, 568-1368, 569-1368, 570-1368, 571-1368, 572-1368, 573-1368, 574 -1368, 575-1368, 576-1368, 577-1368, 578-1368, 579-1368, 580-1368, 581-1368, 582-1368, 583-1368, 584-1368, 585-1368, 586-1368, 587-1368, 588-1368, 589-1368, 590-1368, 591-1368, 592-1368, 593-1368, 594-1368, 595-1368, 596-1368, 597-1368, 598-1368, 599-1368, 600-1368 , 601-1368, 602-1368, 603-1368, 604-1368, 605-1368, 606-1368, 607-1368, 608-1368, 609-1368, 610-1368, 611-1368, 612-1368, 613-1368, 614-1368, 615-1368, 616-1368, 617-1368, 618-1368, 619-1368, 620-1368, 621-1368, 622-1368, 623-1368, 624-1368, 625-1368, 626-1368, 627- 1368, 628-1368, 629-1368, 630-1368, 631-1368, 632-1368, 633-1368, 634-1368, 635-1368, 636-1368, 637-1368, 638-1368, 639-1368, 640-1368, 641-1368, 642-1368, 643-1368, 644-1368, 645-1368, 646-1368, 647-1368, 648-1368, 649-1368, 650-1368, or 651-1368.In some embodiments, the C-terminal portion of the split Cas9 protein comprises a portion of amino acids 574-1368 or 638-1368 of SpCas9.

[0215] "Subject" means a mammal, including, but not limited to, a human or a non-human mammal such as a cow, horse, dog, sheep, or cat. Subjects include livestock, domestic animals raised to produce labor and provide goods such as food, including, but not limited to, cows, goats, chickens, horses, pigs, rabbits, and sheep.

[0216] By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). In one embodiment, such a sequence has at least 60%, 80%, or even 85%, 90%, 95%, or even 99% identity at the amino acid level or nucleic acid to the sequence used for comparison.

[0217] Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program can be used, e.g., -3 and e -100 A probability score between indicates closely related sequences. COBALT can be used, for example, with the following parameters: a) Alignment parameters: Gap penalties -11, -1 and End-Gap penalties -5, -1 b) CDD parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved columns and Recompute on c) Query clustering parameters: Use query clusters on; Word Size 4; Max cluster distance 0.8; Alphabet Regular. The EMBOSS Needle is used, for example, with the following parameters: a) Matrix: BLOSUM62; b) GAP OPEN: 10; c) GAP EXTEND: 0.5; d) OUTPUT FORMAT: pair; e) END GAP PENALTY: false; f) END GAP OPEN: 10; and g) END GAP EXTEND: 0.5.

[0218] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a nucleobase editor. In one embodiment, the target site is deaminated by a deaminase (e.g., a cytidine or adenine deaminase) or a fusion protein comprising same.

[0219] As used herein, the terms "treat," "treating," "treatment," and the like refer to alleviating or ameliorating a disorder and / or its associated symptoms, or achieving a desired pharmacological and / or physiological effect. It will be understood that treating a disorder or condition does not require (nor does complete elimination preclude) the complete elimination of the associated disorder, condition, or symptoms. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, diminishes, eliminates, alleviates, alleviates, reduces the intensity of, or cures, the disease and / or adverse symptoms resulting therefrom. In certain embodiments, the effect is prophylactic, i.e., the effect protects against or prevents the occurrence or recurrence of the disease or condition. To this end, the methods of the present disclosure comprise administering a therapeutically effective amount of a composition as described herein.

[0220] "Uracil glycosylase inhibitor," or alternatively "UGI," refers to an agent that inhibits the uracil excision repair system. In one embodiment, the agent is a protein or fragment thereof that binds to host uracil-DNA glycosylase and prevents the removal of uracil residues from DNA. In one embodiment, UGI is a protein, fragment, or domain thereof that can inhibit the uracil-DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain comprises wild-type UGI or a modified version thereof. In some embodiments, the UGI domain comprises a fragment of an exemplary amino acid sequence provided below. In some embodiments, the UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the exemplary UGI sequence provided below. In some embodiments, the UGI comprises an amino acid sequence homologous to an exemplary UGI amino acid sequence or fragment thereof, as described below. In some embodiments, UGI or a portion thereof has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identity to wild-type UGI or a UGI sequence or a portion thereof, as described below. Exemplary UGIs include the following amino acid sequences: >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD APEYKPWALVIQDSNGENKIKML.

[0221] The term "vector" refers to a means for introducing a nucleic acid sequence into a cell, resulting in a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, and episomes. An "expression vector" is a nucleic acid sequence that comprises a nucleotide sequence that is expressed in a recipient cell. Expression vectors may contain additional nucleic acid sequences to enhance and / or facilitate expression of the introduced sequence, such as initiation sequences, termination sequences, enhancer sequences, promoter sequences, and secretion sequences.

[0222] Any composition or method provided herein can be combined with any one or more of the other compositions and methods provided herein.

[0223] DNA editing has emerged as a viable means to correct disease states by correcting pathogenic mutations at the gene level. Until recently, all DNA editing platforms functioned by inducing DNA double-strand breaks (DSBs) at specific genomic sites and relying on endogenous DNA repair pathways to determine product outcomes in a semi-stochastic manner, resulting in a complex population of genetic products. While precise and user-defined repair outcomes can be achieved via the homology-directed repair (HDR) pathway, numerous challenges have prevented highly efficient repair using HDR in therapeutically relevant cell types. In practice, this pathway is less efficient than the competing, error-prone non-homologous end-joining pathway. Furthermore, HDR is strictly restricted to the G1 and S phases of the cell cycle, preventing accurate repair of DSBs in post-mitotic cells. As a result, it has proven difficult or impossible to modify genome sequences with high efficiency in a user-defined and programmable manner in these populations. [Brief explanation of the drawings]

[0224] [Figure 1]Figures 1A-C show the cis-trans activity of free deaminase. Figure 1A is a schematic showing the experimental design of the cis-trans assay for SpCas9 and deaminase in either the base editor complex format or the unlinked format. Figure 1B is a graph showing the cis-trans activity of rAPOBEC. Figure 1C is a graph showing the cis-trans activity of TadA7.10 and TadA-TadA7.10. [Figure 2]Figures 2A-2F show the cis-trans assay for base editors, an example of a deaminase similarity network, and the screening of 153 deaminases. Figure 2A is a schematic diagram illustrating the experimental design for the cis-trans assay. HEK293T cells were transfected with separate plasmids encoding SaCas9, gRNA for SaCas9, and the target base editor. Figure 2B is a schematic diagram illustrating the similarity network for APOBEC-like deaminases. Dots represent cytidine deaminases screened as next-generation CBEs, and the core next-generation CBE is shown. The shading of the dots represents the average trans / cis ratio, and the size of the dots represents the average cis activity. The method for creating the similarity network for cytidine deaminases shown in Figure 2B is as follows: To focus the search space within the APOBEC1-like protein family, a protein BLAST search was performed against the NCBI nonredundant protein sequence database (nr_v5) using human APOBEC1 as the query sequence. A sequence similarity network (SSN) was generated using the top 1,000 sequences using a protein BLAST-log(E-value) edge threshold of 115. A set of 43 deaminases was selected to sample the sequence space within the SSN. To identify deaminases from other families that may act as base editing enzymes, 80 sequences from the SSN constructed from all deaminases were sampled using the following InterPro annotations: IPR002125 (cytidine and deoxycytidylate deaminase domain), IPR016192 (APOBEC / CMP deaminase, zinc-binding), and IPR016193 (cytidine deaminase-like). This set of 82,043 sequences was first clustered at 55% identity using Cd-HIT3, and then an SSN network was generated using protein BLAST with a -log(E-value) edge threshold of 50. Sequences were selected based on the centrality of the sequences within the cluster in the network. Figure 2C is a graph showing the cis-trans activity of ppBE4 and its mutants. Figure 2D is a graph showing the cis-trans activity of selected editors.Separately, cis-trans activity data were generated based on in cis / in trans assays for three target sites, namely, site 1, site 4, and site 6, as shown in Figure 2E and Figure 2F. Figure 2E is a bar graph showing the cis- and trans-editing activities of the identified CBEs. Shown is a comparison of the cis- and trans-editing frequencies in mammalian cells treated with candidate CBEs. Editor numbers 1 to 36 are the base editors pYY-BEM3.8, pYY-BEM3.9, pYY-BEM3.10, pYY-BEM3.11, pYY-BEM3.12, pYY-BEM3.13, pYY-BEM3.14, pYY-BEM3.15, pYY-BEM3.16, pYY-BEM3.17, pYY-BEM3.18, pYY-BEM3.19, pYY-BEM3.20, pYY-BEM3.21, pYY-BEM3.22, pYY-BEM3.23, pYY-BEM3.24, and pYY-BEM3.25, respectively. The base editing efficiencies are reported for the most edited base at the target site. Figure 2F is a bar graph showing the cis- and trans-editing activities of the identified CBEs. A comparison of cis- and trans-editing frequencies in mammalian cells treated with candidate CBEs is shown.Editor numbers 1 to 37 are rBE4max, mAPOBEC-1, MaAPOBEC-1, hAPOBEC-1, ppAPOBEC-1, OcAPOBEC1, MdAPOBEC-1, mAPOBEC-2, hAPOBEC-2, ppAPOBEC-2, BtAPOBEC-2, mAPOBEC-3, hAPOBEC-3A, hAPOBEC-3B, hAPOBEC-3C, hAPOBEC-3D, and hAPO These are BEC-3F, hAPOBEC-3G, hAPOBEC-4, mAPOBEC-4, rAPOBEC-4, MfAPOBEC-4, hAID, negative control, btAID, mAID, pmCDA-1, pmCDA-2, pmCDA-5, yCD, pYY-BEM3.1, pYY-BEM3.2, pYY-BEM3.3, pYY-BEM3.4, pYY-BEM3.5, pYY-BEM3.6, and pYY-BEM3.7. Base editing efficiencies are reported for the most edited base at the target site. [Figure 3] Figures 3A and 3B show cis-trans activity. Figure 3A is a graph showing the cis-trans activity of ABE7.10. Figure 3B is a graph showing the cis-trans activity of BE4 max. [Figure 4] Figures 4A and 4B show the rAPOBEC1 homology model generated by SWISSMODEL using the hAPOBEC3C structure (PDB ID 3VM8). ssDNA from the hAPOBEC3A structure (PDB ID 5SWW) has been manually docked. Figure 4A is a schematic showing mutations that potentially affect ssDNA binding. Figure 4B is a schematic showing mutations that potentially affect catalytic activity. [Figure 5] 5A-5C show the cis-trans activity of rAPOBEC1 mutants. [Figure 6]Figures 6A to 6E show the cis-trans activity of the rAPOBEC1 double mutant. Figure 6A is a graph showing the in cis and in trans activity of the rAPOBEC1 double mutant. Figure 6B is a graph showing the in cis activity at six sites. Figure 6C is a graph showing the cis / trans ratio. Figure 6D is a graph showing the in cis activity at six sites. Figure 6E is a graph showing the cis / trans ratio. [Figure 7] Figures 7A and 7B show the cis-trans activity of the deaminases in the first round of screening. [Figure 8] 8A to 8C are graphs showing the targeting activity of ppAPOBEC1 against rAPOBEC1. [Figure 9] FIG. 9 is a schematic diagram showing the similarity network of APOBEC-like proteins. [Figure 10] Figures 10A and 10B are graphs showing dose-dependent studies of in cis and in trans activity in TadA-TadA7.10 and rAPOBEC1, respectively.

