Nucleobase editor with reduced off-target deamination reaction and method for modifying nucleobase target sequence using the same

Abstract

The invention features nucleobase editors and multi-effector nucleobase editors with improved editing settings (i.e., minimal off-target deamination reactions), compositions including such editors, and methods of using them to generate modifications in target nucleobase sequences.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is an International PCT application, which claims the benefit of U.S. Provisional Patent Application No. 62 / 799,702 filed on January 31, 2019; No. 62 / 835,456 filed on April 17, 2019; and No. 62 / 941,569 filed on November 27, 2019, the entire contents of each of which are hereby incorporated by reference into this application. Technical Field

[0003] The present application relates to nucleobase editors and multi-effector nucleobase editors with improved editing settings and minimal off-target deamination reactions, compositions comprising such editors, and methods of using the editors and / or compositions to generate modifications in target nucleobase sequences. Background Art

[0004] Targeted editing of nucleic acid sequences, such as targeted cleavage or targeted modification of genomic DNA, is a promising method for studying gene function and has the potential to provide new therapies for human genetic diseases. Currently available base editors include cytidine base editors (e.g., BE4) that convert target C·G base pairs to T·A and adenine base editors (e.g., ABE7.10) that convert A·T to G·C. There is a need in the art for improved base editors that can induce modifications within target sequences with greater specificity and efficiency. Summary of the Invention

[0005] As described below, the invention features nucleobase editors and multi-effector nucleobase editors with improved editing settings (i.e., minimal off-target deamination reactions), compositions comprising such editors, and methods of using them to generate modifications in target nucleobase sequences.

[0006] In one aspect, the present invention provides 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-activity to trans-activity (cis:trans) compared to a standard cytidine base editor.

[0007] In some embodiments, the standard cytidine base editor includes (i) a polynucleotide programmable DNA binding domain and (ii) an APOBEC cytidine deaminase. In some embodiments, the APOBEC cytidine deaminase of the standard cytidine base editor is a rat APOBEC-1 cytidine deaminase (rAPOBEC-1). In some embodiments, the polynucleotide programmable DNA binding domain of the standard cytidine base editor is a Cas9 nickase. In some embodiments, the standard cytidine base editor includes a uracil glycosylase inhibitor (UGI) domain. In some embodiments, the standard cytidine base editor is BE3 or BE4. In some embodiments, the ratio of the increased cis activity to trans activity is increased by at least 2, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 times 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 compared to a standard cytidine base editor.

[0008] In some embodiments, the cytidine base editor has at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, or more fold less trans activity than a standard cytidine base editor.

[0009] 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 some embodiments, the cytidine deaminase is APOBEC1. In some embodiments, the cytidine deaminase is (a) APOBEC-1 from golden hamster (MaAPOBEC-1), Bornean orangutan (PpAPOBEC-1), sycamore rabbit (OcAPOBEC-1), gray short-tailed opossum (MdAPOBEC-1), or American alligator (AmAPOBEC-1), (b) APOBEC-2 from Bornean orangutan (PpAPOBEC-2), cattle (BtAPOBEC-2), or European pig (SsAPOBEC-2). , (c) APOBEC-4 from cynomolgus macaque (MfAPOBEC-4), (d) AID from dog (ClAID) or cattle (BtAID), (e) yeast cytosine deaminase (yCD) from Saccharomyces cerevisiae, (f) APOBEC-3F from golden snub-nosed monkey (RrA3F), 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)-(f).

[0010] In some embodiments, the cytidine deaminase is APOBEC-1 from golden hamster (MaAPOBEC-1), Bornean orangutan (PpAPOBEC-1), ocytophaga (OcAPOBEC-1), gray short-tailed opossum (MdAPOBEC-1), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing proteins. 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 some embodiments, the cytidine deaminase is APOBEC-2 derived from Bornean orangutan (PpAPOBEC-2), cattle (BtAPOBEC-2), or European pig (SsAPOBEC-2), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned proteins. In some embodiments, the cytidine deaminase is APOBEC-4 derived from cynomolgus macaque (MfAPOBEC-4), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned proteins. In some embodiments, the cytidine deaminase is AID from canine (clAID), bovine (BtAID), or a cytidine deaminase having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned protein.

[0011] In some embodiments, the cytidine deaminase is yeast cytosine deaminase (yCD) from Saccharomyces cerevisiae, or a cytidine deaminase having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequence to the aforementioned protein. In some embodiments, the cytidine deaminase is APOBEC-3F from Sichuan golden snub-nosed monkey (RrA3F), or a cytidine deaminase having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequence to the aforementioned protein. In some embodiments, the cytidine deaminase is any cytidine deaminase provided in Table 13, or a cytidine deaminase having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequence to the aforementioned protein. In some embodiments, the cytidine deaminase is APOBEC-3F from golden snub-nosed monkey (RrA3F), APOBEC-1 from American alligator (AmAPOBEC-1), APOBEC-2 from European pig (SsAPOBEC-2), APOBEC-1 from Bornean orangutan (PpAPOBEC-1), or a cytidine deaminase having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing protein.

[0012] In some embodiments, the cytidine deaminase comprises one or more alterations at position R15X, R16X, H21X, R30X, R33X, K34X, R52X, K60X, R118X, H121X, H122X, R126X, R128X, R169X, R198X, T36X, H53X, V62X, L88X, W90X, Y120X, or R132X (as numbered in SEQ ID NO: 1), or corresponding alterations of one or more of said alterations, wherein X is any amino acid.

[0013] In some embodiments, the cytidine deaminase comprises one or more alterations 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 corresponding alterations of one or more of said alterations. In some embodiments, the cytidine deaminase comprises a combination of alterations 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 corresponding alterations thereof. In some embodiments, the cytidine deaminase comprises an alteration at position Y120F and one or more alterations selected from the group consisting of R33A, W90F, K34A, R52A, H122A, and H121A (as numbered in SEQ ID NO: 1), or corresponding alterations to one or more of said alterations. In some embodiments, the cytidine deaminase comprises an alteration at position Y130X or R28X (as numbered in SEQ ID NO: 1), or corresponding alterations to said alterations, wherein X is any amino acid.

[0014] In some embodiments, the cytidine deaminase comprises an alteration at position Y130A or R28A (as numbered in SEQ ID NO: 1), or corresponding alterations thereof. In some embodiments, the cytidine deaminase comprises an alteration at position Y130A and R28A (as numbered in SEQ ID NO: 1), or corresponding alterations thereof. In some embodiments, the cytidine deaminase comprises one or more alterations at position H122X, K34X, R33X, W90X, or R128X (as numbered in SEQ ID NO: 1), or corresponding alterations thereof, wherein X is any amino acid. In some embodiments, the cytidine deaminase comprises one or more alterations selected from the group consisting of H122A, K34A, R33A, W90F, W90A, and R128A (as numbered in SEQ ID NO: 1), or corresponding alterations thereof. In some embodiments, the cytidine deaminase comprises a combination of alterations 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 corresponding alterations thereof.

[0015] In some embodiments, the cytidine deaminase comprises an amino acid sequence that is at least 80% identical to the following amino acid sequence:

[0016] MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVAR LFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR.

[0017] In some embodiments, the cytidine deaminase comprises an amino acid sequence that is at least 80% identical to the following amino acid sequence:

[0018] MKPQIRDHRPNMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEIIKQYLPVPWKKGVFRNQVDPETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTIYTARLYYFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREILQ.

[0019] In some embodiments, the cytidine deaminase comprises an amino acid sequence that is at least 80% identical to the following amino acid sequence:

[0020] MADSSEKMRGQYISRDTFEKNYKPIDGTKEAHLLCEIKWGKYGKPWLHWCQNQRMNIHAEDYFMNNIFKAKKHPVHCYVTWYLSWSPCADCASKIVKFLEERPYLKLTIYVAQLYYHTEEENRKGLRLLRSKKVIIRVMDISDYNYCWKVFVSNQNGNEDYWPLQFDPWVKENYSRLLDIFWESKCRSPNPW.

[0021] In some embodiments, the cytidine deaminase comprises an amino acid sequence that is at least 80% identical to the following amino acid sequence:

[0022] MDPQRLRQWPGPGPASRGGYGQRPRIRNPEEWFHELSPRTFSFHFRNLRRFASGRNRSYICCQVEGKNCFFQGIFQNQVPPDPPCHAELCFLSWFQSWGLSPDEHYYVTWFISWSPCCECAAKVAQFLEENRNVSLSLSAA RLYYFWKSESREGLRRLSDLGAQVGIMSFQDFQHCWNNFVHNLGMPFQPWKKLHKNYQRLVTELKQILREEPATYGSPQAQGKVRIGSTAAGLRHSHSHTRSEAHLRPNHSSRQHRILNPPREARARTCVLVDASWICYR.

[0023] In some embodiments, the cytidine deaminase comprises an H122A change. In some embodiments, the cytidine base editor of any of the above aspects further comprises at least one adenosine deaminase or a catalytically active fragment thereof. In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is a modified adenosine deaminase that does not exist in nature. In some embodiments, the cytidine base editor comprises two adenosine deaminases, which are the same or different. In some embodiments, the two adenosine deaminases are capable of forming heterodimers or homodimers. In some embodiments, the adenosine deaminase domain is wild-type TadA and TadA7.10.

[0024] In some embodiments, the adenosine deaminase comprises a C-terminal deletion starting from a residue selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 157. In some embodiments, 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 some embodiments, 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, at least one nucleobase editor domain further comprises an abasic nucleobase editor. In some embodiments, the cytidine base editor of any 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.

[0025] 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 a nuclease-inactivated Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease-active Cas9. In some embodiments, the polynucleotide programmable DNA binding domain comprises a catalytic domain capable of cleaving the reverse complementary strand 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 a Cas9 nickase (nCas9). In some embodiments, the nCas9 comprises the amino acid substitution D10A or its corresponding amino acid substitution.

[0026] In some embodiments, the cytidine base editor of any of the above aspects further comprises one or more uracil DNA glycosylase inhibitors (UGI). In some embodiments, the one or more UGIs are derived from Bacillus subtilis phage PBS1 and inhibit human UDG activity. In some embodiments, the cytidine base editor comprises two uracil DNA glycosylase inhibitors (UGI). In some embodiments, the cytidine base editor of any of the above aspects further comprises one or more linkers.

[0027] Provided herein are cells comprising a cytidine base editor according to any of the above aspects. In some embodiments, the cell is a bacterial cell, a plant cell, an insect cell, or a mammalian cell.

[0028] Provided herein is a molecular complex comprising a cytidine base editor of any aspect described above and one or more guide RNA sequences, tracrRNA sequences, or target DNA sequences.

[0029] Provided herein is a method for editing a nucleobase of a nucleic acid sequence, the method comprising contacting the nucleic acid sequence with a cytidine base editor of any aspect described above and converting a first nucleobase of the DNA sequence to a second nucleobase.

[0030] In some embodiments, the method further comprises contacting the nucleic acid sequence with a guide polynucleotide to effectuate the conversion. In some embodiments, the first nucleobase is cytosine and the second nucleobase is thymidine.