[0225] [Figure 11] Figure 11 is a graph showing off-target editing of selected CBEs. SNVs were identified by exome sequencing. [Figure 12] Figures 12A and 12B are graphs showing quantification of base editor mRNA and protein, respectively, from HEK293T cells transfected with base editor plasmids. [Figure 13] Figure 13 is a graph showing targeted RNA sequencing for selected editors. Three regions of 200-300 bp were sequenced. [Figure 14] FIG. 14 is a graph showing selected CBE-guided off-target editing. [Figure 15] 15A to 15E show the editing window of the selected editor. [Figure 16]FIG. 16 is a graph showing the indel rates of selected CBEs at 10 target sites. [Figure 17] Figures 17A-17D show diagrams and graphs for unguided ssDNA deamination and cis / trans assays. Figure 17A shows potential ssDNA formation in the genome during transcription or translation. Figure 17B shows the experimental design for the in cis / in trans assay. HEK293T cells were transfected with separate constructs encoding SaCas9, a gRNA for SaCas9, and a base editor. In cis and in trans activity was measured in different transfectants but at the target site defined by the NGGRRT PAM sequence. Figure 17C shows the in cis / in trans activity of BE4 with rAPOBEC1. Figure 17D shows the ABE7.10 variant at 34 genomic sites. The leftmost bar of each genomic site on the x-axis indicates on-target editing in cis. The rightmost bar of each genomic site on the x-axis indicates trans editing. Base editing efficiency is reported for the most edited base at the target site. Values ​​and error bars reflect the mean and standard deviation (s.d.) of independent biological replicates. [Figure 18] Figure 18 is a bar graph showing identified next-generation CBEs with enhanced cis activity and reduced trans activity compared to BE4 with rAPOBEC1. Shown is a comparison of in cis and in trans editing frequencies at 10 genomic sites in mammalian cells treated with next-generation CBEs (BE4 with PpAPOBEC1 [wt, H122], RrA3F [wt, F130L], AmAPOBEC1, and SsAPOBEC2 [wt, R54Q]). Base editing efficiencies are reported for the most edited base at the target site. Values ​​and error bars reflect the mean and standard deviation (SD) of four independent biological replicates. [Figure 19]Figures 19A-19E show allele frequencies and graphs for next-generation CBEs with reduced DNA and RNA off-target editing compared to BE4 in mammalian cells. Figure 19A shows whole transcriptome sequencing and targeted RNA sequencing (Figure 19B) of Hek293T cells expressing an off-target deamination-minimizing cytosine base editor. Figure 19C shows the percentage of C→T editing at known guiding off-target sites. Figure 19D shows the percentage of C→T editing in an in vitro enzymatic assay on a single-stranded DNA substrate. C→U editing of core next-generation CBEs on ssDNA substrates. Dots represent the NC local sequence context of editing. The black line indicates the average editing efficiency of the target cytosine in the substrate. Figure 29E shows the time course of product formation in an in vitro enzymatic assay from cell lysates containing selected CBEs. The sequences of the oligos used in Figures 19D and 19E are listed in the table provided in Example 5 below. Values ​​and error bars reflect the mean and sd of independent triplicate biological replicates (Fig. 19 A, B, C) or duplicate replicates (Fig. 19 D, E). [Figure 20] Figure 20 graphically depicts the cis / trans editing activity of BE4 with the rAPOBEC1 mutants shown in Figures 4A and 4B at site 1. Base editing efficiencies are reported for the most edited base at the target site. In trans efficiencies are shown at the left edge of each target site on the x-axis; in cis efficiencies are shown in the right bar for each target on the x-axis. Values ​​and error bars reflect the mean and SD of independent biological replicates.

[0226] [Figure 21] Figure 21 shows the cis / trans editing activity of BE4-rAPOBEC1 with HiFi mutations at 10 target sites. Values ​​and error bars reflect the mean and SD of four independent biological replicates. [Figure 22]Figures 22A and 22B show graphs and sequence alignments related to the cis / trans editing activity and sequence alignment of CBEs tested in the first round of screening. In cis / in trans editing activity (Figure 22A) and sequence alignment (Figure 22B) at site 10 of selected CBEs. Amino acid residues flanking the HiFi mutations in rAPOBEC1 are highlighted. Values ​​and error bars reflect the mean and sd of independent biological replicates. [Figure 23] Figure 23 shows the in cis / in trans activity of BE4-PpAPOBEC1 and BE4-PpAPOBEC with HiFi mutations at 10 target sites. Base editing efficiencies are reported for the most edited base at the target site. Values ​​and error bars reflect the mean and SD of four independent biological replicates. [Figure 24] Figure 24 shows a heatmap illustrating the leading base preference of the CBE shown in Figure 18 B. The values ​​used to generate the heatmap reflect the average of four independent biological replicates. [Figure 25] Figure 25 shows the editing window of the CBE shown in Figure 18B at 10 target sites. Values ​​reflect the average of four independent biological replicates. In cis and in trans editing are displayed in the heatmaps in the leftmost and rightmost panels, respectively. [Figure 26] Figure 26 is a table showing the indel rates of the CBEs shown in Figure 18B at the 10 target sites. The values ​​used to generate the heatmap reflect the average of four independent biological replicates. [Figure 27]Figures 27A-27D show homology models of four cytidine deaminases selected based on existing crystal structures. Figure 27A: The homology model of PpAPOBEC1 is based on the predicted APOBEC3G structure (PDB ID 5K81). Figure 27B: RrA3F is based on the Vif-binding domain of hAPOBEC3F (PDB ID 3WUS). Figure 27C: AmAPOBEC1 is based on the hAPOBEC3B N-terminal domain (PDB ID 5TKM). Figure 27D: SsAPOBEC2 is based on the Vif-binding domain of hAPOBEC3F (PDB ID 3WUS). [Figure 28] Figures 28A-28D are graphs showing the guided off-target editing of selected next-generation CBEs. Figure 28A: Editing efficiency of next-generation CBEs at HEK2, HEK3, and HEK4 sites, and Figure 28B: Editing efficiency of next-generation CBEs at reported guided off-target editing sites for HEK2 sgRNA, HEK3 sgRNA, and Figure 28D: HEK4 sgRNA. Base editing efficiency is reported for the most edited base at the target site. Values ​​and error bars reflect the mean and SD of three independent biological replicates. [Figure 29] Figure 29 is a graph showing the C→T editing efficiency of selected CBEs on ssDNA substrates in an in vitro enzymatic assay. Editing efficiency was measured for all 25 cytidines in two ssDNA substrates and grouped by NC sequence context. The sequences of the two substrates used are listed in Table 18 herein. Values ​​and error bars reflect the mean and SD of data obtained from two independent biological replicates. [Figure 30] Figure 30 shows a graph showing quantification of CBE protein concentration in HEK293T cells transfected with base editor expression plasmids. Base editor protein concentration was quantified by measuring total Cas9 protein concentration and total protein amount in cell lysates. BE protein concentration was normalized to BE4-rAPOBEC1. Values ​​and error bars reflect the mean and SD of data obtained from two or more independent biological replicates. [Figure 31] Figure 31 presents a graph showing the off-target deamination activity of CBEs as determined by whole genome sequencing (WGS). Relative mutation rates are shown as odds ratios. DETAILED DESCRIPTION OF THE INVENTION

[0227] The present invention provides nucleobase editors and multi-effector nucleobase editors with improved editing profiles that minimize off-target deamination, compositions comprising such editors, and methods of using the same to generate modifications in target nucleobase sequences.

[0228] [Nucleobase Editor] Disclosed herein is a base editor, nucleobase editor, or multi-effector nucleobase editor for editing, modifying, or altering a target nucleotide sequence of a polynucleotide. Described herein is a nucleobase editor, nucleobase editor, or multi-effector nucleobase editor that includes a polynucleotide-programmable nucleotide binding domain (e.g., Cas9) and at least one nucleobase editing domain (adenosine deaminase and / or cytidine deaminase). When the polynucleotide-programmable nucleotide binding domain (e.g., Cas9) is combined with a bound guide polynucleotide (e.g., gRNA), it can specifically bind to the target polynucleotide sequence (through complementary base pairing between the bases of the bound guide nucleic acid and the bases of the target polynucleotide sequence), thereby allowing the base editor to localize to the target nucleic acid sequence desired to be edited.

[0229] Polynucleotide-programmable nucleotide-binding domains It should be understood that polynucleotide programmable nucleotide binding domain can also comprise nucleic acid programmable protein that binds to RNA.For example, polynucleotide programmable nucleotide binding domain can be bound to nucleic acid that guides polynucleotide programmable nucleotide binding domain to RNA.Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, but they are not specifically listed in the present disclosure.

[0230] The polynucleotide-programmable nucleotide-binding domain of a base editor can itself comprise one or more domains. For example, a polynucleotide-programmable nucleotide-binding domain can comprise one or more nuclease domains. In certain embodiments, the nuclease domain of a polynucleotide-programmable nucleotide-binding domain can comprise an endonuclease or exonuclease. As used herein, the term "exonuclease" refers to a protein or polypeptide capable of digesting nucleic acids (e.g., RNA or DNA) from free ends, and the term "endonuclease" refers to a protein or polypeptide capable of catalyzing (e.g., cleaving) an internal region of a nucleic acid (e.g., DNA or RNA). In certain embodiments, an endonuclease can cleave one strand of a double-stranded nucleic acid. In certain embodiments, an endonuclease can cleave both strands of a double-stranded nucleic acid molecule. In certain embodiments, a polynucleotide-programmable nucleotide-binding domain can be a deoxyribonuclease. In certain embodiments, a polynucleotide-programmable nucleotide-binding domain can be a ribonuclease.

[0231] In certain embodiments, the nuclease domain of a polynucleotide-programmable nucleotide-binding domain can cleave zero, one, or two strands of a target polynucleotide. In certain embodiments, the polynucleotide-programmable nucleotide-binding domain can comprise a nickase domain. As used herein, the term "nickase" refers to a polynucleotide-programmable nucleotide-binding domain that comprises a nuclease domain that can cleave only one strand of a double-stranded nucleic acid molecule (e.g., DNA). In certain embodiments, a nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide-programmable nucleotide-binding domain by introducing one or more mutations into the active polynucleotide-programmable nucleotide-binding domain. For example, if the polynucleotide-programmable nucleotide-binding domain comprises a nickase domain derived from Cas9, the nickase domain derived from Cas9 can comprise a D10A mutation and a histidine at position 840. In such embodiments, residue H840 retains catalytic activity, thereby enabling single-strand cleavage of a nucleic acid duplex. In another example, a Cas9-derived nickase domain can include an H840A mutation, while the amino acid residue at position 10 remains D. In some embodiments, a nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide-programmable nucleotide-binding domain by removing all or a portion of a nuclease domain that is not required for nickase activity. For example, if a polynucleotide-programmable nucleotide-binding domain includes a nickase domain from Cas9, the nickase domain from Cas9 can include a deletion of all or a portion of the RuvC domain or the HNH domain.

[0232] The amino acid sequence of an exemplary catalytically active Cas9 is as follows:

[0233] Thus, a base editor comprising a polynucleotide-programmable nucleotide-binding domain comprising a nickase domain can generate a single-stranded DNA break (nick) in a specific polynucleotide target sequence (e.g., as determined by the complementary sequence of a bound guide nucleic acid). In some embodiments, the strand of a nucleic acid double-stranded target polynucleotide sequence cleaved by a base editor comprising a nickase domain (e.g., a nickase domain derived from Cas9) is the strand not edited by the base editor (i.e., the strand cleaved by the base editor is the strand opposite the strand containing the base to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., a nickase domain derived from Cas9) can cleave the strand of a DNA molecule targeted for editing. In such embodiments, the non-target strand is not cleaved.

[0234] Also provided herein are base editors comprising a catalytically dead (i.e., incapable of cleaving a target polynucleotide sequence) polynucleotide-programmable nucleotide-binding domain. As used herein, the terms "catalytically dead" and "nuclease-inactive" are used interchangeably to refer to a polynucleotide-programmable nucleotide-binding domain having one or more mutations and / or deletions that result in an inability to cleave a strand of nucleic acid. In some embodiments, a catalytically dead polynucleotide-programmable nucleotide-binding domain base editor can lack nuclease activity as a result of specific point mutations in one or more nuclease domains. For example, in the case of a base editor comprising a Cas9 domain, Cas9 can contain both the D10A and H840A mutations. Such mutations inactivate both nuclease domains, resulting in the loss of nuclease activity. In other embodiments, a catalytically dead polynucleotide-programmable nucleotide-binding domain can contain one or more deletions of all or part of a catalytic domain (e.g., the RuvC1 and / or HNH domain). In further embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (e.g., D10A or H840A) as well as a deletion of all or part of the nuclease domain.

[0235] Also contemplated herein are mutations that can generate catalytically dead polynucleotide-programmable nucleotide-binding domains from previously functional versions of the polynucleotide-programmable nucleotide-binding domain. For example, in the case of catalytically dead Cas9 ("dCas9"), variants are provided that have mutations other than D10A and H840A, resulting in nuclease-inactivated Cas9. Such mutations include, for example, other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or RuvC1 subdomain). Additional suitable nuclease-inactive dCas9 domains will be apparent to those skilled in the art based on this disclosure and knowledge in the art, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference).

[0236] Non-limiting examples of polynucleotide-programmable nucleotide-binding domains that can be incorporated into base editors include domains derived from CRISPR proteins, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some embodiments, the base editor comprises a polynucleotide-programmable nucleotide-binding domain comprising a natural or modified protein, or a portion thereof, that can bind to a nucleic acid sequence via a binding guide nucleic acid during CRISPR (i.e., Clustered Regularly Interspaced Short Palindromic Repeats)-mediated modification of the nucleic acid. Such proteins are referred to herein as "CRISPR proteins." Accordingly, disclosed herein are base editors that comprise a polynucleotide-programmable nucleotide-binding domain comprising all or a portion of a CRISPR protein (i.e., a base editor that comprises all or a portion of a CRISPR protein as a domain (which is also referred to as the "CRISPR protein-derived domain" of the base editor). The CRISPR protein-derived domain incorporated into the base editor can be modified compared to a wild-type or natural CRISPR protein. For example, as described below, a domain derived from a CRISPR protein can contain one or more mutations, insertions, deletions, rearrangements, and / or recombinations compared to a wild-type or naturally occurring CRISPR protein.

[0237] CRISPR is an adaptive immune system that provides defense against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of the pre-crRNA requires a transcoding small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. tracrRNA guides ribonuclease 3 processing of the pre-crRNA. Cas9 / crRNA / tracrRNA then endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. Target strands not complementary to the crRNA are first endonucleolytically cleaved and then exonucleolytically trimmed 3'-5'. In nature, both proteins and RNAs are required for DNA binding and cleavage. However, a single guide RNA ("sgRNA," or simply "gRNA") can be engineered to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif (the PAM or protospacer adjacent motif) in the CRISPR repeat sequence to help distinguish between "self" and "non-self."

[0238] In some embodiments, the methods described herein can utilize engineered Cas proteins. Guide RNAs (gRNAs) are short synthetic RNAs consisting of a scaffold sequence required for Cas binding and a user-defined approximately 20-base spacer that defines the genomic target to be modified. Thus, one skilled in the art can vary the genomic target of Cas protein specificity, which is determined in part by how specific the gRNA targeting sequence is for the genomic target relative to other parts of the genome.

[0239] In some embodiments, the gRNA scaffold sequence is: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU.

[0240] In some embodiments, the domain derived from a CRISPR protein incorporated into the base editor is an endonuclease (e.g., a deoxyribonuclease or ribonuclease) capable of binding to a target polynucleotide when combined with a bound guide nucleic acid. In some embodiments, the domain derived from a CRISPR protein incorporated into the base editor is a nickase capable of binding to a target polynucleotide when combined with a bound guide nucleic acid. In some embodiments, the domain derived from a CRISPR protein incorporated into the base editor is a catalytically dead domain capable of binding to a target polynucleotide when combined with a bound guide nucleic acid. In some embodiments, the target polynucleotide that binds to the CRISPR protein-derived domain of the base editor is DNA, and in some embodiments, the target polynucleotide that binds to the CRISPR protein-derived domain of the base editor is RNA.

[0241] CAs proteins that can be used herein include class 1 and class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also called Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Cssx16, Cx16, Cx, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i, CARF, DinG, their homologs, or their variants.Unmodified CRISPR enzyme, like Cas9, can have the DNA cleavage activity with two functional endonuclease regions, RuvC and HNH. CRISPR enzymes can induce cleavage of one or both strands of a target sequence, such as within the target sequence and / or within the complementary strand of the target sequence. For example, CRISPR enzymes can induce cleavage of one or both strands at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence.

[0242] Vectors can be used that encode CRISPR enzymes that are mutated relative to the corresponding wild-type enzyme so that they lack the ability to cleave one or both strands of a target polynucleotide containing the target sequence. Cas9 can refer to a polypeptide that has at least, or at least about, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from S. pyogenes). Cas9 can refer to a polypeptide having at most, or at most, about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., from S. pyogenes). Cas9 can refer to wild-type or modified forms of the Cas9 protein, which can include amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0243] In some embodiments, the base editor CRISPR protein-derived domain is derived from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningitidis (NCBI Ref: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus.

[0244] [Cas9 domain, a nucleobase editor] The sequence and structure of Cas9 nuclease are well known to those skilled in the art (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes," Ferretti et al., Proc. Natl. Acad. Sci. USA 98:4658-4663(2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III," Deltcheva E. et al., Nature 471:602-607(2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," Jinek M. et al., Science 337:816-821(2012), the entire contents of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, including Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference.

[0245] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting exemplary Cas9 domains are provided herein. The Cas9 domain can be a nuclease-active Cas9 domain, a nuclease-inactive Cas9 domain (dCas9), or a Cas9 nickase (nCas9). In some embodiments, the Cas9 domain is a nuclease-active domain. For example, the Cas9 domain can be a Cas9 domain that cleaves both strands of a double-stranded nucleic acid (e.g., both strands of a double-stranded DNA molecule). In some embodiments, the Cas9 domain comprises any one of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences described herein.In some embodiments, the Cas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical stretches of amino acid residues compared to any one of the amino acid sequences described herein.

[0246] In some embodiments, proteins comprising a fragment of Cas9 are provided. For example, in some embodiments, the protein comprises one of the following two Cas9 domains: (1) the gRNA binding domain of Cas9; (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or a fragment thereof are referred to as "Cas9 variants." Cas9 variants share homology with Cas9 or a fragment thereof. For example, Cas9 variants are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 mutant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In certain embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of the corresponding wild-type Cas9.In some embodiments, the fragments are at least 100 amino acids in length. In some embodiments, the fragments are at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0247] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but rather comprise only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0248] The Cas9 protein can bind to a guide RNA, which guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a Cas9 domain, such as a nuclease-active Cas9, Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Examples of nucleic acid-programmable DNA-binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b / C2C1, and Cas12c / C2C3. In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, nucleotide and amino acid sequences are as follows): TIFF0007600122000008.tif172166 (single underline: HNH domain; double underline: RuvC domain)

[0249] In some embodiments, wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence: TIFF0007600122000009.tif172165 (single underline: HNH domain; double underline: RuvC domain)

[0250] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2 (nucleotide sequence is as follows) and Uniprot Reference Sequence: Q99ZW2 (amino acid sequence is as follows): TIFF0007600122000010.tif168163 (single underline: HNH domain; double underline: RuvC domain)

[0251] In some embodiments, Cas9 is Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1) or Neisseria meningitidis (NCBI Ref: YP_002342100.1), or represents Cas9 from any other organism.