[0031] In one aspect, the present invention provides 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 (i) APOBEC-1 from golden hamster (MaAPOBEC-1), Bornean orangutan (PpAPOBEC-1), burrowing rabbit (OcAPOBEC-1), gray short-tailed opossum (MdAPOBEC-1), or American alligator (AmAPOBEC-1), (ii) APOBEC-2 from Bornean orangutan (PpAPOBEC-2), domestic cattle (BtAPOBEC-2), or (iii) APOBEC-3 from swinhoe's brood (S ... BEC-2), or European pig (SsAPOBEC-2), (iii) APOBEC-4 from cynomolgus macaque (MfAPOBEC-4), (iv) AID from dog (ClAID) or cattle (BtAID), (v) yeast cytosine deaminase (yCD) from Saccharomyces cerevisiae, (vi) APOBEC-3F from golden snub-nosed monkey (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) to (viii).

[0032] In one aspect, the present invention provides 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 golden hamster (MaAPOBEC-1), Bornean orangutan (PpAPOBEC-1), cave rabbit (OcAPOBEC-1), gray short-tailed opossum (MdAPOBEC-1), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing protein.

[0033] In one aspect, the present invention provides 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 Bornean orangutan (PpAPOBEC-2), domestic cattle (BtAPOBEC-2), or European pigs (SsAPOBEC-2), or a cytidine deaminase having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing proteins.

[0034] In one aspect, the present invention provides 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-4 from cynomolgus macaque (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 to the aforementioned protein.

[0035] In one aspect, the present invention provides 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 AID from canine (ClAID), cattle (BtAID), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned protein.

[0036] In one aspect, the present invention provides 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 a yeast cytosine deaminase (yCD) from Saccharomyces cerevisiae, or a cytidine deaminase having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned protein.

[0037] On the one hand, the present invention provides 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-3F, which is from Sichuan golden snub-nosed monkey (RrA3F), or a cytidine deaminase having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned protein.

[0038] In one aspect, the present invention provides 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 any one of the cytidine deaminases provided in Table 13, or a cytidine deaminase having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned protein.

[0039] On the one hand, the present invention provides 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-3F (RrA3F) from Sichuan golden snub-nosed monkey, APOBEC-1 (AmAPOBEC-1) from American alligator, APOBEC-2 from European pig (SsAPOBEC-2), APOBEC-1 from Bornean orangutan (PpAPOBEC-1), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing protein.

[0040] In some embodiments, the cytidine deaminase comprises one or more alterations at position R15X, R16X, H21X, R30X, R33X, K34X, R52X, K60X, R118X, H121X, H122X, R126X, R128X, R169X, R198X, T36X, H53X, V62X, L88X, W90X, Y120X, or R132X (as numbered in SEQ ID NO: 1), or corresponding alterations of one or more of said alterations, wherein X is any amino acid. In some embodiments, the cytidine deaminase comprises one or more alterations 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 corresponding alterations of one or more of said alterations. In some embodiments, the cytidine deaminase comprises a combination of alterations 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 corresponding alterations thereof. In some embodiments, the cytidine deaminase comprises a combination of alterations selected from the group consisting of Y120F and one or more alterations selected from the group consisting of R33A, W90F, K34A, R52A, H122A, and H121A (as numbered in SEQ ID NO: 1), or corresponding alterations of one or more of said alterations.

[0041] In some embodiments, the cytidine deaminase comprises one or more alterations at position Y130X or R28X (as numbered in SEQ ID NO: 1), or corresponding alterations to one or more of said alterations, wherein X is any amino acid. In some embodiments, the cytidine deaminase comprises one or more alterations selected from the group consisting of Y130A and R28A (as numbered in SEQ ID NO: 1), or corresponding alterations to one or more of said alterations. In some embodiments, the cytidine deaminase comprises one or more alterations at position H122X, K34X, R33X, W90X, or R128X (as numbered in SEQ ID NO: 1), or corresponding alterations to one or more of said alterations, wherein X is any amino acid. In some embodiments, the cytidine deaminase comprises one or more alterations selected from the group consisting of H122A, K34A, R33A, W90F, W90A, and R128A (as numbered in SEQ ID NO: 1), or corresponding alterations of one or more of said alterations.

[0042] In some embodiments, the cytidine deaminase comprises a combination of alterations 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 corresponding alterations of one or more of said alterations. In some embodiments, the cytidine deaminase comprises an H122A alteration (as numbered in SEQ ID NO: 1), or corresponding alterations of said alterations. In some embodiments, the cytidine deaminase is rAPOBEC1 and comprises one or more alterations 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 corresponding alterations of one or more of said alterations. In some embodiments, the cytidine deaminase comprises a combination of alterations 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 corresponding alterations of one or more of said alterations.

[0043] In one aspect, the present invention provides 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 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.

[0044] In some embodiments, the APOBEC3 family member is selected from the group consisting of APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3G, and APOBEC3H. In some embodiments, the APOBEC2 family member is SsAPOBEC2.

[0045] Provided herein is a fusion protein comprising a polynucleotide-programmable DNA-binding domain and at least one nucleobase editor domain comprising APOBEC1, wherein the APOBEC1 is selected from the group consisting of ppAPOBEC1, AmAPOBEC1 (BEM3.31), ocAPOBEC1, SsAPOBEC2 (BEM3.39), hAPOBEC3A, maAPOBEC1, and mdAPOBEC1.

[0046] In some embodiments, the cytidine deaminase comprises one or more alterations at position R15X, R16X, H21X, R30X, R33X, K34X, R52X, K60X, R118X, H121X, H122X, R126X, R128X, R169X, R198X, T36X, H53X, V62X, L88X, W90X, Y120X, or R132X (as numbered in SEQ ID NO: 1), or corresponding alterations of one or more of said alterations, wherein X is any amino acid. In some embodiments, the one or more alterations 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 corresponding alterations of one or more of said alterations. In some embodiments, the cytidine deaminase comprises a combination of alterations 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 corresponding alterations of one or more of said alterations. In some embodiments, the cytidine deaminase comprises a combination of alterations selected from the group consisting of Y120F and one or more alterations selected from the group consisting of R33A, W90F, K34A, R52A, H122A, and H121A (as numbered in SEQ ID NO: 1), or corresponding alterations of one or more of said alterations.

[0047] 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 comprises one or more alterations at position R15X, R16X, H21X, R30X, R33X, K34X, R52X, K60X, R118X, H121X, H122X, R126X, R128X, R169X, R198X, T36X, H53X, V62X, L88X, W90X, Y120X, or R132X (as numbered in SEQ ID NO: 1), or corresponding alterations of one or more of said alterations, wherein X is any amino acid.

[0048] In some embodiments, the cytidine deaminase comprises one or more alterations 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 corresponding alterations of one or more of said alterations. In some embodiments, the cytidine deaminase comprises a combination of alterations 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 corresponding alterations of one or more of said alterations. In some embodiments, the cytidine deaminase comprises an alteration at position Y120F and one or more alterations selected from the group consisting of R33A, W90F, K34A, R52A, H122A, and H121A (as numbered in SEQ ID NO: 1), or corresponding alterations of one or more of said alterations.

[0049] 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 comprises one or more alterations at positions Y130X and R28X (as numbered in SEQ ID NO: 1), or corresponding alterations thereof, wherein X is any amino acid.

[0050] In some embodiments, the cytidine deaminase comprises one or more alterations selected from the group consisting of Y130A and R28A (as numbered in SEQ ID NO: 1), or corresponding alterations of one or more of said alterations. In some embodiments, the cytidine deaminase comprises alterations Y130A and R28A.

[0051] 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 comprises one or more alterations at position H122X, K34X, R33X, W90X, or R128X (as numbered in SEQ ID NO: 1), or corresponding alterations of one or more of said alterations, wherein X is any amino acid.

[0052] In some embodiments, the cytidine deaminase comprises one or more alterations selected from the group consisting of H122A, K34A, R33A, W90F, W90A, and R128A (as numbered in SEQ ID NO: 1), or corresponding alterations to one or more of said alterations. In some embodiments, the cytidine deaminase comprises a combination of alterations 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 corresponding alterations to one or more of said alterations. In some embodiments, the cytidine deaminase is selected from the group consisting of:

[0053] 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 some embodiments, the cytidine deaminase is APOBEC1. In some embodiments, the cytidine deaminase is rAPOBEC1. In some embodiments, the cytidine deaminase is hAPOBEC3A. In some embodiments, the cytidine deaminase is ppAPOBEC1.

[0054] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA binding domain and a cytidine deaminase, wherein the cytidine deaminase comprises an amino acid sequence that is at least 80% identical to the following amino acid sequence:

[0055] MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVAR LFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR.

[0056] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA binding domain and a cytidine deaminase, wherein the cytidine deaminase comprises an amino acid sequence that is at least 80% identical to the following amino acid sequence:

[0057] MKPQIRDHRPNMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEIIKQYLPVPWKKGVFRNQVDPETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTIYTARLYYFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREILQ.

[0058] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA binding domain and a cytidine deaminase, wherein the cytidine deaminase comprises an amino acid sequence that is at least 80% identical to the following amino acid sequence:

[0059] MADSSEKMRGQYISRDTFEKNYKPIDGTKEAHLLCEIKWGKYGKPWLHWCQNQRMNIHAEDYFMNNIFKAKKHPVHCYVTWYLSWSPCADCASKIVKFLEERPYLKLTIYVAQLYYHTEEENRKGLRLLRSKKVIIRVMDISDYNYCWKVFVSNQNGNEDYWPLQFDPWVKENYSRLLDIFWESKCRSPNPW.

[0060] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA binding domain and a cytidine deaminase, wherein the cytidine deaminase comprises an amino acid sequence that is at least 80% identical to the following amino acid sequence:

[0061] MDPQRLRQWPGPGPASRGGYGQRPRIRNPEEWFHELSPRTFSFHFRNLRRFASGRNRSYICCQVEGKNCFFQGIFQNQVPPDPPCHAELCFLSWFQSWGLSPDEHYYVTWFISWSPCCECAAKVAQFLEENRNVSLSLSAA RLYYFWKSESREGLRRLSDLGAQVGIMSFQDFQHCWNNFVHNLGMPFQPWKKLHKNYQRLVTELKQILREEPATYGSPQAQGKVRIGSTAAGLRHSHSHTRSEAHLRPNHSSRQHRILNPPREARARTCVLVDASWICYR.

[0062] In some embodiments, the cytidine deaminase comprises an H122A alteration.

[0063] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA binding domain and a cytidine deaminase, wherein the cytidine deaminase is an APOBEC1 deaminase and comprises an H122A alteration.

[0064] In one aspect, provided herein is a fusion protein comprising a polynucleotide-programmable DNA binding domain and a cytidine deaminase, wherein the cytidine deaminase is rAPOBEC1 and comprises one or more alterations 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 some embodiments, the cytidine deaminase comprises a combination of alterations 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.

[0065] In one aspect, the present invention provides a fusion protein comprising a polynucleotide-programmable DNA binding domain and at least one nucleobase editor domain comprising APOBEC1, wherein the APOBEC1 is selected from the group consisting of: ppAPOBEC1, AmAPOBEC1 (BEM3.31), ocAPOBEC1, SsAPOBEC2 (BEM3.39), hAPOBEC3A, maAPOBEC1, and mdAPOBEC1.

[0066] In some embodiments, the APOBEC1 comprises one or more alterations at position R15X, R16X, H21X, R30X, R33X, K34X, R52X, K60X, R118X, H121X, H122X, R126X, R128X, R169X, R198X, T36X, H53X, V62X, L88X, W90X, Y120X, or R132X (as numbered in SEQ ID NO: 1), or corresponding alterations of one or more of said alterations, wherein X is any amino acid.

[0067] In some embodiments, the one or more alterations 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 corresponding alterations of one or more of said alterations. In some embodiments, the APOBEC1 comprises a combination of alterations 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 corresponding alterations of one or more of said alterations. In some embodiments, the APOBEC1 comprises an alteration at position Y120F and one or more alterations selected from the group consisting of R33A, W90F, K34A, R52A, H122A, and H121A (as numbered in SEQ ID NO: 1), or corresponding alterations of one or more of said alterations.

[0068] In some embodiments, the fusion protein of any of the above aspects further comprises at least one adenosine deaminase or a catalytically active fragment thereof. In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is a modified adenosine deaminase that does not exist in nature. In some embodiments, the fusion protein comprises two adenosine deaminases, which are the same or different. In some embodiments, the two adenosine deaminases are capable of forming heterodimers or homodimers. In some embodiments, the two adenosine deaminase domains are wild-type TadA and TadA7.10.

[0069] In some embodiments, the adenosine deaminase comprises a C-terminal deletion starting from a residue selected from the group consisting of 149, 150, 151, 152, 153, 154, 155, 156, and 157. In some embodiments, 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 some embodiments, 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, at least one nucleobase editor domain further comprises an abasic nucleobase editor.

[0070] In some embodiments, the fusion protein of any 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.

[0071] 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 variants thereof. In some embodiments, the polynucleotide programmable DNA binding domain comprises a nuclease-inactivated Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease-active Cas9.

[0072] In some embodiments, the polynucleotide-programmable DNA binding domain includes a catalytic domain capable of cleaving the reverse complementary strand of a nucleic acid sequence. In some embodiments, the polynucleotide-programmable DNA binding domain does not include a catalytic domain capable of cleaving a nucleic acid sequence.

[0073] In some embodiments, the Cas9 is dCas9. In some embodiments, the Cas9 is a Cas9 nickase (nCas9). In some embodiments, the nCas9 comprises the amino acid replacement D10A or its corresponding amino acid replacement. In some embodiments, the fusion protein of any of the above aspects further comprises one or more uracil DNA glycosylase inhibitors (UGI). In some embodiments, the one or more UGIs are derived from Bacillus subtilis phage PBS1 and inhibit human UDG activity. In some embodiments, the fusion protein comprises two uracil DNA glycosylase inhibitors (UGI). In some embodiments, the fusion protein of any of the above aspects further comprises one or more linkers. In some embodiments, the fusion protein deaminates the nucleobases in the target nucleotide sequence, and wherein the deamination reaction has an increased ratio of cis activity to trans activity (cis: trans) compared to a standard cytidine base editor.

[0074] In some embodiments, the standard cytidine base editor comprises (i) a polynucleotide-programmable DNA-binding domain and (ii) an APOBEC cytidine deaminase.

[0075] In some embodiments, the APOBEC cytidine deaminase of the standard cytidine base editor is rat APOBEC-1 cytidine deaminase (rAPOBEC-1). In some embodiments, the polynucleotide programmable DNA binding domain of the standard cytidine base editor is a Cas9 nickase. In some embodiments, the standard cytidine base editor includes a uracil glycosylase inhibitor (UGI) domain. In some embodiments, the standard cytidine base editor is BE3 or BE4. In some embodiments, the increased cis activity to trans activity ratio is increased by at least 2, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 times or more. In some embodiments, the cytidine base editor has at least 50%, 60%, 70%, 80%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, or higher cis activity compared to the standard cytidine base editor. In some embodiments, the cytidine base editor has at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, or more fold less trans activity than a standard cytidine base editor.

[0076] In one aspect, the present invention provides a polynucleotide molecule encoding a fusion protein according to any one of the above aspects. In some embodiments, the polynucleotide molecule is codon-optimized.

[0077] Provided herein are expression vectors comprising the polynucleotide molecules described above. In some embodiments, the expression vector is a mammalian expression vector. In some embodiments, the vector is a viral vector selected from the group consisting of an adeno-associated virus (AAV) vector, a retroviral vector, an adenoviral vector, a lentiviral vector, a Sendai virus vector, and a herpes virus vector. In some embodiments, the vector comprises a promoter.

[0078] Provided herein are cells comprising the polynucleotides or vectors described above. In some embodiments, the cell is a bacterial cell, a plant cell, an insect cell, a human cell, or a mammalian cell.

[0079] Provided herein is a molecular complex comprising a fusion protein of any of the above aspects and one or more guide RNA sequences, tracrRNA sequences, or target DNA sequences.

[0080] Provided herein is a kit comprising the fusion protein, the polynucleotide, the vector, or the molecular complex according to any one of the above aspects.

[0081] Provided herein is a method for editing the nucleobases of a nucleic acid sequence, the method comprising contacting the nucleic acid sequence with a base editor (which comprises a fusion protein of any of the above aspects) and converting the first nucleobase of the DNA sequence to a second nucleobase. In some embodiments, the first nucleobase is cytosine and the second nucleobase is thymidine.

[0082] Provided herein is a method for editing the nucleobase of a nucleic acid sequence, the method comprising contacting the nucleic acid sequence with a base editor (which includes the fusion protein of any of the above aspects) and converting the first nucleobase of the 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 the 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 thymidine and the fourth nucleobase is cytosine.

[0083] Provided herein is a method for optimal base editing, the method comprising contacting a target nucleobase in a target nucleotide sequence with a cytidine base editor, the 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 in the target nucleotide sequence with a lower pseudo-deamination reaction than a positive cytidine base editor comprising rAPOBEC1. In some embodiments, the cytidine base editor deaminates the target nucleobase with higher efficiency than a positive cytidine base editor. In some embodiments, the positive cytidine base editor further comprises a uracil glycosylase inhibitor (UGI) domain, and in some embodiments, the positive cytidine base editor is BE3 or BE4. In some embodiments, the cytidine base editor generates at least 20%, 30%, 50%, 70%, or 90% fewer spurious deamination reactions as measured by a cis / trans deamination assay compared to a true cytidine base editor. 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 compared to a true cytidine base editor. In some embodiments, the cytidine base editor has at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, or more fold less trans activity than a true cytidine base editor. In some embodiments, the cytidine deaminase is (a) APOBEC-1 from golden hamster (MaAPOBEC-1), Bornean orangutan (PpAPOBEC-1), sycamore rabbit (OcAPOBEC-1), gray short-tailed opossum (MdAPOBEC-1), or American alligator (AmAPOBEC-1), (b) APOBEC-2 from Bornean orangutan (PpAPOBEC-2), cattle (BtAPOBEC-2), or European pig (SsAPOBEC-2). , (c) APOBEC-4 from cynomolgus macaque (MfAPOBEC-4), (d) AID from dog (ClAID) or cattle (BtAID), (e) yeast cytosine deaminase (yCD) from Saccharomyces cerevisiae, (f) APOBEC-3F from golden snub-nosed monkey (RrA3F), 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)-(f).

[0084] In some embodiments, the cytidine deaminase is AID from dog (ClAID), cattle (BtAID), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned protein. In some embodiments, the cytidine deaminase is APOBEC-3F from golden snub-nosed monkey (RrA3F), or a cytidine deaminase having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned protein.

[0085] In some embodiments, 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 (as numbered in SEQ ID NO: 1), or corresponding alterations thereof, wherein X is any amino acid. In some embodiments, 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 corresponding alterations thereof.

[0086] In some embodiments, the cytidine deaminase comprises a combination of alterations 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 corresponding combinations of combinations of said alterations.

[0087] In some embodiments, the cytidine deaminase comprises an alteration at position Y120F and one or more alterations selected from the group consisting of R33A, W90F, K34A, R52A, H122A, and H121A (as numbered in SEQ ID NO: 1), or corresponding alterations of one or more of said alterations. In some embodiments, the cytidine deaminase comprises an alteration at position Y130X or R28X (as numbered in SEQ ID NO: 1), or corresponding alterations of said alterations, wherein X is any amino acid. In some embodiments, the cytidine deaminase comprises an alteration at position Y130A or an alteration at R28A (as numbered in SEQ ID NO: 1), or corresponding alterations of said alterations. In some embodiments, the cytidine deaminase comprises an alteration at position Y130A and an alteration at R28A (as numbered in SEQ ID NO: 1), or corresponding alterations of said alterations.

[0088] In some embodiments, the cytidine deaminase comprises alterations at positions H122X, K34X, R33X, W90X, and R128X (as numbered in SEQ ID NO: 1), or corresponding alterations thereof, wherein X is any amino acid. In some embodiments, the cytidine deaminase comprises alterations selected from the group consisting of H122A, K34A, R33A, W90F, W90A, and R128A (as numbered in SEQ ID NO: 1), or corresponding alterations thereof. In some embodiments, the cytidine deaminase comprises a combination of alterations 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 corresponding alterations thereof.

[0089] In some embodiments, the cytidine deaminase comprises an amino acid sequence that is at least 80% identical to the following amino acid sequence:

[0090] MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVAR LFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR.

[0091] In some embodiments, the cytidine deaminase comprises an amino acid sequence that is at least 80% identical to the following amino acid sequence:

[0092] MKPQIRDHRPNMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEIIKQYLPVPWKKGVFRNQVDPETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTIYTARLYYFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREILQ.

[0093] In some embodiments, the cytidine deaminase comprises an amino acid sequence that is at least 80% identical to the following amino acid sequence:

[0094] MADSSEKMRGQYISRDTFEKNYKPIDGTKEAHLLCEIKWGKYGKPWLHWCQNQRMNIHAEDYFMNNIFKAKKHPVHCYVTWYLSWSPCADCASKIVKFLEERPYLKLTIYVAQLYYHTEEENRKGLRLLRSKKVIIRVMDISDYNYCWKVFVSNQNGNEDYWPLQFDPWVKENYSRLLDIFWESKCRSPNPW.

[0095] In some embodiments, the cytidine deaminase comprises an amino acid sequence that is at least 80% identical to the following amino acid sequence:

[0096] MDPQRLRQWPGPGPASRGGYGQRPRIRNPEEWFHELSPRTFSFHFRNLRRFASGRNRSYICCQVEGKNCFFQGIFQNQVPPDPPCHAELCFLSWFQSWGLSPDEHYYVTWFISWSPCCECAAKVAQFLEENRNVSLSLSAA RLYYFWKSESREGLRRLSDLGAQVGIMSFQDFQHCWNNFVHNLGMPFQPWKKLHKNYQRLVTELKQILREEPATYGSPQAQGKVRIGSTAAGLRHSHSHTRSEAHLRPNHSSRQHRILNPPREARARTCVLVDASWICYR.

[0097] In some embodiments, the cytidine deaminase comprises an H122A alteration. 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.

[0098] In one aspect, provided herein is a cytidine deaminase comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of:

[0099] MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVAR LFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR;

[0100] MKPQIRDHRPNMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEIIKQYLPVPWKKGVFRNQVDPETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTIYTARLYYFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREILQ;

[0101] MADSSEKMRGQYISRDTFEKNYKPIDGTKEAHLLCEIKWGKYGKPWLHWCQNQRMNIHAEDYFM and

[0102] MDPQRLRQWPGPGPASRGGYGQRPRIRNPEEWFHELSPRTFSFHFRNLRRFASGRNRSYICCQVEGKNCFFQGIFQNQVPPDPPCHAELCFLSWFQSWGLSPDEHYYVTWFISWSPCCECAAKVAQFLEENRNVSLSLSAA RLYYFWKSESREGLRRLSDLGAQVGIMSFQDFQHCWNNFVHNLGMPFQPWKKLHKNYQRLVTELKQILREEPATYGSPQAQGKVRIGSTAAGLRHSHSHTRSEAHLRPNHSSRQHRILNPPREARARTCVLVDASWICYR.