[0252] It is understood that additional Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including variants and homologs thereof, are within the scope of this disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is nuclease-dead Cas9 (dCas9). In some embodiments, the Cas9 protein is Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is nuclease-active Cas9.

[0253] In some embodiments, the Cas9 domain is a nuclease-inactive Cas9 domain (dCas9). For example, the dCas9 domain can bind to a double-stranded nucleic acid molecule (e.g., via a gRNA molecule) without cleaving either strand of the double-stranded nucleic acid molecule. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10X mutation and a H840X mutation in the amino acid sequence described herein, or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid change. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10A mutation and a H840A mutation in the amino acid sequence described herein, or a corresponding mutation in any of the amino acid sequences described herein. As an example, the nuclease-inactive Cas9 domain comprises the following amino acid sequence provided in the cloning vector pPlatTET-gRNA 2 (Accession No. BAV54124):

[0254] The amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows: (See, e.g., Qi et al., "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression." Cell. 2013; 152(5):1173-83, the entire contents of which are incorporated herein by reference.)

[0255] Additional suitable nuclease-inactive dCas9 domains will be apparent to those skilled in the art based on this disclosure and knowledge in the art, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference).

[0256] In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase, referred to as a "nCas9" protein (for "nickase" Cas9). A nuclease-inactivated Cas9 protein may also be interchangeably referred to as a "dCas9" protein (for nuclease-"dead" Cas9) or catalytically inactive Cas9. Methods for generating Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains are known (see, e.g., Jinek et al., Science. 337:816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152(5): 1173-83, the contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can suppress the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28;152(5):1173-83 (2013)).

[0257] In some embodiments, the dCas9 domain comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to any of the dCas9 domains provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any of the amino acid sequences described herein.

[0258] In some embodiments, the dCas9 corresponds to, or comprises part or all of, a Cas9 amino acid sequence with one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises D10A and H840A mutations or corresponding mutations in another Cas9.

[0259] In some embodiments, the dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A): TIFF0007600122000011.tif171165 (single underline: HNH domain; double underline: RuvC domain)

[0260] 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.

[0261] In other embodiments, dCas9 variants are provided that have mutations other than D10A and H840A, e.g., that result in nuclease-inactivated Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, dCas9 variants or homologs are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants of dCas9 are provided that have amino acid sequences that are shorter or longer by about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids or more.

[0262] In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase can be a Cas9 protein that can cleave only one strand of a double-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, the Cas9 nickase cleaves the target strand of a double-stranded nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is base-paired (complementary) to a gRNA (e.g., an sgRNA) bound to the Cas9. In some embodiments, the Cas9 nickase comprises a D10A mutation and has a histidine at position 840. In some embodiments, the Cas9 nickase cleaves the non-target, non-base-edited strand of a double-stranded nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is not base-paired to a gRNA (e.g., an sgRNA) bound to the Cas9. In some embodiments, the Cas9 nickase comprises a H840A mutation and has an aspartic acid residue at position 10, or a corresponding mutation. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the Cas9 nickases provided herein. Additional suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the art, and are within the scope of this disclosure.

[0263] The amino acid sequence of an exemplary catalytic Cas9 nickase (nCas9) is as follows:

[0264] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., nanoarchaea), which constitute the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, the programmable nucleotide-binding protein can be a CasX or CasY protein, as described, for example, in Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genome-resolved metagenomics, many CRISPR-Cas systems have been identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was discovered as part of an active CRISPR-Cas system in the little-studied nanoarchaea. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, have been discovered, which are among the most compact systems discovered to date. In some embodiments, in the base editor systems described herein, Cas9 is replaced by CasX or a variant of CasX. In some embodiments, in the base editor systems described herein, Cas9 is replaced by CasY or a variant of CasY. It should be understood that other RNA-guided DNA-binding proteins can also be used as nucleic acid programmable DNA-binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0265] In some embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) of any of the fusion proteins provided herein can be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide-binding protein is a naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide-binding protein comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to any CasX or CasY protein described herein. It should be understood that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.

[0266] The amino acid sequence of an exemplary CasX ((uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53) tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx protein OS = Sulfolobus islandicus (strain HVE10 / 4) GN = SiH_0402 PE=4 SV=1) is as follows: MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTSNIILPLSGNDKNPWTETLKCYNFPTTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0267] Exemplary CasX (>tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS = Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4 SV=1) の amino acid sequence is as follows: MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTSNIILPLSGNDKNPWTETLKCYNFPTTTVALSEVFKNFSQVKECEEVSAPSFVKPFEYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0268] Deltaproteobacteria CasX MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQ KWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0269] The amino acid sequence of an exemplary CasY ((ncbi.nlm.nih.gov / protein / APG80656.1) >APG80656.1 CRISPR-associated protein CasY (uncultured Parcubacteria group bacterium)) is as follows:

[0270] The Cas9 nuclease contains two functional endonuclease domains, RuvC and HNH. Upon binding to target DNA, Cas9 undergoes a conformational change that positions the nuclease domain to cleave the opposite strand of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (approximately 3–4 nucleotides upstream of the PAM sequence). The resulting DSB can be repaired by one of two general repair pathways: (1) the efficient but error-prone non-homologous end joining (NHEJ) pathway; or (2) the less efficient but high-fidelity homology-directed repair (HDR) pathway.

[0271] The "efficiency" of non-homologous end joining (NHEJ) and / or homology-directed repair (HDR) can be calculated by any convenient method. For example, in certain embodiments, efficiency can be expressed as the percentage of successful HDR. For example, a test nuclease assay can be used to generate cleavage products, and the percentage can be calculated using the ratio of product to substrate. For example, a test nuclease enzyme can be used that directly cleaves DNA containing the newly incorporated restriction sequence as a result of successful HDR. The more substrate cleaved, the higher the rate of HDR (the more efficient the HDR). As an illustrative example, the rate (percentage) of HDR can be calculated using the following formula: [(cleavage product) / (substrate + cleavage product)] (e.g., (b + c) / (a ​​+ b + c), where "a" is the band intensity of the DNA substrate, and "b" and "c" are the cleavage products).

[0272] In one embodiment, efficiency can be expressed as the success rate of NHEJ. For example, a T7 endonuclease I assay can be used to generate cleavage products, and the ratio of product to substrate can be used to calculate the percentage of NHEJ. T7 endonuclease I cleaves mismatched heteroduplex DNA resulting from hybridization of wild-type and mutant DNA strands (NHEJ generates small random insertions or deletions (indels) at the initial cleavage site). More cleavages indicate a higher rate of NHEJ (more efficient NHEJ). As an illustrative example, the rate (percentage) of NHEJ can be calculated using the formula (1-(1-(b+c) / (a+b+c)) 1 / 2 ) × 100, where "a" is the band intensity of the DNA substrate, and "b" and "c" are the cleavage products (Ran et al., Cell. 2013 Sep. 12; 154(6):1380-9; and Ran et al., Nat Protoc. 2013 Nov.; 8(11): 2281-2308).

[0273] The NHEJ repair pathway is the most active repair mechanism, frequently resulting in small nucleotide insertions or deletions (indels) at DSB sites. The random nature of NHEJ-mediated DSB repair has important practical implications, as cell populations expressing Cas9 and gRNA or guide polynucleotides can result in a diverse array of mutations. In most embodiments, NHEJ generates small indels in the target DNA, resulting in amino acid deletions, insertions, or frameshift mutations that result in premature stop codons within the open reading frame (ORF) of the target gene. The ideal end result is a loss-of-function mutation within the target gene.

[0274] While NHEJ-mediated DSB repair often disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to generate specific nucleotide changes ranging from single nucleotide changes to large insertions such as the addition of fluorophores or tags.

[0275] To utilize HDR for gene editing, a DNA repair template containing the desired sequence can be delivered to the cell type of interest along with a gRNA and Cas9 or Cas9 nickase. The repair template can contain additional homologous sequences immediately upstream and downstream of the desired edit and its target (called left and right homologous arms). The length of each homologous arm can depend on the magnitude of the change being introduced; larger insertions require longer homologous arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. HDR efficiency is generally low (less than 10% corrected alleles), even in cells expressing Cas9, gRNA, and an exogenous repair template. Because HDR occurs between the S and G2 phases of the cell cycle, synchronizing cells can increase HDR efficiency. Chemical or genetic inhibition of genes involved in NHEJ can also increase HDR frequency.

[0276] In some embodiments, Cas9 is a modified Cas9. A given gRNA target sequence may have additional sites of partial homology throughout the genome. These sites are called off-targets and need to be considered when designing gRNA. In addition to optimizing gRNA design, CRISPR specificity can also be improved by modifying Cas9. Cas9 generates double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase, a D10A mutant of SpCas9, retains one nuclease domain and generates DNA nicks instead of DSBs. Nickases can also be combined with HDR-mediated gene editing for specific gene editing.

[0277] In some embodiments, the Cas9 is a variant Cas9 protein. A variant Cas9 polypeptide has an amino acid sequence that differs by a single amino acid (e.g., by a deletion, insertion, substitution, or fusion) compared to the amino acid sequence of a wild-type Cas9 protein. In some instances, the variant Cas9 polypeptide has an amino acid change (e.g., a deletion, insertion, or substitution) that reduces the nuclease activity of the Cas9 polypeptide. For example, in some instances, the variant Cas9 polypeptide has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 protein. In some embodiments, the variant Cas9 protein does not have substantial nuclease activity. When a subject Cas9 protein is a variant Cas9 protein that does not have substantial nuclease activity, it may be referred to as "dCas9."

[0278] In some embodiments, the variant Cas9 protein has reduced nuclease activity, e.g., the variant Cas9 protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the endonuclease activity of a wild-type Cas9 protein (e.g., a wild-type Cas9 protein).

[0279] In some embodiments, a variant Cas9 protein can cleave the complementary strand of a guide target sequence but has a reduced ability to cleave the non-complementary strand of a double-stranded guide target sequence. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, the variant Cas9 protein has D10A (aspartic acid to alanine at amino acid position 10), and thus can cleave the complementary strand of a double-stranded guide target sequence but has a reduced ability to cleave the non-complementary strand of a double-stranded guide target sequence (thus, when this variant Cas9 protein cleaves a double-stranded target nucleic acid, it generates a single-strand break (SSB) instead of a double-strand break (DSB)) (see, e.g., Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21).