[0103] The description and examples herein illustrate the embodiments disclosed herein in detail. It should be understood that the disclosure is not limited to the specific embodiments described herein and therefore can vary. Those skilled in the art will recognize the existence of many variations and modifications disclosed herein, which are encompassed by the scope disclosed herein.

[0104] Unless otherwise indicated, the practice of some embodiments disclosed herein uses conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, Fourth Edition (2012); Contemporary Protocols Series - Molecular Biology (FM Ausubel et al., eds.); Enzymology Methods Series (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)); Antibodies, a Laboratory Manual, edited by Harlow and Lane (1988); and Animal Cell Culture: A Manual of Basic Techniques and Professional Applications, Sixth Edition (RI Freshney, ed. (2010)).

[0105] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0106] Although various features of the present disclosure may be described in the context of a single embodiment, these features may also be provided individually or in any suitable combination. Conversely, although the present disclosure may be described herein in the context of a single embodiment 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.

[0107] The features of the present disclosure are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description which lists exemplary embodiments in which the principles of the present disclosure are applied, and in view of the accompanying drawings as described later herein.

[0108] definition

[0109] The following definitions supplement existing definitions in the art and are directed to the current application without imputing blame on any related or unrelated cases, such as any commonly owned patents or applications. Although any methods and materials similar or equivalent to those described herein can be used to test the practice of the present disclosure, preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0110] Unless defined otherwise, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art to which the present invention belongs. The following references provide the common definitions of many terms used in the present invention to the technician: " Microbiology and Molecular Biology Dictionary (1994 Second Edition)" by Singleton et al.; " Cambridge Scientific and Technical Dictionary" (Walker compiles, 1988); " Genetics Vocabulary (Fifth Edition)" (R. Rieger et al. (compiles), Springer-Verlag (1991)); and " HarperCollins Biology Dictionary (1991)" by Hale and Marham.

[0111] In this application, unless otherwise specifically stated, the use of the singular includes the plural. It must be noted that, as used in this specification, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" and is understood to be inclusive, unless otherwise stated. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is not limiting.

[0112] As used in this specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of containing, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with any method or composition disclosed herein, and vice versa. In addition, the compositions disclosed herein can be used to implement the methods disclosed herein.

[0113] The term "about" or "approximately" means within an acceptable error range (as determined by one of ordinary skill in the art) for a particular value, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" can mean within 1 standard deviation or more than 1 standard deviation. Alternatively, "about" can mean a range within 20%, within 10%, within 5%, or within 1% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within an order of magnitude, such as within 5 times or within 2 times the value. When describing a particular value in the application and claims, unless otherwise indicated, it should be assumed that the term "about" means within an acceptable error range for the particular value.

[0114] 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0115] References in the specification to "some embodiments," "one embodiment," "one embodiment," or "other embodiments" mean that the particular features, structures, or characteristics described in conjunction with the embodiments are included in at least some of the embodiments disclosed herein, but not necessarily all of the embodiments disclosed herein.

[0116] "A base-free base editor" means an agent that can excise a nucleobase and insert a DNA nucleobase (A, T, C, or G). A base-free base editor includes a nucleic acid glycosylase polypeptide or a fragment thereof. In one embodiment, the nucleic acid glycosylase is a mutant of human uracil DNA glycosylase, comprising Asp at amino acid 204 (or the corresponding position in uracil DNA glycosylase) in the following sequence (e.g., replacing Asn at amino acid 204), and having cytosine-DNA glycosylase activity or an active fragment thereof. In one embodiment, the nucleic acid glycosylase is a mutant of human uracil DNA glycosylase, comprising Ala, Gly, Cys, or Ser (e.g., replacing Tyr at amino acid 147) at amino acid 147 (or the corresponding position in uracil DNA glycosylase) in the following sequence, and having thymine-DNA glycosylase activity, or an active fragment thereof. The sequence of an exemplary human uracil-DNA glycosylase, isoform 1, is as follows:

[0117]

[0118] The sequence of human uracil-DNA glycosylase, isoform 2, is as follows:

[0119]

[0120] In other embodiments, the base-free (base) editor is any one of the base-free (base) editors described in PCT / JP2015 / 080958 and US20170321210 (which are incorporated herein by reference). In a specific embodiment, the base-free (base) editor includes a mutation, which is located at a position that is bold and underlined in the above sequence, or at a corresponding amino acid in any other base-free (base) editor or uracil deglycosylase known in the art. In one embodiment, the base-free (base) editor includes mutations at Y147, N204, L272, and / or R276, or corresponding positions. In another embodiment, the base-free (base) editor includes a Y147A or Y147G mutation, or a corresponding mutation. In another embodiment, the base-free (base) editor includes an N204D mutation, or a corresponding mutation. In another embodiment, the base-free editor comprises an L272A mutation, or a corresponding mutation. In another embodiment, the base-free editor comprises an R276E or R276C mutation, or a corresponding mutation.

[0121] "Adenosine deaminase" means a polypeptide or fragment thereof that can catalyze the hydrolytic deamination reaction of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination reaction of adenosine to inosine or deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination reaction of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) can be from any organism, such as bacteria.

[0122] In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is a TadA variant. In some embodiments, the TadA variant is TadA*7.10. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to a naturally occurring deaminase. For example, deaminase domains are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), the entire contents of each of which are incorporated herein by reference. See also Komor, AC et al. “Programmable editing of target bases 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 incorporation of phage Mu Gam protein generate C:G-to-T:A base editors with higher efficiency and higher product purity,” Science Advances 3: eaao4774 (2017), and Rees, HA et al. “Base editing: precision chemistry on the genome and transcriptome of living cells,” Nat Rev Genet. 2018 Dec; 19(12): 770-788. doi: 10.1038 / s41576-018-0059-1, the entire contents of which are hereby incorporated by reference.

[0123] In some embodiments, the adenosine deaminase comprises an alteration in the following sequence:

[0124] MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG LHDPTAHAEIMALRQGGLVM QNYRLIDATL YVTFEPCVMC AGAMIHSRIG RVVFGVRNAK TGAAGSLMDV LHYPGMNHRVEITEGILADE CAALLCYFFR MPRQVFNAQK KAQSSTD

[0125] (also known as TadA*7.10).

[0126] In a specific embodiment, the adenosine deaminase heterodimer comprises a TadA*7.10 domain and an adenosine deaminase domain selected from one of the following:

[0127] Staphylococcus aureus (S. aureus) TadA:

[0128] MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN

[0129] Bacillus subtilis TadA:

[0130] MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE

[0131] Salmonella typhimurium TadA:

[0132] MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV

[0133] Shewanella putrefaciens TadA:

[0134] MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE

[0135] Haemophilus influenzae F3031 TadA:

[0136] MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTΑΗAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK

[0137] Caulobacter crescentus TadA:

[0138] MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI

[0139] Geobacter sulfurreducens TadA:

[0140] MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP

[0141] TadA*7.10

[0142] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0143] "Administration" herein refers to providing one or more compositions described herein to a patient or subject. For example, but not limited to, administration of the composition, such as injection, can be performed by intravenous (iv) injection, subcutaneous (sc) injection, intradermal (id) injection, intraperitoneal (ip) injection, or intramuscular (im) injection. One or more of the above routes can be used. Parenteral administration can be, for example, by bolus injection or time-dependent progressive infusion. In some embodiments, parenteral administration comprises intravascular, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, intradermal, intraperitoneal, transtracheal, subcutaneous, transdermal, intraarticular, subcapsular, subarachnoid, and intrasternal infusion or injection. Alternatively, or simultaneously, administration can be by oral route.

[0144] "Agent" means any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0145] "Alteration" means a change (e.g., an increase or decrease) in the structure, expression level, or activity of a gene or polypeptide as detected by standard methods known in the art, such as those described herein. As used herein, an alteration comprises a change in the sequence of a polynucleotide or polypeptide or a change in expression level, such as a 10% change, a 25% change, a 40% change, a 50% or greater change.

[0146] "Ameliorate" means to reduce, inhibit, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

[0147] "Analog" means a molecule that is not identical but has similar functional or structural characteristics. For example, a polynucleotide analog or polypeptide analog retains the biological activity of the corresponding naturally occurring polynucleotide or polypeptide while having modifications that enhance the analog's function relative to the naturally occurring polynucleotide or polypeptide. Such modifications may increase the analog's affinity for DNA, potency, specificity, protease or nuclease resistance, membrane permeability, and / or half-life without altering, for example, ligand binding. Analogs may contain non-natural nucleotides or amino acids.

[0148] "Base editor (BE)" or "nucleobase editor (NBE)" means an agent that binds to a polynucleotide and has nucleobase modification activity. In several embodiments, the base editor includes a nucleobase modification polypeptide (e.g., a deaminase) and a nucleic acid-programmable nucleotide binding domain, which is connected to a guide polynucleotide (e.g., a guide RNA). In several embodiments, the agent is a biomolecular complex comprising a protein domain with base editing activity, i.e., a domain capable of modifying bases (e.g., A, T, C, G, or U) in a nucleic acid molecule (e.g., DNA). In some embodiments, the polynucleotide-programmable DNA binding domain is fused to or linked to one or more deaminase domains. In one embodiment, the agent is a fusion protein comprising one or more domains with base editing activity. In another embodiment, the protein domain with base editing activity is linked to the aforementioned guide RNA (e.g., via an RNA binding motif on the guide RNA and an RNA binding domain fused to the deaminase). In some embodiments, the domain with base editing activity is capable of deaminating bases in nucleic acid molecules. In some embodiments, the base editor is capable of deaminating 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) (eg, 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, which includes a naturally occurring protein domain having base editing activity and / or programmable DNA binding activity. For example, a standard cytidine base editor may contain a cytidine deaminase, such as an APOBEC cytidine deaminase or an AID deaminase. In some embodiments, the standard cytidine deaminase contains an APOBEC1 cytidine deaminase, such as rAPOBEC1. In some embodiments, the standard cytidine base editor further comprises an additional domain associated or linked to the cytidine deaminase, for example, one or more UGI domains that may be linked to the cytidine deaminase. In some embodiments, the base editor is an adenosine base editor (ABE) and a cytidine base editor (CBE).

[0149] In some embodiments, the base editor is a nuclease-inactivated Cas9 (dCas9) fused to an adenosine deaminase and / or a cytidine deaminase. In some embodiments, the Cas9 is a circular permutation array Cas9 (e.g., spCas9 or saCas9). Circular permutation array Cas9 is known in the art and is described, for example, in Oakes et al., Cell 176, 254–267, 2019. In some embodiments, the base editor is fused to an inhibitor of base excision repair, for example, a UGI domain or a dISN domain. In some embodiments, the fusion protein comprises a Cas9 nickase fused to one or more deaminases and an inhibitor of base excision repair, such as a UGI or dISN domain). In other embodiments, the base editor is a base-free base editor.

[0150] In some embodiments, an adenosine base editor is generated by cloning an adenosine deaminase variant into a scaffold comprising a circular permutation array Cas9 (e.g., spCAS9 or saCAS9) and a bipartite nuclear localization sequence. Circular permutation array Cas9 is known in the art and is described in, for example, Oakes et al., Cell 176, 254–267, 2019. Exemplary circular permutations are as follows, where bold sequences represent sequences derived from Cas9, italic sequences represent linker sequences, and underlined sequences represent bipartite nuclear localization sequences.

[0151] CP5 (with MSP "NGC = Pam variant with mutation, normal Cas9 prefers NGG" PID = protein interaction domain and "D10A" nickase):

[0152]

[0153]

[0154] 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 an inhibitor of base excision repair (BER). In some embodiments, the inhibitor of base excision repair is a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of base excision repair is an inosine base excision repair inhibitor.

[0155] Details of base editors are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), the entire contents of each of which are incorporated herein by reference. See also Komor, AC et al., “Programmable editing of target bases 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 incorporating bacteriophage Mu Gam protein generates C:G-to-T:A base editors with higher efficiency and higher product purity,” Science Advances 3:eaao4774 (2017), and Rees, HA et al., “Base editing: precision chemistry on the genome and transcriptome of living cells,” Nat Rev Genet. 2018 Dec; 19(12): 770-788. doi: 10.1038 / s41576-018-0059-1, the entire contents of which are hereby incorporated by reference.

[0156] For example, the adenine base editor (ABE) used in the base editing compositions, systems, and methods described herein has a nucleic acid sequence (8877 base pairs) as provided below (Addgene, Watertown, MA.; Gaudelli NM et al., Nature. 2017 Nov 23; 551(7681): 464-471. doi: 10.1038 / nature24644; Koblan LW et al., Nat Biotechnol. 2018 Oct; 36(9): 843-846. doi: 10.1038 / nbt.4172.). Polynucleotide sequences having at least 95% or higher identity to the ABE nucleic acid sequence are also encompassed.

[0157]

[0158] For example, the cytidine base editor (CBE) used in the base editing compositions, systems, and methods described herein has the following nucleic acid sequence (8877 base pairs) as 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 encompassed.

[0159] 1 ATATGCCAAG TACGCCCCCT ATTGACGTCA ATGACGGTAA ATGGCCCGCC TGGCATTATG

[0160] 61 CCCAGTACAT GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA TTAGTCATCG