[0280] In some embodiments, a variant Cas9 protein can cleave the non-complementary strand of a double-stranded guide target sequence, but has a reduced ability to cleave the complementary strand of the guide target sequence. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif). As a non-limiting example, in some embodiments, the variant Cas9 protein has an H840A (histidine to alanine at amino acid position 840) mutation, and thus can cleave the non-complementary strand of a guide target sequence, but has a reduced ability to cleave the complementary strand of a guide target sequence (thus, when this variant Cas9 protein cleaves a double-stranded guide target sequence, an SSB occurs instead of a DSB). Such a Cas9 protein has a reduced ability to cleave a guide target sequence (e.g., a single-stranded guide target sequence), but retains the ability to bind to the guide target sequence (e.g., a single-stranded guide target sequence).

[0281] In some embodiments, the variant Cas9 protein has a reduced ability to cleave both the complementary and non-complementary strands of double-stranded target DNA. As a non-limiting example, in some embodiments, the variant Cas9 protein has both the D10A and H840A mutations, resulting in a polypeptide with a reduced ability to cleave both the complementary and non-complementary strands of double-stranded target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0282] As another non-limiting example, in some embodiments, a variant Cas9 protein has a W476A and a W1126A mutation such that the polypeptide has a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0283] As another non-limiting example, in some embodiments, a variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations such that the polypeptide has a reduced ability to cleave target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0284] As another non-limiting example, in some embodiments, a variant Cas9 protein has H840A, W476A, and W1126A mutations, resulting in a polypeptide with reduced ability to cleave target DNA (e.g., single-stranded target DNA) while retaining the ability to bind to target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, a variant Cas9 protein has H840A, D10A, W476A, and W1126A mutations, resulting in a polypeptide with reduced ability to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA) while retaining the ability to bind to target DNA (e.g., single-stranded target DNA). In some embodiments, a variant Cas9 has a restored catalytic His residue at position 840 of the Cas9 HNH domain (A840H).

[0285] As another non-limiting example, in some embodiments, a variant Cas9 protein has H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, such that the polypeptide has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind to target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, a variant Cas9 protein has D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, such that the polypeptide has a reduced ability to cleave target DNA (e.g., single-stranded target DNA). Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA). In some embodiments, when a variant Cas9 protein has the W476A and W1126A mutations, or when a variant Cas9 protein has the P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, the variant Cas9 protein does not efficiently bind to a PAM sequence. Thus, in some such embodiments, when such a variant Cas9 protein is used in a binding method, the method does not require a PAM sequence. In other words, in some embodiments, when such a variant Cas9 protein is used in a binding method, the method may include a guide RNA, but the method can be performed in the absence of a PAM sequence (thus, binding specificity is provided by the target segment of the guide RNA). To achieve the above effect, other residues may be mutated (i.e., inactivating one or other nuclease moiety). By way of non-limiting example, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 can be altered (i.e., substituted). Mutations other than alanine substitutions are also suitable.

[0286] In some embodiments, a variant Cas9 protein with reduced catalytic activity (e.g., when the Cas9 protein has a D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 mutation, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A) can bind to target DNA in a site-specific manner (because it is guided to the target DNA sequence by the guide RNA), so long as it retains the ability to interact with the guide RNA.

[0287] 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.

[0288] In some embodiments, a modified SpCas9 was used that contained the amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and has specificity for a modified PAM5'-NGC-3'.

[0289] Alternatives to S. pyogenes Cas9 include RNA-guided endonucleases from the Cpf1 family that exhibit cleavage activity in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease in a class II CRISPR / Cas system. This adaptive immune mechanism is found in Prevotella and Francisella bacteria. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the limitations of the CRISPR / Cas9 system. Unlike Cas9 nuclease, Cpf1-mediated DNA cleavage results in a double-strand break with a short 3' overhang. The staggered cleavage pattern of Cpf1 opens up the possibility of directional gene transfer, similar to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Like the Cas9 variants and orthologs described above, Cpf1 can also expand the number of CRISPR targetable sites to AT-rich regions or AT-rich genomes lacking the NGG PAM site preferred by SpCas9. The Cpf1 locus contains an α / β mixed domain, a RuvC-I region followed by a helical region, a RuvC-II region, and a zinc finger-like domain. The Cpf1 protein possesses a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, Cpf1 lacks the HNH endonuclease domain, and the N-terminus of Cpf1 lacks the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain organization demonstrated that Cpf1 is functionally unique and classified as a class 2, type V CRISPR system. The Cpf1 locus encoded Cas1, Cas2, and Cas4 proteins that were more similar to type I and type III systems than to type II systems. Functional Cpf1 does not require trans-activating CRISPR RNA (tracrRNA); therefore, it requires only CRISPR (crRNA).Cpf1 is not only smaller than Cas9, but also possesses a smaller sgRNA molecule (approximately half the number of nucleotides of Cas9), which is beneficial for genome editing. In contrast to the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex cleaves target DNA or RNA by identifying a protospacer adjacent to the motif 5'-YTN-3'. After identifying the PAM, Cpf1 introduces a sticky-end-like DNA double-strand break with a 4- or 5-nucleotide overhang.

[0290] [Nucleic acid-programmable DNA-binding protein] Certain aspects of the present disclosure provide fusion proteins comprising a domain that acts as a nucleic acid-programmable DNA-binding protein, which can be used to target proteins such as base editors to specific nucleic acid (e.g., DNA or RNA) sequences. In certain embodiments, the fusion protein comprises a nucleic acid-programmable DNA-binding protein domain and one or more deaminase domains. Non-limiting examples of nucleic acid-programmable DNA-binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also called Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, and Cse 1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3 , Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins include Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, homologs thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, even though they may not be specifically listed herein.See, e.g., Makarova et al. "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR J. 2018 Oct;1:325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., "Functionally diverse type V CRISPR-Cas systems" Science. 2019 Jan 4;363(6422):88-91. doi: 10.1126 / science.aav7271 (the entire contents of each are incorporated herein by reference).

[0291] An example of a nucleic acid programmable DNA-binding protein with PAM specificity distinct from Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats (Cpf1) from Prevotella and Francisella1. Like Cas9, Cpf1 is also a class 2 CRISPR effector. Cpf1 has been shown to mediate robust DNA interference with characteristics distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA and utilizing T-rich protospacer adjacent motifs (TTN, TTTN, or YTN). Furthermore, Cpf1 cleaves DNA with staggered double-strand breaks. Of the 16 Cpf1 family proteins, two enzymes from Acidaminococcus and Lachnospiraceae have been shown to have efficient genome editing activity in human cells. The Cpf1 protein is known in the art and has been previously described, for example, in Yamano et al., "Crystal structure of Cpf1 in complex with guide RNA and target DNA." Cell (165) 2016, pp. 949-962, the entire contents of which are incorporated herein by reference.

[0292] Nuclease-inactive Cpf1 (dCpf1) variants, which can be used as guide nucleotide sequence-programmable DNA-binding protein domains, are also useful in the compositions and methods of the present invention. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9, but does not have the HNH endonuclease domain, and the N-terminus of Cpf1 does not have the alpha-helix recognition lobe of Cas9. Zetsche et al., Cell, 163, 759-771, 2015 (incorporated herein by reference) showed that the RuvC-like domain of Cpf1 is responsible for cleaving both DNA strands, and inactivating the RuvC-like domain inactivates Cpf1 nuclease activity. For example, mutations corresponding to D917A, E1006A, or D1255A in Francisella novicida Cpf1 inactivate Cpf1 nuclease activity. In some embodiments, dCpf1 of the present disclosure includes mutations corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It is understood that any mutation that inactivates the RuvC domain of Cpf1, e.g., a substitution mutation, deletion, or insertion, can be used in accordance with the present disclosure.

[0293] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein can be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In certain aspects, the Cpf1 protein is a nuclease-inactive Cpf1 (dCpf1). In some embodiments, the Cpf1, nCpf1, or dCpf1 comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a Cpf1 sequence disclosed herein. In some embodiments, the dCpfl comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a Cpfl sequence disclosed herein, and includes mutations corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It should be understood that Cpfl from other bacterial species may also be used in accordance with the present disclosure.

[0294] Wild-type Francisella novicida Cpf1 (D917, E1006, and D1255 are bold and underlined). MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKL ALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDKAIKENKGEGYKKIVYKKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI DRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDY KNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN.

[0295] Francisella novicida Cpf1 D917A (A917, E1006, and D1255 are bold and underlined.) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKL ALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDKAIKENKGEGYKKIVYKKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI ARGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDY KNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN.

[0296] Francisella novicida Cpf1 E1006A (D917, A1006, and D1255 are bold and underlined.) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKL ALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDKAIKENKGEGYKKIVYKKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI DRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF A DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDY KNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN.

[0297] Francisella novicida Cpf1 D1255A (D917, E1006, and A1255 are bold and underlined.) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKL ALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDKAIKENKGEGYKKIVYKKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI DRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDY KNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

[0298] Francisella novicida Cpf1 D917A / E1006A (A917, A1006, and D1255 are bold and underlined.) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKL ALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDKAIKENKGEGYKKIVYKKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI ARGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF A DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDY KNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN.

[0299] Francisella novicida Cpf1 D917A / D1255A (A917, E1006, and A1255 are bold and underlined.) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKL ALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDKAIKENKGEGYKKIVYKKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI ARGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDY KNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN.

[0300] Francisella novicida Cpf1 E1006A / D1255A (D917, A1006, and A1255 are bold and underlined.) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKL ALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDKAIKENKGEGYKKIVYKKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI DRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF A DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDY KNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN.

[0301] Francisella novicida Cpf1 D917A / E1006A / D1255A (A917, A1006, and A1255 are bold and underlined.) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKL ALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDKAIKENKGEGYKKIVYKKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI ARGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF A DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDY KNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN.

[0302] In some embodiments, one of the Cas9 domains present in the fusion protein can be replaced with a guide nucleotide sequence-programmable DNA-binding protein domain that does not require a PAM sequence.

[0303] 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 domain comprises an N579A mutation, or a corresponding mutation in any of the amino acid sequences provided herein.

[0304] In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to nucleic acid sequences with a non-canonical PAM, and in some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to nucleic acid sequences with an NNGRRT or NNGRRT PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of the 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 the E781K, N967K, and R1014H mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SaCas9 domain comprises the E781K, N967K, or R1014H mutations, or corresponding mutations in any of the amino acid sequences provided herein.

[0305] Exemplary SaCas9 Amino Acid Sequences KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQ KKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRKLVPKKVDLSQQK EIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEE NSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNLDVKVKSINGGFTSFLRRKWKFKKERNKGY KHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLK KLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIK KENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG. Residue N579, shown above in bold and underlined, can be mutated (e.g., to A579) to generate a SaCas9 nickase.

[0306] Exemplary SaCas9n Amino Acid Sequences KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQ KKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRKLVPKKVDLSQQK EIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEE ASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNLDVKVKSINGGFTSFLRRKWKFKKERNKGY KHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLK KLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIK KENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG. Residue A579, which can be mutated from N579 to generate SaCas9 nickase, is bold and underlined.

[0307] Exemplary SaKKH Cas9 Amino Acid Sequences Residue A579, which can be mutated from N579 to generate SaCas9 nickase, is shown in bold and underlined. Residues K781, K967, and H1014, which can be mutated from E781, N967, and R1014 to generate SaKKH Cas9, are shown in italics and underlined.

[0308] In one embodiment, napDNAbp is a circular permutant. In the sequences below, plain text represents the adenosine deaminase sequence, bolded sequences represent Cas9-derived sequences, italicized sequences represent linker sequences, and underlined sequences represent bipartite nuclear localization sequences. TIFF0007600122000013.tif179169

[0309] In some embodiments, a nucleic acid programmable DNA-binding protein (napDNAbp) is the single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, but are not limited to, Cas9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Typically, microbial CRISPR-Cas systems are divided into class 1 and class 2 systems. Class 1 systems have a multi-subunit effector complex, while class 2 systems have a single protein effector. For example, Cas9 and Cpf1 are class 2 effectors. In addition to Cas9 and Cpf1, three distinct Class 2 CRISPR-Cas systems (Cas12b / C2c1 and Cas12c / C2c3) have been described by Shmakov et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems," Mol. Cell, 2015 Nov. 5; 60(3): 385-397 (the entire contents of which are incorporated herein by reference). The effectors of two systems, Cas12b / C2c1 and Cas12c / C2c3, contain a RuvC-like endonuclease domain related to Cpf1. The third system contains an effector with two predicted HEPN RNase domains. Unlike CRISPR RNA production by Cas12b / C2c1, mature CRISPR RNA production is tracrRNA-independent. Cas12b / C2c1 relies on both CRISPRRNA and tracrRNA for DNA cleavage.

[0310] The crystal structure of Alicyclobacillus acidoterrastris Cas12b / C2c1 (AacC2c1) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See, e.g., Liu et al., "C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism," Mol. Cell, 2017 Jan. 19; 65(2):310-322, the entire contents of which are incorporated herein by reference. The crystal structure of Alicyclobacillus acidoterrestris C2c1 bound to target DNA as a ternary complex has also been reported. See, e.g., Yang et al., "PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease," Cell, 2016 Dec. 15; 167(7):1814-1828, the entire contents of which are incorporated herein by reference. Catalytically competent conformations of AacC2c1, along with both target and non-target DNA strands, are independently trapped within a single RuvC catalytic pocket, and Cas12b / C2c1-mediated cleavage results in staggered seven-nucleotide cuts in the target DNA. Structural comparison between the Cas12b / C2c1 ternary complex and previously identified Cas9 and Cpf1 counterparts demonstrates the diversity of the mechanisms used by CRISPR-Cas9 systems.

[0311] In some embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) of any of the fusion proteins provided herein can be a Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp is a Cas12b / C2c1 protein. In some embodiments, the napDNAbp is a Cas12c / C2c3 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 Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp is a naturally occurring Cas12b / C2c1 or Cas12c / C2c3 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 of the napDNAbp sequences provided herein. It should be understood that Cas12b / C2c1 or Cas12c / C2c3 from other bacterial species can also be used in accordance with the present disclosure.

[0312] The amino acid sequence of Cas12b / C2c1 ((uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2|C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS = Alicyclobacillus acido-terrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) GN=c2c1 PE=1 SV=1) is as follows:

[0313] AacCas12b (Alicyclobacillus acidiphilus) - WP_067623834

[0314] BhCas12b (Bacillus hisashii) NCBI Reference Sequence: WP_095142515

[0315] Contains a variant called BvCas12b V4 (S893R / K846R / E837G changes compared to the wild type).

[0316] BhCas12b (V4) is expressed as follows: 5' mRNA Cap---5'UTR---bhCas12b---STOP sequence---3'UTR---120 polyA tail 5'UTR: GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC 3' UTR (TriLink standard UTR) GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGA

[0317] Nucleic acid sequence of bhCas12b (V4)

[0318] In some embodiments, the Cas12b is BvCas12B, which is a variant of BhCas12b and contains the following changes relative to BhCas12B: S893R, K846R, and E837G. BvCas12b (Bacillus sp. V3-13) NCBI Reference Sequence: WP_101661451.1

[0319] [Guide polynucleotide] In one embodiment, the guide polynucleotide is a guide RNA. The RNA / Cas complex can help "guide" the Cas protein to the target DNA. Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. The target strand not complementary to the crRNA is first endonucleolytically cleaved and then exonucleolytically trimmed in the 3'-5' direction. In nature, both proteins and RNAs are typically required for DNA binding and cleavage. However, single guide RNAs ("sgRNAs," or simply "gRNAs") can be engineered to incorporate both crRNA and tracrRNA aspects into a single RNA species. See, for example, Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif (PAM or protospacer-adjacent motif) in CRISPR repeat sequences to help distinguish self from non-self. The sequence and structure of Cas9 nuclease are well known to those of skill in the art (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes," Ferretti, JJ et al., Natl. Acad. Sci. USA 98:4658-4663(2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III," Deltcheva E. et al., Nature 471:602-607(2011); and "Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," Jinek M. et al., Science 337:816-821(2012), the entire contents of which are incorporated herein by reference).Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those skilled 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. In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase.

[0320] In some embodiments, the guide polynucleotide is at least one single guide RNA ("sgRNA" or "gRNA"). In some embodiments, the guide polynucleotide is at least one tracrRNA, and in some embodiments, the guide polynucleotide does not require a PAM sequence to guide a polynucleotide-programmable DNA-binding domain (e.g., Cas9 or Cpf1) to a target nucleotide sequence.

[0321] The polynucleotide-programmable nucleotide-binding domain (e.g., a CRISPR-derived domain) of the base editor disclosed herein can recognize a target polynucleotide sequence by associating with a guide polynucleotide. The guide polynucleotide (e.g., a gRNA) is typically single-stranded and can be programmed to site-specifically bind (i.e., via complementary base pairing) to a target sequence of the polynucleotide, thereby guiding the base editor with the guide nucleic acid to the target sequence. The guide polynucleotide can be DNA. The guide polynucleotide can be RNA. In some embodiments, the guide polynucleotide comprises a natural nucleotide (e.g., adenosine). In some embodiments, the guide polynucleotide comprises a non-natural nucleotide (e.g., a peptide nucleic acid or a nucleotide analog). In some embodiments, the targeted region of the guide nucleic acid sequence can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The targeted region of the guide nucleic acid can be between 10 and 30 nucleotides in length, or between 15 and 25 nucleotides in length, or between 15 and 20 nucleotides in length.

[0322] In some embodiments, a guide polynucleotide comprises two or more separate polynucleotides that can interact with each other, e.g., through complementary base pairing (e.g., a dual guide polynucleotide). For example, a guide polynucleotide can comprise a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). For example, a guide polynucleotide can comprise one or more trans-activating CRISPR RNAs (tracrRNAs).

[0323] In Type II CRISPR systems, targeting of nucleic acids by a CRISPR protein (e.g., Cas9) typically requires complementary base pairing between a first RNA molecule (crRNA) that contains a sequence that recognizes the target sequence and a second RNA molecule (trRNA) that contains repeats that form a scaffolding region that stabilizes the guide RNA-CRISPR protein complex. Such dual-guide RNA systems can be utilized as guide polynucleotides to direct the base editors disclosed herein to target polynucleotide sequences.

[0324] In some embodiments, the base editors provided herein utilize a single guide polynucleotide (e.g., a gRNA). In some embodiments, the base editors provided herein utilize dual (double) guide polynucleotides (e.g., dual gRNAs). In some embodiments, the base editors provided herein utilize one or more guide polynucleotides (e.g., multiple gRNAs). In some embodiments, a single guide polynucleotide is utilized for the different base editors described herein. For example, a single guide polynucleotide can be utilized for a cytidine base editor and an adenosine base editor.

[0325] In other embodiments, a guide polynucleotide can include both a polynucleotide targeting portion of a nucleic acid and a scaffold portion of a nucleic acid in a single molecule (i.e., a single-molecule guide nucleic acid). For example, a single-molecule guide polynucleotide can be a single guide RNA (sgRNA or gRNA). As used herein, the term "guide polynucleotide sequence" contemplates any single-, double-, or multi-molecule nucleic acid that can interact with a base editor and guide the base editor to a target polynucleotide sequence.

[0326] Typically, a guide polynucleotide (e.g., a crRNA / trRNA complex or a gRNA) comprises a "polynucleotide target segment" that includes a sequence capable of recognizing and binding to a target polynucleotide sequence, and a "protein-binding segment" that stabilizes the guide polynucleotide within the polynucleotide-programmable nucleotide-binding domain component of a base editor. In some embodiments, the polynucleotide target segment of a guide polynucleotide recognizes and binds to a DNA polynucleotide, thereby facilitating base editing in DNA. In other embodiments, the polynucleotide target segment of a guide polynucleotide recognizes and binds to an RNA polynucleotide, thereby facilitating base editing in RNA. Here, "segment" refers to a portion or region of a molecule, e.g., a stretch of contiguous nucleotides in a guide polynucleotide. A segment can also refer to a region / section of a complex, and thus, a segment can comprise regions of two or more molecules. For example, if a guide polynucleotide comprises multiple nucleic acid molecules, the protein-binding segment can comprise all or part of multiple separate molecules hybridized, e.g., along regions of complementarity. In some embodiments, the protein-binding segment of a DNA-targeting RNA comprising two separate molecules can comprise: (i) 40-75 base pairs of a first RNA molecule that is 100 base pairs in length; and (ii) 10-25 base pairs of a second RNA molecule that is 50 base pairs in length. The definition of "segment," unless otherwise defined in a particular context, is not limited to a specific number of total base pairs, is not limited to a specific number of base pairs from a given RNA molecule, is not limited to a specific number of separate molecules within a complex, and can include regions of any full-length RNA molecule and can include regions having complementarity to other molecules.