[0161] 121 CTATTACCAT GGTGATGCGG TTTTGGCAGT ACATCAATGG GCGTGGATAG CGGTTTGACT

[0162] 181 CACGGGGATT TCCAAGTCTC CACCCCATTG ACGTCAATGG GAGTTTGTTT TGGCACCAAA

[0163] 241 ATCAACGGGA CTTTCCAAAA TGTCGTAACA ACTCCGCCCC ATTGACGCAA ATGGGCGGTA

[0164] 301 GGCGTGTACG GTGGGAGGTC TATATAAGCA GAGCTGGTTT AGTGAACCGT CAGATCCGCT

[0165] 361 AGAGATCCGC GGCCGCTAAT ACGACTCACT ATAGGGAGAG CCCGCCACCAT GAGCTCAGAG

[0166] 421 ACTGGCCCAG TGGCTGTGGA CCCCACATTG AGACGGCGGA TCGAGCCCCA TGAGTTTGAG

[0167] 481 GTATTCTTCG ATCCGAGAGA GCTCCGCAAG GAGACCTGCC TGCTTTACGA AATTAATTGG

[0168] 541 GGGGGCCGGC ACTCCATTTG GCGACATACA TCACAGAACA CTAACAAGCA CGTCGAAGTC

[0169] 601 AACTTCATCG AGAAGTTCAC GACAGAAAGA TATTTCTGTC CGAACACAAG GTGCAGCATT

[0170] 661 ACCTGGTTTC TCAGCTGGAG CCCATGCGGC GAATGTAGTA GGGCCATCAC TGAATTCCTG

[0171] 721 TCAAGGTATC CCCACGTCAC TCTGTTTATT TACATCGCAA GGCTGTACCA CCACGCTGAC

[0172] 781 CCCCGCAATC GACAAGGCCT GCGGGATTTG ATCTCTTCAG GTGTGACTAT CCAAATTATG

[0173] 841 ACTGAGCAGG AGTCAGGATA CTGCTGGAGA AACTTTGTGA ATTATAGCCC GAGTAATGAA

[0174] 901 GCCCACTGGC CTAGGTATCC CCATCTGTGG GTACGACTGT ACGTTCTTGA ACTGTACTGC

[0175] 961 ATCATACTGG GCCTGCCTCC TTGTCTCAAC ATTCTGAGAA GGAAGCAGCC ACAGCTGACA

[0176] 1021 TTCTTTACCA TCGCTCTTCA GTCTTGTCAT TACCAGCGAC TGCCCCCACACATCTCTGG

[0177] 1081 GCCACCGGGT TGAAATCTGG TGGTTCTTCT GGTGGTTCTA GCGGCAGCGAGACTCCCGGG

[0178] 1141 ACCTCAGGT CCGCCACACC CGAAAGTTCT GGTGGTTCTT CTGGTGGTTCTGATAAAAAAG

[0179] 1201 TATTCTATTG GTTTAGCCAT CGGCACTAAT TCCGTTGGAT GGGCTGTCATAACCGATGAA

[0180] 1261 TACAAAGTAC CTTCAAAGAA ATTTAAGGTG TTGGGGAACA CAGACCGTCATTCGATTAAA

[0181] 1321 AAGAATCTTA TCGGTGCCCT CCTATTCGAT AGTGGCGAAA CGGCAGAGGCGACTCGCCTG

[0182] 1381 AAACGAACCG CTCGGAGAAG GTATACACGT CGCAAGAACC GAATATGTTACTTACAAGAA

[0183] 1441 ATTTTTAGCA ATGAGATGGC CAAAGTTGAC GATTCTTTCT TTCACCGTTTGGAAGAGTCC

[0184] 1501 TTCCTTGTCG AAGAGGACAA GAAACATGAA CGGCACCCCA TCTTTGGAAAACATAGTAGAT

[0185] 1561 GAGGTGGCAT ATCATGAAAA GTACCCAACG ATTTATCACC TCAGAAAAAAGCTAGTTGAC

[0186] 1621 TCAACTGATA AAGCGGACCT GAGGTTAATC TACTTGGCTC TTGCCCATATGATAAAGTTC

[0187] 1681 CGTGGGCACT TTCTCATTGA GGGTGATCTA AATCCGGACA ACTCGGATGTCGACAAACTG

[0188] 1741 TTCATCCAGT TAGCAAAAC CTATATCAG TTGTTTGAAG AGAACCCTATAAAATGCAAGT

[0189] 1801 GGCGTGGATG CGAAGGCTAT TCTTAGCGCC CGCCTCTCTA AATCCCGACGGCTAGAAAAC

[0190] 1861 CTGATCGCAC AATTACCCGG AGAGAAGAAA AATGGGTTGT TCGGTAACCTTATAGCGCTC

[0191] 1921 TCACTAGGCC TGACACCAAA TTTTAAGTCG AACTTCGACT TAGCTGAAGATGCCAAATTG

[0192] 1981 CAGCTTAGTA AGGACACGTA CGATGACGAT CTCGACAATC TACTGGCACAAATTGGAGAT

[0193] 2041 CAGTATGCGG ACTTATTTTT GGCTGCCAAA AACCTTAGCG ATGCAATCCTCCTATCTGAC

[0194] 2101 ATACTGAGAG TTAATACTGA GATTACCAAG GCGCCGTTAT CCGCTTCAATGATCAAAAAGG

[0195] 2161 TACGATGAAC ATCACCAAGA CTTGACACTT CTCAAGGCCC TAGTCCGTCAGCAACTGCCT

[0196] 2221 GAGAAATATA AGGAAATATT CTTTGATCAG TCGAAAAACG GGTACGCAGGTTATTGAC

[0197] 2281 GGCGGAGCGA GTCAAGAGGA ATTCTACAAG TTTATCAAAC CCATATTAGAGAAGATGGAT

[0198] 2341 GGGACGGAAG AGTTGCTTGT AAAACTCAAT CGCGAAGATC TACTGCGAAAGCAGCGGACT

[0199] 2401 TTCGACAACG GTAGCATTCC ACATCAAATC CACTTAGGCG AATTGCATGCTATACTTAGA

[0200] 2461 AGGCAGGAGG ATTTTTATCC GTTCCTCAAA GACAATCGTG AAAAGATTGAGAAAATCCTA

[0201] 2521 ACCTTTCGCA TACCTTACTA TGTGGGACCC CTGGCCCGAG GGAACTCTCGGTTCGCATGG

[0202] 2581 ATGACAAGAA AGTCCGAAGA AACGATTACT CCATGGAATT TTGAGAAGTTGTCGATAAA

[0203] 2641 GGTGCGTCAG CTCAATCGTT CATCGAGAGG ATGACCAACT TTGACAAGAATTTACCGAAC

[0204] 2701 GAAAAAGTAT TGCCTAAGCA CAGTTTACTT TACGAGTATT TCACAGTGTACAATGAACTC

[0205] 2761 ACGAAAGTTA ATTATGTCAC TGAGGGCATG CGTAAACCCG CCTTTCTAAGCGGAGAACAG

[0206] 2821 AAGAAAGCAA OWNERCT GTTATTCAAG ACCAACCGCA AAGTGACAGTTAAGCAATTG

[0207] 2881 AAAGAGGACT ACTTTAAGAA AATTGAATGC TTCGATTCTG TCGAGATCTCCGGGGTAGAA

[0208] 2941 GATCGATTTA ATGCGTCACT TGGTACGTAT CATGACCTCC TAAAGATAATTAAAGATAAG

[0209] 3001 GACTTCCTGG ATAACGAAGA GAATGAAGAT ATCTTAGAAG ATATAGTGTTGACTCTTACC

[0210] 3061 CTCTTTGAAG ATCGGGAAAT GATTGAGGAA AGACTAAAA CATACGCTCACCTGTTCGAC

[0211] 3121 GATAAGGTTA TGAAACAGTT AAAGAGGCGT CGCTATACGG GCTGGGGACGATTGTCGCGG

[0212] 3181 AAACTATCA ACGGGATAAG AGACAAGCAA AGTGGTAAAA CTATTCTCGATTTTCTAAAG

[0213] 3241 AGCGACGGCT TCGCCAATAG GAACTTTATG CAGCTGATCC ATGATGACTCTTTAACCTTC

[0214] 3301 AAAGAGGATA TACAAAAGGC ACAGGTTTCC GGACAAGGGG ACTCATTGCACGAACATATT

[0215] 3361 GCGAATCTTG CTGGTTCGCC AGCCATCAAA AAGGGCATAC TCCAGACAGTCAAAGTAGTG

[0216] 3421 GATGAGCTAG TTAAGGTCAT GGGACGTCAC AAACCGGAAA ACATTGTAATCGAGATGGCA

[0217] 3481 CGCGAAAATC AAACGACTCA GAAGGGGCAA AAAAACAGTC GAGAGCGGATGAAGAGAATA

[0218] 3541 GAAGAGGGTA TTAAAGAACT GGGCAGCCAG ATCTTAAAGG AGCATCCTGTGGAAAATACC

[0219] 3601 CAATTGCAGA ACGAGAAACT TTACCTCTAT TACCTACAAA ATGGAAGGGACATGTATGTT

[0220] 3661 GATCAGGAAC TGGACATAAA CCGTTTATCT GATTACGACG TCGATCACATTGTACCCCAA

[0221] 3721 TCCTTTTTGA AGGACGATTC AATCGACAAT AAAGTGCTTA CACGCTCGGATAAGAACCGA

[0222] 3781 GGGAAAAGTG ACAATGTTCC AAGCGAGGAA GTCGTAAAGA AAATGAAGAACTATTGGCGG

[0223] 3841 CAGCTCCTAA ATGCGAAACT GATAACGCAA AGAAAGTTCG ATAACTTAACTAAAGCTGAG

[0224] 3901 AGGGGTGGCT TGTCTGAACT TGACAAGGCC GGATTTATTA AACGTCAGCTCGTGGAAACC

[0225] 3961 CGCCAAATCA CAAAGCATGT TGCACAGATA CTAGATTCCC GAATGAATACGAAATACGAC

[0226] 4021 GAGAACGATA AGCTGATTCG GGAAGTCAAA GTAATCACTT TAAAGTCAAAATTGGTGTCG

[0227] 4081 GACTTCAGAA AGGATTTTCA ATTCTATAAA GTTAGGGAGA TAAATAACTACCACCATGCG

[0228] 4141 CACGACGCTT ATCTTAATGC CGTCGTAGGG ACCGCACTCA TTAAGAAATACCCGAAGCTA

[0229] 4201 GAAAGTGAGT TTGTGTATGG TGATTACAAA GTTTATGACG TCCGTAAGATGATCGCGAAA

[0230] 4261 AGCGAACAGG AGATAGGCAA GGCTACAGCC AAATACTTCT TTTATTCTAACATTATGAAT

[0231] 4321 TTCTTTAAGA CGGAAATCAC TCTGGCAAAC GGAGAGATAC GCAAACGACCTTTAATTGAA

[0232] 4381 ACCAATGGGG AGACAGGTGA AATCGTATGG GATAAGGGCC GGGACTTCGCGACGGTGAGA

[0233] 4441 AAAGTTTTGT CCATGCCCCA AGTCAACATA GTAAAGAAAA CTGAGGTGCAGACCGGAGGG

[0234] 4501 TTTTCAAAGG AATCGATTCT TCCAAAAAGG AATAGTGATA AGCTCATCGCTCGTAAAAAG

[0235] 4561 GACTGGGACC CGAAAAAGTA CGGTGGCTTC GATAGCCCTA CAGTTGCCTATTCTGTCCTA

[0236] 4621 GTAGTGGCAA AAGTTGAGAA GGGAAAATCC AAGAAACTGA AGTCAGTCAAAGAATTATTG

[0237] 4681 GGGTAACGA TTATGGAGCG CTCGTCTTTT GAAAAGAACC CCATCGACTTCCTTGAGGCG

[0238] 4741 AAAGGTTACA AGGAAGTAAA AAAGGATCTC ATAATTAAAC TACCAAAGTATAGTCTGTTT

[0239] 4801 GAGTTAGAAA ATGGCCGAAA ACGGATGTTG GCTAGCGCCG GAGAGCTTCAAAAGGGGAAC

[0240] 4861 GAACTCGCAC TACCGTCTAA TACCGTGAAT TTCCTGTATT TAGCGTCCCATTACGAGAAG

[0241] 4921 TTGAAAGGTT CACCTGAAGA TAACGAACAG AAGCAACTTT TTGTTGAGCAGCACAAACAT

[0242] 4981 TATCTCGACG AAATCATAGA GCAAATTTCG GAATTCAGTA AGAGAGTCATCCTAGCTGAT

[0243] 5041 GCCAATCTGG ACAAAGTATT AAGCGCATAC AACAAGCACA GGTAAACCCATACGTGAG

[0244] 5101 CAGGCGGAAA ATTACK TTTGTTTACT CTTACCAACC TCGGCGCTCCAGCCGCATTC

[0245] 5161 AAGTATTTTG ACACAACGAT AGATCGCAAA CGATACACTT CTACCAAGGAGGTGCTAGAC

[0246] 5221 GCGACACTGA TTCACCAATC CATCACGGGA TTATGAAA CTCGGATAGATTTGTCACAG

[0247] 5281 CTTGGGGGTG ACTCTGGTGG TTCTGGAGGA TCTGGTGGTT CTACTAATCTGTCAGATATT

[0248] 5341 ATTGAAAAGG AGACCGGTAA GCAACTGGTT ATCCAGGAAT CCATCCTCATGCTCCCAGAG

[0249] 5401 GAGGTGGAAG AAGTCATTGG GAACAAGCCG GAAAGCGATA TACTCGTGCACACCGCCTAC

[0250] 5461 GACGAGAGCA CCGACGAGAA TGTCATGCTT CTGACTAGCG ACGCCCCTGAATACAAGCCT

[0251] 5521 TGGGCTCTGG TCATACAGGA TAGCAACGGT GAGAACAAGA TTAAGATGCTCTCTGGTGGT

[0252] 5581 TCTGGAGGAT CTGGTGGTTC TACTAATCTG TCAGATATTA TTGAAAAGGAGACCGGTAAG

[0253] 5641 CAACTGGTTA TCCAGGAATC CATCCTCATG CTCCCAGAGG AGGTGGAAGAAGTCATTGGG

[0254] 5701 AACAAGCCGG AAAGCGATAT ACTCGTGCAC ACCGCCTACG ACGAGAGCACCGACGAGAAT

[0255] 5761 GTCATGCTTC TGACTAGCGA CGCCCCTGAA TACAAGCCTT GGGCTCTGGTCATACAGGAT

[0256] 5821 AGCAACGGTG AGAACAAGAT TAAGATGCTC TCTGGTGGTT CTCCCAAGAAGAAGAGGAAA

[0257] 5881 GTCTAACCGG TCATCATCAC CATCACCATT GAGTTTAAAC CCGCTGATCAGCCTCGACTG

[0258] 5941 TGCCTTCTAG TTGCCAGCCA TCTGTTGTTT GCCCCTCCCC CGTGCCTTCCTTGACCCTGG

[0259] 6001 AAGGTGCCAC TCCCACTGTC CTTTCCTAAT AAAATGAGGA AATTGCATCGCATTGTCTGA

[0260] 6061 GTAGGTGTCA TTCTATTCTG GGGGGTGGGG TGGGGCAGGA CAGCAAGGGGGAGGATTGGG

[0261] 6121 AAGACAATAG CAGGCATGCT GGGGATGCGG TGGGCTCTAT GGCTTCTGAGGCGGAAAGAA

[0262] 6181 CCAGCTGGGG CTCGATACCG TCGACCTCTA GCTAGAGCTT GGCGTAATCATGGTCATAGC

[0263] 6241 TGTTTCCTGT GTGAAATTGT TATCCGCTCA CAATTCCACA CAACATACGAGCCGGAAGCA

[0264] 6301 TAAAGTGTAA AGCCTAGGGT GCCTAATGAG TGAGCTAACT CACATTAATTGCGTTGCGCT

[0265] 6361 CACTGCCCGC TTTCCAGTCG GGAAACCTGT CGTGCCAGCT GCATTAATGAATCGGCCAAC

[0266] 6421 GCGCGGGGAG AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTCACTGACTCGC

[0267] 6481 TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCGGTAATACGGT

[0268] 6541 TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGCCAGCAAAAGG

[0269] 6601 CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGCCCCCCTGACG

[0270] 6661 AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGACTATAAAGAT

[0271] 6721 ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACCCTGCCGCTTA

[0272] 6781 CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCATAGCTCACGCT

[0273] 6841 GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTGCACGAACCCC

[0274] 6901 CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCCAACCCGGTAA

[0275] 6961 GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGAGCGAGGTATG

[0276] 7021 TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACTAGAAGAACAG

[0277] 7081 TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTTGGTAGCTCTT

[0278] 7141 GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAGCAGCAGATTA

[0279] 7201 CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGGTCTGACGCTC

[0280] 7261 AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAAAGGATCTTCA

[0281] 7321 CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATATATGAGTAAA

[0282] 7381 CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCGATCTGTCTAT

[0283] 7441 TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATACGGGAGGGCT

[0284] 7501 TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCGGCTCCAGATT

[0285] 7561 TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCTGCAACTTTAT

[0286] 7621 CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGTTCGCCAGTTA

[0287] 7681 ATAGTTTGCG CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGCTCGTCGTTTG

[0288] 7741 GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGATCCCCCATGT

[0289] 7801 TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGTAAGTTGGCCG

[0290] 7861 CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTCATGCCATCCG

[0291] 7921 TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAATAGTGTATGC

[0292] 7981 GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCACATAGCAGAA

[0293] 8041 CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCAAGGATCTTAC

[0294] 8101 CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCTTCAGCATCTT

[0295] 8161 TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCCGCAAAAAAGG

[0296] 8221 GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAATATTATTGAA

[0297] 8281 GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATTTAGAAAAATA

[0298] 8341 AACAAATAGGGGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTCGACGGATCGG

[0299] 8401 GAGATCGATC TCCCGATCCC CTAGGGTCGA CTCTCAGTAC AATCTGCTCTGATGCCGCAT

[0300] 8461 AGTTAAGCCA GTATCTGCTC CCTGCTTGTG TGTTGGAGGT CGCTGAGTAGTGCGCGAGCA

[0301] 8521 AAATTTAAGC TACAACAAGG CAAGGCTTGA CCGACAATTG CATGAAGAATCTGCTTAGGG

[0302] 8581 TTAGGCGTTT TGCGCTGCTT CGCGATGTAC GGGCCAGATA TACGCGTTGACATTGATTAT

[0303] 8641 TGACTAGTTA TTAATAGTAA TCAATTACGG GGTCATTAGT TCATAGCCCATATATGGAGT

[0304] 8701 TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG ACCGCCCAACGACCCCCGCC

[0305] 8761 CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACTTTCCATTGAC

[0306] 8821 GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATC

[0307] In some embodiments, the cytidine base editor is BE4, which has a nucleic acid sequence selected from one of the following:

[0308] Original BE4 nucleic acid sequence:

[0309]

[0310]

[0311]

[0312] BE4 codon optimized 1 nucleic acid sequence:

[0313]

[0314]

[0315]

[0316] BE4 codon-optimized 2 nucleic acid sequence:

[0317] ATGAGCAGCGAGACAGGCCCTGTGGCTGTGGATCCTACACTGCGGAGAAGAATCGAGCCCCACGAGTTCGAGGTGTTCTTCGACCCCAGAGAGCTGCGGAAAGAGACATGCCTGCTGTACGAGATCAACTGGGGCGGCAGACACTCTATCTGGCGGCACACAAGCCAGAACACCAACAAGCACGTGGAAGTGAACTTTATCGAGAAGTTTACGACCGAGCGGTACTTCTGCCCCAACACCAGATGCAGCATCACCTGGTTTCTGAGCTGGTCCCCTTGCGGCGAGTGCAGCAGAGCCATCACCGAGTTTCTGTCCAGATATCCCCACGTGACCCTGTTCATCTATATCGCCCGGCTGTACCACCACGCCGATCCTAGAAATAGACAGGGACTGCGCGACCTGATCAGCAGCGGAGTGACCATCCAGATCATGACCGAGCAAGAGAGCGGCTACTGCTGGCGGAACTTCGTGAACTACAGCCCCAGCAACGAAGCCCACTGGCCTAGATATCCTCACCTGTGGGTCCGACTGTACGTGCTGGAACTGTACTGCATCATCCTGGGCCTGCCTCCATGCCTGAACATCCTGAGAAGAAAGCAGCCTCAGCTGACCTTCTTCACAATCGCCCTGCAGAGCTGCCACTACCAGAGACTGCCTCCACACATCCTGTGGGCCACCGGACTTAAGAGCGGAGGATCTAGCGGCGGCTCTAGCGGATCTGAGACACCTGGCACAAGCGAGT

[0318]

[0319] "Base editing activity" means the action of chemically changing a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, for example, converting the target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, for example, converting A·T to G·C. In another embodiment, the base editing activity is cytidine deaminase activity (for example, converting the target C·G to T·A) and adenosine or adenine deaminase activity (for example, converting A·T to G·C).

[0320] The term "base editor system" refers to a system for editing a nucleobase of a target nucleotide sequence. In several 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 a nucleobase in a target nucleotide sequence; and (2) one or more guide polynucleotides (e.g., guide RNA) connected to 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 a base-free (base) editor.

[0321] In some embodiments, a base editor system may include more than one base editing component. 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.

[0322] The deaminase domain and the polynucleotide programmable nucleotide binding component of the base editor system can be covalently or non-covalently associated with each other, or associated with each other by any combination of their association 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 some embodiments, a polynucleotide programmable nucleotide binding domain can be fused to 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-covalent interaction with or association with a deaminase domain. For example, in some embodiments, the deaminase domain can include additional heterologous portions or domains that can interact, associate, or form a complex with an additional heterologous portion or domain that is part of a polynucleotide programmable nucleotide binding domain. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous moiety may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous moiety may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous moiety may be capable of binding to a polynucleotide linker. The additional heterologous moiety may be a protein domain. In some embodiments, the additional heterologous moiety may be a Kappa homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, the telomerase Ku binding motif and Ku protein, the telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0323] The base editor system may further include a guide polynucleotide component. It should be understood that the components of the base editor system can be associated with each other via covalent bonds, non-covalent interactions, or any combination of their association and interaction. In some embodiments, 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 portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding domain) that can interact, associate, or form a complex with a portion or segment (e.g., a polynucleotide motif) of a guide polynucleotide. In some embodiments, the additional heterologous portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) can be fused to or linked to the deaminase domain. In some embodiments, the additional heterologous portion may be able to bind to, interact with, associate with, or form a complex with a polypeptide. In some embodiments, the additional heterologous portion may be able to bind to, interact with, associate with, or form a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be able to bind to a guide polynucleotide. In some embodiments, the additional heterologous moiety may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous moiety may be capable of binding to a polynucleotide linker. The additional heterologous moiety may be a protein domain. In some embodiments, the additional heterologous moiety may be a Kappa homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0324] In some embodiments, the base editor system may further include an inhibitor of a base excision repair (BER) component. It should be understood that the components of the base editor system can be associated with each other via covalent bonds, non-covalent interactions, or any combination of their association and interaction. The inhibitor of the BER component may include a BER inhibitor. In some embodiments, the inhibitor of BER can be a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of BER can be an inosine BER inhibitor. In some embodiments, the inhibitor of BER can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused to or linked to an inhibitor of BER. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused to or linked to one or more deaminase domains and an inhibitor of BER. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the inhibitor of BER to a target nucleotide sequence by non-covalent interaction or association with an inhibitor of BER. For example, in some embodiments, the inhibitor of a BER component can include an additional heterologous moiety or domain that is capable of interacting, associating, or forming a complex with an additional heterologous moiety or domain that is part of a polynucleotide-programmable nucleotide binding domain.

[0325] In some embodiments, the inhibitor of BER can be targeted to a target nucleotide sequence via a guide polynucleotide. For example, in some embodiments, the inhibitor of BER can include an additional heterologous portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding domain) that is capable of interacting, associating, or forming a complex with a portion or segment of a guide polynucleotide (e.g., a polynucleotide motif). In some embodiments, an additional heterologous portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding domain) of a guide polynucleotide can be fused to or linked to the inhibitor of BER. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterility alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0326] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease comprising a Cas9 protein or a fragment thereof (e.g., a protein comprising an active, inactivated, or partially active DNA-cleaving domain of Cas9, and / or a gRNA-binding domain of Cas9). Cas9 nucleases are sometimes also referred to as Casn1 nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)-associated nucleases. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). The CRISPR cluster contains spacer sequences, i.e., sequences that are complementary to the previous mobile element and target invading nucleic acids. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, the correct processing of pre-crRNA requires trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. The tracrRNA serves as a guide for the processing of ribonuclease 3-assisted pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA cuts the linear or circular dsDNA target complementary to the aforementioned spacer sequence in a manner of endonucleolytic cleavage. The target strand that is not complementary to the crRNA is first cut in a manner of endonucleolytic cleavage and then trimmed 3′-5′ in a manner of exonucleolytic cleavage. In nature, DNA-binding and cleavage usually require proteins and two types of RNA. However, single guide RNAs ("sgRNA", or "gNRAs" for short) can be engineered to incorporate all aspects of both crRNA and tracrRNA into a single RNA species. See, for example, Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are hereby incorporated herein by reference. Cas9 recognizes short motifs in CRISPR repeat sequences (i.e., PAM or protospacer adjacent motifs) to help distinguish self from non-self.The Cas9 nuclease sequence and structure are well known to those skilled in the art (see, for example, "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E. et al., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M. et al., Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference). Heterologs of Cas9 have been described in several species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on the present disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0327] An exemplary Cas9 is Streptococcus pyogenes Cas9 (spCas9), the amino acid sequence of which is provided below:

[0328]

[0329] (Single underline: HNH domain; double underline: RuvC domain)

[0330] Nuclease-inactivated Cas9 proteins are interchangeably referred to as "dCas9" proteins (for nuclease-"dead" Cas9) or catalytically inactivated Cas9. Methods for generating Cas9 proteins (or fragments thereof) with inactivated DNA cleavage domains are known (see, for example, Jinek et al., Science. 337: 816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152 (5): 1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to comprise two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cuts the strand complementary to the gRNA, while the RuvC1 subdomain cuts the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337:816-821 (2012); Qi et al., Cell. 28; 152(5):1173-83 (2013)). In some embodiments, the Cas9 nuclease has an inactivated (e.g., inactivated) DNA cleavage domain, that is, the Cas9 is a nickase, referred to as a "nCas9" protein (from "nickase" Cas9). In some embodiments, a protein comprising a fragment of Cas9 is provided. For example, in some embodiments, the protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, a protein comprising Cas9 or a fragment thereof is referred to as a "Cas9 variant". Cas9 variants share homology with Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9.In some embodiments, the Cas9 variant 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-cleaving domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding wild-type Cas9 fragment. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of the corresponding wild-type Cas9.

[0331] In some embodiments, the length of the fragment is at least 100 amino acids. In some embodiments, the length of the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids.

[0332] 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 below).

[0333]

[0334]

[0335]

[0336]

[0337] (Single underline: HNH domain; double underline: RuvC domain)

[0338] In some embodiments, the wild-type Cas9 corresponds to or includes the following nucleotide and / or amino acid sequence:

[0339]

[0340]

[0341]

[0342]

[0343] (Single underline: HNH domain; double underline: RuvC domain)

[0344] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI reference sequence: NC_002737.2) (nucleotide sequence below); and Uniprot reference sequence: Q99ZW2 (amino acid sequence below).

[0345]

[0346]

[0347] (Single underline: HNH domain; double underline: RuvC domain)

[0348] In some embodiments, Cas9 refers to Cas9 from the following: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheriae (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma aphididae (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanensis (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Campylobacter contortus I (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: NC_018721.1). 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.

[0349] In some embodiments, the dCas9 corresponds to or includes a portion or all of a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain includes D10A and H840A mutations, or corresponding mutations in another Cas9. In some embodiments, the dCas9 includes the amino acid sequence of dCas9 (D10A and H840A):

[0350]

[0351]

[0352] (Single underline: HNH domain; double underline: RuvC domain).

[0353] In some embodiments, the Cas9 domain includes a D10A mutation, and 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.

[0354] In other embodiments, dCas9 variants are provided having mutations other than D10A and H840A, which, for example, result in nuclease-inactivated Cas9 (dCas9). Such mutations, for example, include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions within the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs of dCas9 are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants of dCas9 are provided that have an amino acid sequence that is shorter or longer by about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or more.

[0355] In some embodiments, the Cas9 fusion protein as provided herein includes the full-length amino acid sequence of the Cas9 protein, for example, one of the Cas9 sequences provided herein. However, in other embodiments, the fusion protein as provided herein does not include the full-length Cas9 sequence, but only includes one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and the sequences of additional suitable Cas9 domains and fragments will be apparent to those skilled in the art.

[0356] It will be understood that additional Cas9 proteins (e.g., nuclease-inactivated Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including variants and homologs thereof, are within the scope of the present disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is a nuclease-inactivated Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease-active Cas9.

[0357] Exemplary catalytically inactive Cas9 (dCas9):

[0358]

[0359] Exemplary catalytic Cas9 nickase (nCas9):

[0360]

[0361] Exemplary catalytically active Cas9:

[0362]

[0363] In some embodiments, Cas9 refers to Cas9 from an archaea (e.g., nanoarchaea), which constitute a domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, Cas refers to CasX or CasY, which have been 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 hereby incorporated by reference. Using genome-resolved metagenomics, several CRISPR-Cas systems have been identified, including Cas9, which was first reported in the Archaeal domain of life. This distinct Cas9 protein was discovered as part of an active CRISPR-Cas system in the little-studied genus 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 refers to CasX or a variant of CasX. In some embodiments, Cas9 refers to CasY or a variant of CasY. It should be understood that other RNA-guided DNA binding proteins may be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0364] In a specific embodiment, the napDNAbps useful in the methods of the present invention comprise circular permutation arrays, which are known in the art and described, for example, by Oakes et al., Cell 176, 254–267, 2019. Exemplary circular permutation arrays are as follows, where bold sequences represent sequences derived from Cas9, italic sequences represent linker sequences, and underlined sequences represent bipartite nuclear localization sequences.

[0365] CP5 (with MSP "NGC = Pam variant with mutation, normal Cas9 prefers NGG" PID = protein interaction domain and "D10A" nickase):

[0366]

[0367]

[0368] Non-limiting examples of polynucleotide-programmable nucleotide-binding domains that can be incorporated into base editors include CRISPR protein-derived domains, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs).

[0369] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can be 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 amino acid sequence included in the napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp is a naturally occurring CasX or CasY protein. In some embodiments, the amino acid sequence comprised by the napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any CasX or CasY protein described herein. It should be understood that Cas12b / C2c1, CasX, and CasY from other bacterial species can also be used according to the present disclosure.

[0370] Cas12b / C2c1(uniprot.org / uniprot / T0D7A2#2)

[0371] 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

[0372]

[0373] CasX

[0374] (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

[0375] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0376] >tr|F0NH53|F0NH53_SULIR CRISPR-associated protein, Casx OS=Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4 SV=1 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

[0377] CasX of Deltaproteobacteria

[0378] MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEK

[0379] RNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQKWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0380] CasY (ncbi.nlm.nih.gov / protein / APG80656.1) > APG80656.1 CRISPR - associated protein CasY [uncultured Parcubacteria bacterium]

[0381]

[0382] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid having a common property. A functional way to define the 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)). Based on this analysis, groups of amino acids can be defined in which the amino acids within the group are preferentially interchanged, and therefore their effects on the overall protein structure are also most similar to each other (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include amino acid replacements of amino acids, such as arginine for lysine and vice versa, so that a positive charge is retained; glutamic acid for aspartic acid and vice versa, so that a negative charge is retained; threonine for serine, so that free –OH is retained; and glutamine for asparagine, so that free –NH2 is retained.