[0327] A guide RNA or guide polynucleotide can comprise two or more RNAs, such as a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). A guide RNA or guide polynucleotide can sometimes comprise a single-stranded RNA formed by the fusion of portions (e.g., functional portions) of a crRNA and a tracrRNA, or a single guide RNA (sgRNA). A guide RNA or guide polynucleotide can also be a duplex RNA comprising a crRNA and a tracrRNA. Furthermore, the crRNA can hybridize with a target DNA.

[0328] As mentioned above, guide RNA or guide polynucleotide can be an expression product.For example, DNA encoding guide RNA can be a vector containing a sequence encoding guide RNA.Guide RNA or guide polynucleotide can be introduced into cells by transfecting isolated guide RNA or plasmid DNA containing a sequence encoding guide RNA and a promoter into cells.Guide RNA or guide polynucleotide can also be introduced into cells by other methods, such as using virus-mediated gene delivery.

[0329] Guide RNA or guide polynucleotide can be isolated.For example, guide RNA can be transfected into cell or organism in the form of isolated RNA.Guide RNA can be prepared by in vitro transcription using any in vitro transcription system known in the art.Guide RNA can be introduced into cell in the form of isolated RNA, not in the form of a plasmid that contains the sequence that codes for guide RNA.

[0330] A guide RNA or guide polynucleotide may comprise three regions: a first region at the 5' end that may be complementary to a target site in a chromosomal sequence, a second internal region that may form a stem-loop structure, and a third 3' region that may be single-stranded. The first region of each guide RNA may be different so that each guide RNA guides the fusion protein to a specific target site. Furthermore, the second and third regions of each guide RNA may be identical in all guide RNAs.

[0331] The first region of the guide RNA or guide polynucleotide can be complementary to a sequence at a target site in a chromosomal sequence such that the first region of the guide RNA can base-pair with the target site. In some embodiments, the first region of the guide RNA can comprise about 10 to 25 nucleotides (i.e., 10 nucleotides to nucleotides; or about 10 nucleotides to 25 nucleotides; or 10 nucleotides to 25 nucleotides; or about 10 nucleotides to 25 nucleotides) or more. For example, the base-pairing region between the first region of the guide RNA and the target site in the chromosomal sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides in length, or approximately that length. Sometimes, the first region of the guide RNA can be 19, 20, or 21 nucleotides in length, or about 19, 20, or 21 nucleotides in length.

[0332] The guide RNA or guide polynucleotide can also include a second region that forms a secondary structure. For example, the secondary structure formed by the guide RNA can include a stem (or hairpin) and a loop. The lengths of the loop and stem can vary. For example, the length of the loop can range from (approximately) 3 to 10 nucleotides, and the length of the stem can range from (approximately) 6 to 20 base pairs. The stem can include one or more bulges of 1 to 10 or approximately 10 nucleotides. The total length of the second region can range from about 16 to 60 nucleotides in length. For example, the loop can be (approximately) 4 nucleotides in length, and the stem can be (approximately) 12 base pairs.

[0333] The guide RNA or guide polynucleotide can also include a third region at the 3' end, which can be essentially single-stranded. For example, the third region can be non-complementary to any chromosomal sequence in the target cell, or to the remainder of the guide RNA. Furthermore, the length of the third region can vary. The third region can be about 4 nucleotides or more in length. For example, the length of the third region can range from (about) 5 to 60 nucleotides in length.

[0334] The guide RNA or guide polynucleotide can target any exon or intron of the gene target. In some embodiments, the guide can target exon 1 or 2 of the gene; in other embodiments, the guide can target exon 3 or 4 of the gene. The composition can include multiple guide RNAs that all target the same exon, or in some embodiments, multiple guide RNAs that target different exons. Both exons and introns of the gene can be targeted.

[0335] The guide RNA or guide polynucleotide may target a nucleic acid sequence of (approximately) 20 nucleotides. The target nucleic acid may be less than (approximately) 20 nucleotides. The target nucleic acid may be at least (approximately) 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or any length between 1 and 100 nucleotides in length. The target nucleic acid may be at most about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50 nucleotides in length, or any length between 1 and 100 nucleotides in length. The target nucleic acid sequence may be (approximately) 20 bases immediately 5' to the first nucleotide of the PAM. The guide RNA may target a nucleic acid sequence. The target nucleic acid can be at least (approximately) 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, or 1-100 nucleotides.

[0336] A guide polynucleotide, e.g., a guide RNA, can refer to a nucleic acid that can hybridize to another nucleic acid, e.g., a target nucleic acid or a protospacer in the genome of a cell. A guide polynucleotide can be RNA. A guide polynucleotide can be DNA. A guide polynucleotide can be programmed or designed to site-specifically bind to a nucleic acid sequence. A guide polynucleotide can include one polynucleotide strand and be referred to as a single guide polynucleotide. A guide polynucleotide can include two polynucleotide strands and be referred to as a dual guide polynucleotide. A guide RNA can be introduced into a cell or embryo as an RNA molecule. For example, an RNA molecule can be transcribed in vitro and / or chemically synthesized. RNA can be transcribed from a synthetic DNA molecule, e.g., a gBlocks® gene fragment. A guide RNA can also be introduced into a cell or embryo as an RNA molecule. A guide RNA can also be introduced into a cell or embryo in the form of a non-RNA nucleic acid molecule, e.g., a DNA molecule. For example, DNA encoding the guide RNA can be operably linked to a promoter regulatory sequence for expression of the guide RNA in the cell or embryo of interest. The RNA coding sequence can be operably linked to a promoter sequence recognized by RNA polymerase III (Pol III). Plasmid vectors that can be used to express guide RNAs include, but are not limited to, px330 vectors and px333 vectors. In some embodiments, a plasmid vector (e.g., px333 vector) can contain DNA sequences encoding at least two guide RNAs.

[0337] Methods for selecting, designing, and validating guide polynucleotides (e.g., guide RNAs) and targeting sequences are described herein and are known to those skilled in the art. For example, to minimize the potential substrate promiscuity of the deaminase domain (e.g., the AID domain) in a nucleobase editor system, the number of residues that could unintentionally be targeted for deamination (e.g., off-target C residues potentially located on ssDNA within the target nucleic acid locus) can be minimized. Furthermore, software tools can be used to optimize the gRNA corresponding to the target nucleic acid sequence, for example, to minimize total off-target activity across the genome. For example, for each possible targeting domain option using S. pyogenes Cas9, all off-target sequences containing up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base pairs (e.g., preceding a selected PAM such as NAG or NGG) can be identified across the genome. First regions of gRNAs complementary to the target site can be identified, and all first regions (e.g., crRNAs) can be ranked according to their total predicted off-target score; the top-ranked targeting domains represent those likely to have the greatest on-target activity and the least off-target activity. Candidate targeting gRNAs can be functionally evaluated using methods known in the art and / or described herein.

[0338] As a non-limiting example, target DNA hybridizing sequences in the crRNA of a guide RNA for use with Cas9 can be identified using a DNA sequence search algorithm. gRNA design can be performed using custom gRNA design software based on the public tool cas-offinder, such as that described in Bae S., Park J., & Kim J.-S. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473-1475 (2014). This software scores guides after calculating their genome-wide off-target propensity. Typically, matches ranging from a perfect match to seven mismatches are considered for guides ranging in length from 17 to 24. Once off-target sites are computationally determined, an aggregate score is calculated for each guide and summarized in a tabular output using a web interface. In addition to identifying potential target sites adjacent to the PAM sequence, the software also identifies all PAM-flanking sequences that differ from the selected target site by 1, 2, 3, or more than 3 nucleotides. A target nucleic acid sequence, such as a genomic DNA sequence for a target gene, can be obtained and repetitive elements screened using publicly available tools, such as the RepeatMasker program. RepeatMasker searches for repetitive elements and low-complexity regions in the input DNA sequence. The output is a detailed annotation of the repeats present in a given query sequence.

[0339] After identification, the first regions of the guide RNA (e.g., crRNA) can be ranked in a hierarchy based on their distance to the target site, their orthogonality, and the presence of a 5' nucleotide for a close match to a related PAM sequence (e.g., a 5' G based on the identification of a close match in the human genome with a related PAM (e.g., an NGG PAM for Streptococcus pyogenes, an NNGRRT or NNGRRV PAM for Staphylococcus aureus). As used herein, orthogonality refers to the number of sequences in the human genome that contain a minimal number of mismatches to the target sequence. "High level of orthogonality" or "good orthogonality" can refer, for example, to a 20-mer targeting domain that has no identical sequences in the human genome other than the intended target and no sequences containing one or two mismatches in the target sequence. Targeting domains with good orthogonality can be selected to minimize off-target DNA cleavage.

[0340] In some embodiments, a reporter system can be used to detect base editing activity and test candidate guide polynucleotides. In some embodiments, the reporter system can include a reporter gene-based assay in which base editing activity results in expression of a reporter gene. For example, the reporter system can include a reporter gene containing an inactivated start codon, e.g., a mutation of 3'-TAC-5' to 3'-CAC-5' on the template strand. Upon successful deamination of the target C, the corresponding mRNA is transcribed as 5'-AUG-3' rather than 5'-GUG-3', allowing translation of the reporter gene. Suitable reporter genes will be apparent to those of skill in the art. Non-limiting examples of reporter genes include genes encoding green fluorescent protein (GFP), red fluorescent protein (RFP), luciferase, secreted alkaline phosphatase (SEAP), or any other gene whose expression is detectable and apparent to those of skill in the art. The reporter system can be used to test many different gRNAs, for example, to determine which residues in the target DNA sequence are targeted by their respective deaminases. sgRNAs targeting the non-template strand can also be tested to assess the off-target effects of specific base-editing proteins (e.g., Cas9 deaminase fusion proteins). In some embodiments, such gRNAs can be designed so that the mutated start codon does not base-pair with the gRNA. The guide polynucleotide can comprise standard ribonucleotides, modified ribonucleotides (e.g., pseudouridine), ribonucleotide isomers, and / or ribonucleotide analogs. In some embodiments, the guide polynucleotide can comprise at least one detectable label. The detectable label can be a fluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluor, Halo Tag, or a suitable fluorescent dye), a detection tag (e.g., biotin, digoxigenin, etc.), a quantum dot, or a gold particle.

[0341] The guide polynucleotide can be chemically synthesized, enzymatically synthesized, or a combination thereof. For example, the guide RNA can be synthesized using standard phosphoramidite-based solid-phase synthesis. Alternatively, the guide RNA can be synthesized in vitro by operably linking DNA encoding the guide RNA to a promoter regulatory sequence recognized by a phage RNA polymerase. Examples of suitable phage promoter sequences include T7, T3, and SP6 promoter sequences, or variants thereof. In embodiments in which the guide RNA comprises two separate molecules (e.g., crRNA and tracrRNA), the crRNA can be chemically synthesized and the tracrRNA can be enzymatically synthesized.

[0342] In some embodiments, a base editor system can include multiple guide polynucleotides, e.g., gRNAs. For example, a gRNA can target one or more target loci (e.g., at least 1 gRNA, at least 2 gRNAs, at least 5 gRNAs, at least 10 gRNAs, at least 20 gRNAs, at least 30 gRNAs, at least 50 gRNAs) included in the base editor system. The multiple gRNA sequences can be arranged in tandem, preferably separated by direct repeats.

[0343] The DNA sequence encoding the guide RNA or guide polynucleotide may be part of a vector. Furthermore, the vector may include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selectable marker sequences (e.g., GFP or antibiotic resistance genes such as puromycin), origins of replication, etc. The DNA molecule encoding the guide RNA may be linear. The DNA molecule encoding the guide RNA or guide polynucleotide may be circular.

[0344] In some embodiments, one or more components of the base editor system can be encoded by a DNA sequence. Such DNA sequences can be introduced into an expression system (e.g., a cell) together or separately. For example, DNA sequences encoding a polynucleotide programmable nucleotide binding domain and a guide RNA can be introduced into a cell, and each DNA sequence can be part of a separate molecule (e.g., one vector containing the coding sequence for the polynucleotide programmable nucleotide binding domain and a second vector containing the guide RNA coding sequence), or both can be part of the same molecule (e.g., one vector containing the coding (and regulatory) sequences for both the polynucleotide programmable nucleotide binding domain and the guide RNA).

[0345] The guide polynucleotide can include one or more modifications to provide a nucleic acid with new or enhanced characteristics. The guide polynucleotide can include a nucleic acid affinity tag. The guide polynucleotide can include synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides.

[0346] In some embodiments, the gRNA or guide polynucleotide may contain modifications. The modifications may be made to any position in the gRNA or guide polynucleotide. Multiple modifications may be made to a single gRNA or guide polynucleotide. The gRNA or guide polynucleotide may be subjected to quality control after modification. In some embodiments, quality control may include PAGE, HPLC, MS, or any combination thereof.

[0347] Modifications of the gRNA or guide polynucleotide can be substitutions, insertions, deletions, chemical modifications, physical modifications, stabilization, purification, or any combination thereof.

[0348] The gRNA or guide polynucleotide may also be modified with any of the following amino acids: 5' adenylate, 5' guanosine triphosphate cap, 5'N7-methylguanosine triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' thiophosphate, 5' phosphate, 5' thiophosphate, Cis-Syn thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, d spacer, PC spacer, r spacer, spacer 18, spacer 9, 3'-3' modification, 5'-5' modification, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesterol TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, double biotin, psoralen, psoralen C2, psoralen C6, TINA, 3'DABCYL, black hole quencher 1, black hole quencher 2, DABCYL The nucleic acids may also be modified with SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, a carboxyl linker, a thiol linker, a 2'-deoxyribonucleoside analog purine, a 2'-deoxyribonucleoside analog pyrimidine, a ribonucleoside analog, a 2'-O-methylribonucleoside analog, a sugar-modified analog, a wobble / universal base, a fluorescent dye label, 2'-fluoroRNA, 2'-O-methylRNA, a methylphosphonate, a phosphodiester DNA, a phosphodiester RNA, a phosphothioester DNA, a phosphorothioate RNA, a UNA, pseudolysine-5'-triphosphate, a 5'-methyllysine-5'-triphosphate, or any combination thereof.

[0349] In some embodiments, the modifications are permanent. In other embodiments, the modifications are temporary. In some embodiments, multiple modifications are made to the gRNA or guide polynucleotide. Modifications to the gRNA or guide polynucleotide can change the physicochemical properties of the nucleotides, such as their conformation, polarity, hydrophobicity, chemical reactivity, base pairing interactions, or a combination thereof.

[0350] The PAM sequence can be any PAM sequence kn...

Claims

1. (i) a nucleic acid programmable DNA binding protein (napDNAbp) domain; and (ii) a cytidine deaminase comprising an amino acid sequence having at least 95% sequence identity to any of the following amino acid sequences: a) MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVARLFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR, or a variant of the above amino acid sequence comprising an alteration selected from the group consisting of R33A, K34A, R52A, W90F, and H122A; b) MKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEIIKQYLPVPWKKGVFRNQVDPETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTIYTARLYYFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREILQ, or a variant of the above amino acid sequence comprising the F130L modification; c) MADSSEKMRGQYISRDTFEKNYKPIDGTKEAHLLCEIKWGKYGKPWLHWCQNQRMNIHAEDYFMNNIFKAKKHPVHCYVTWYLSWSPCADCASKIVKFLEERPYLKLTIYVAQLYYHTEEENRKGLRLLRSKKVIIRVMDISDYNYCWKVFVSNQNGNEDYWPLQFDPWVKENYSRLLDIFWESKCRSPNPW, and d) MDPQRLRQWPGPGPASRGGYGQRPRIRNPEEWFHELSPRTFSFHFRNLRFASGRNRSYICCQVEGKNCFFQGIFQNQVPPDPPCHAELCFLSWFQSWGLSPDEHYYVTWFISWSPCCECAAKVAQFLEENRNVSLSLSAARLYYFWKSESREGLRRLSDLGAQVGIMSFQDFQHCWNNFVHNLGMPFQPWKKLHKNYQRLVTELKQILREEPATYGSPQAQGKVRIGSTAAGLRHSHSHTRSEAHLRPNHSSRQHRILNPPREARARTCVLVDASWICYR, or a variant of the above amino acid sequence containing the R54Q modification; and a cytidine base editor fusion protein comprising an increased ratio of cis to trans activity (in cis:in trans) compared to a standard cytidine base editor fusion protein comprising rat APOBEC-1 cytidine deaminase (rAPOBEC-1) and a Cas9 nickase.

2. 2. The cytidine base editor fusion protein of claim 1, wherein the canonical cytidine base editor fusion protein is BE3 or BE4.

3. 3. The cytidine base editor fusion protein of claim 1 or 2, further comprising at least one adenosine deaminase or a catalytically active fragment thereof.

4. the adenosine deaminase is TadA deaminase; and / or The adenosine deaminase is a non-naturally occurring modified adenosine deaminase, TadA deaminase. The cytidine base editor fusion protein of claim 3.

5. the cytidine base editor fusion protein comprises two adenosine deaminases, which may be the same or different; and / or the cytidine base editor fusion protein comprises two adenosine deaminases capable of forming a heterodimer or a homodimer; and / or the cytidine base editor fusion protein comprises two adenosine deaminase domains, wild-type TadA and TadA7.10; The cytidine base editor fusion protein of claim 3.

6. (i) the cytidine base editor fusion protein further comprises one or more nuclear localization signals (NLS); and / or (ii) the cytidine base editor fusion protein comprises an N-terminal NLS and / or a C-terminal NLS; and / or (iii) the cytidine base editor fusion protein further comprises a bipartite NLS; and / or (iv) the cytidine base editor fusion protein further comprises an N-terminal bipartite NLS and / or a C-terminal bipartite NLS. The cytidine base editor fusion protein of any one of claims 1 to 5.

7. (i) the napDNAbp domain is Cas9; or (ii) the napDNAbp domain is Staphylococcus aureus Cas9 (SaCas9), Streptococcus pyogenes Cas9 (SpCas9), or a variant thereof; or (iii) the napDNAbp domain comprises a nuclease-inactive Cas9 (dCas9), a Cas9 nickase (nCas9), a nCas9 containing the amino acid substitution D10A or a corresponding amino acid substitution, or a nuclease-active Cas9; or (iv) the napDNAbp domain comprises a catalytic domain capable of cleaving the reverse complement of a nucleic acid sequence or does not comprise a catalytic domain capable of cleaving a nucleic acid sequence; The cytidine base editor fusion protein of any one of claims 1 to 6.

8. (i) further comprising one or more uracil DNA glycosylase inhibitors (UGI); and / or (ii) further comprising one or more UGIs derived from Bacillus subtilis bacteriophage PBS1 and inhibiting human UDG activity; The cytidine base editor fusion protein of any one of claims 1 to 7.

9. A cell comprising the cytidine base editor fusion protein of any one of claims 1 to 8.

10. 9. A molecular complex comprising the cytidine base editor fusion protein of any one of claims 1 to 8 and one or more of a guide RNA, a tracrRNA, and a target DNA molecule.

11. 9. An in vitro or ex vivo method of editing a nucleobase of a nucleic acid molecule comprising contacting the nucleic acid molecule with a cytidine base editor fusion protein of any one of claims 1 to 8 and converting a first nucleobase of the nucleic acid molecule to a second nucleobase, wherein the first nucleobase is cytosine and the second nucleobase is thymidine, and wherein the nucleic acid molecule is a DNA molecule.

12. A polynucleotide molecule encoding the cytidine base editor fusion protein of any one of claims 1 to 8.

13. The polynucleotide molecule of claim 12, which is codon optimized.

14. An expression vector comprising the polynucleotide molecule of claim 12 or 13.

15. the expression vector is a mammalian expression vector; or The vector is a viral vector selected from the group consisting of an adeno-associated virus (AAV), a retroviral vector, an adenoviral vector, a lentiviral vector, a Sendai virus vector, and a herpes virus vector; The expression vector of claim 14.

16. A cell comprising a polynucleotide molecule according to claim 12 or 13 or a vector according to claim 14 or 15.

17. 17. A kit comprising a cytidine base editor fusion protein according to any one of claims 1 to 8, a polynucleotide molecule according to claim 12 or 13, a vector according to claim 14 or 15, or a molecular complex according to claim 10.

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