[0383] 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 by a start codon near the 5' end and a stop codon near the 3' end. A coding sequence may also be referred to as an open reading frame.

[0384] "Cytidine deaminase" means a polypeptide or fragment thereof that is capable of catalyzing a deamination reaction that converts an amino group to a carbonyl group. In one embodiment, the cytidine deaminase converts cytosine to uracil, or converts 5-methylcytosine to thymine. The cytidine deaminase provided herein (e.g., an engineered cytidine deaminase, an evolved cytidine deaminase) can be from any organism, such as bacteria.

[0385] In some embodiments, the cytidine deaminase of the base editor can include all or part of a deaminase of the apolipoprotein B mRNA editing complex (APOBEC) family. APOBEC is a family of evolutionarily conserved cytidine deaminases. Members of this family are C-to-U editing enzymes. In some embodiments, the cytidine deaminase includes, but is not limited to, a member of the APOBEC family, including, but not limited to, APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D ("APOBEC3E" now refers to this), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, activation-induced (cytidine) deaminase (AID), hAPOBEC1, derived from Homo sapiens, rAPOBEC1, derived from brown rat / Norway rat (Rattus norvegicus), ppAPOBEC1, derived from Bornean orangutan (Pongo pygmaeus), AmAPOBEC1 (BEM3.31), derived from American alligator (Alligator mississippiensis), ocAPOBEC1, derived from Oryctolagus cuniculus, SsAPOBEC2 (BEM3.39), derived from European pig (Sus scrofa), hAPOBEC3A, which is derived from Homo sapiens, maAPOBEC1, which is derived from Mesocricetus auratus, mdAPOBEC1, which is derived from Monodelphis domestica; cytidine deaminase 1 (CDA1), hA3A, which is APOBEC3A derived from Homo sapiens, RrA3F (BEM3.14), which is APOBEC3F derived from Rhinopithecus roxellana; PmCDA1, which is derived from Petromyzon marinus (sea lamprey cytosine deaminase 1, "PmCDA1"); AID (activation-induced cytidine deaminase; AICDA), which is derived from mammals (e.g., humans, pigs, cattle, horses, monkeys, etc.); hAID, which is derived from Homo sapiens; and FENRY.

[0386] The term "deaminase" or "deaminase domain", as used herein, 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 reaction of cytidine to uridine or deoxycytidine to deoxyuridine. In some embodiments, the deaminase or deaminase domain is a cytosine deaminase, which catalyzes the hydrolytic deamination reaction of cytosine to uracil. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination reaction of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination reaction of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase, which catalyzes the hydrolytic deamination reaction of adenosine to inosine or deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination reaction of adenosine in deoxyribonucleic acid (DNA). The deaminases provided herein (e.g., engineered adenosine deaminases, evolved adenosine deaminases) can be from any organism, such as bacteria. In some embodiments, the deaminases are from bacteria, such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Caulobacter crescentus.

[0387] "Detection" refers to identifying the presence, absence, or amount of an analyte to be detected. In one embodiment, sequence changes in a polynucleotide or polypeptide are detected. In another embodiment, the presence of an insertion / deletion is detected.

[0388] "Detectable label" means a composition that, when linked to a target molecule, enables the target molecule to be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in enzyme-linked immunosorbent assays (ELISAs)), biotin, digoxigenin, or haptens.

[0389] "Disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0390] The term "effective amount", as used herein, refers to the amount of a bioactive agent sufficient to cause a desired biological response. The effective amount of one (or several) active agents used to implement the present invention to treat a disease varies depending on the mode of administration, the age, weight, and overall health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. This amount is referred to as an "effective" amount. In one embodiment, an effective amount is an amount of a base editor of the present invention (e.g., a fusion protein comprising a programmable DNA binding protein, a nucleobase editor, and a gRNA) sufficient to introduce a change in a target gene in a cell (e.g., a cell in vivo or in vitro). In some embodiments, an effective amount of a fusion protein provided herein (e.g., a multi-effector nucleobase editor comprising an nCas9 domain and one or more deaminase domains (e.g., adenosine deaminase, cytidine deaminase)) may refer to an amount of the fusion protein sufficient to induce the editing process of a target site (which is specifically bound and edited by the multi-effector nucleobase editor). In one embodiment, an effective amount is the amount of base editor required to achieve a therapeutic effect (e.g., reducing or controlling a disease or its symptoms or condition). Such a therapeutic effect does not require sufficient to change the target gene in all cells of a subject, tissue, or organ, but only requires changing about 1%, 5%, 10%, 25%, 50%, 75% or more of the target gene in existing cells in the subject, tissue, or organ.

[0391] In some embodiments, an effective amount of a fusion protein provided herein (e.g., a nucleobase editor comprising an nCas9 domain and one or more deaminase domains (e.g., adenosine deaminase, cytidine deaminase)) refers to an amount of the fusion protein sufficient to induce editing of a target site that is specifically bound and edited by a multi-effector nucleobase editor as described herein. One skilled in the art will appreciate that the effective amount of an agent (e.g., a fusion protein, a nuclease, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a 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 being used.

[0392] "Fragment" means a portion of a polypeptide or nucleic acid molecule. This portion contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0393] "Guide RNA" or "gRNA" means a polynucleotide that can be specific for a target sequence and can form a complex with a polynucleotide-programmable nucleotide binding domain protein (e.g., Cas9 or Cpf1). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). The gRNA can exist as a complex with two or more RNAs or as a single RNA molecule. A gRNA that exists as a single RNA molecule can be referred to as a single-guide RNA (sgRNA), although "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or as a complex with two or more molecules. Typically, a gRNA that exists as a single RNA species includes two domains: (1) a domain that shares homology with the target nucleic acid (e.g., and guides the Cas9 complex to bind to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence called tracrRNA and comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in U.S. provisional patent application USSN 61 / 874,682, entitled “Switchable Cas9 Nucleases and Uses Thereof,” filed on September 6, 2013, and in U.S. provisional patent application USSN 61 / 874,746, entitled “Delivery System For Functional Nucleases,” filed on September 6, 2013, the entire contents of each of which are incorporated herein by reference. In some embodiments, a gRNA includes two or more domains (1) and (2) and can be referred to as an “extended gRNA.” As described herein, an extended gRNA will bind to two or more Cas9 proteins and bind to a target nucleic acid at two or more distinct regions. The gRNA includes a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site and provides sequence specificity of the nuclease:RNA complex.

[0394] "Hybridization" means hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0395] 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 some embodiments, the IBR is an inhibitor of inosine base excision repair. Exemplary inhibitors of base repair include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGG1, hNEIL1, T7 Endol, T4PDG, UDG, hSMUG1, and hAAG. In some embodiments, the IBR is an inhibitor of EndoV or hAAG. In some embodiments, the IBR is a catalytically inactivated EndoV or a catalytically inactivated hAAG. In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, the base repair inhibitor is a catalytically inactivated EndoV or a catalytically inactivated hAAG.

[0396] In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI refers to a protein that can inhibit the uracil-DNA glycosylase base-excision repair enzyme. In some embodiments, the UGI domain includes wild-type UGI or a fragment of wild-type UGI. In some embodiments, the UGI protein provided herein includes a fragment of UGI and a protein homologous to UGI or a UGI fragment. In some embodiments, the base repair inhibitor is an inhibitor of inosine base excision repair. In some embodiments, the base repair inhibitor is a "catalytically inactivated inosine-specific nuclease" or an "inactivated inosine-specific nuclease." Without wishing to be bound by any particular theory, catalytically inactivated inosine glycosylases (e.g., alkyladenine glycosylase (AAG)) can bind to inosine, but cannot create abasic sites or remove the inosine, thereby spatially blocking the newly formed inosine moiety and exempting it from the DNA damage / repair mechanism. In some embodiments, the catalytically inactivated inosine-specific nuclease can be capable of binding to inosine in nucleic acids but does not cut the nucleic acid. Non-limiting exemplary catalytically inactive inosine-specific nucleases include catalytically inactive alkyl adenine glycosylase (AAG nuclease), for example, from humans, and catalytically inactive endonuclease V (EndoV nuclease), for example, from Escherichia coli. In some embodiments, the catalytically inactive AAG nuclease comprises an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0397] "Increase" means a positive change of at least 10%, 25%, 50%, 75%, or 100%.

[0398] An "intein" is a fragment of a protein that is able to excise itself and join the remaining fragments (exteins) together with peptide bonds in a process called protein splicing. Inteins are also called "protein introns." The process by which an intein excises itself and joins the remaining parts of a protein is referred to herein as "protein splicing" or "intein-mediated protein splicing." In some embodiments, the inteins of a precursor protein (a protein containing an intein that precedes intein-mediated protein splicing) come 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 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."

[0399] Other intein systems may also be used. For example, synthetic inteins based on the dnaE intein, namely the Cfa-N (e.g., cleaved intein-N) and Cfa-C (e.g., cleaved intein-C) intein pair, have been described (e.g., in 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 in accordance with the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, and Cne Prp8 intein (e.g., as described in U.S. Pat. No. 8,394,604, incorporated herein by reference).

[0400] Exemplary nucleotide and amino acid sequences of inteins are provided.

[0401] DnaE intein-N DNA:

[0402] TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTCTGCCAATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCGATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGGGGAGAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAGGGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTTCCTAAT

[0403] DnaE intein - N protein:

[0404] CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN

[0405] DnaE intein - C DNA:

[0406] ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGATATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAGCTTCTAAT

[0407] Intein - C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN

[0408] Cfa - N DNA:

[0409] TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCCTATTGGAAAGATTGTCGAAGAGAGAATTGAATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGGCACAATCGCGGCGAACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACGAGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAATAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTGCCA

[0410] Cfa-N protein:

[0411] CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP

[0412] Cfa-C DNA:

[0413] ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAGGAAAGTAAAGATAATATCTCGAAAAAGTCTTGGTACCCAAAATGTCTATGATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTAGCCAGCAAC

[0414] Cfa-C protein:

[0415] MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN

[0416] Intein-N and intein-C can be fused to the N-terminal portion of the broken Cas9 and the C-terminal portion of the broken Cas9, respectively, to connect the N-terminal portion of the broken Cas9 and the C-terminal portion of the broken Cas9. For example, in some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of the broken Cas9, i.e., forming a structure of N-[N-terminal portion of the broken Cas9]-[intein-N]--C. In some embodiments, intein-C is fused to the N-terminus of the C-terminal portion of the broken Cas9, i.e., forming a structure of N-[intein-C]--[C-terminal portion of the broken Cas9]-C. The mechanism of intein-mediated protein splicing for connecting proteins to which intein peptides are fused (e.g., broken Cas9) is known in the art, for example, as described in Shah et al., Chem Sci. 2014; 5(1):446-461, incorporated herein by reference. Methods for designing and using inteins are known in the art and described, for example, in WO2014004336, WO2017132580, US20150344549, and US20180127780, the entire contents of each of which are incorporated herein by reference.

[0417] The terms "isolated," "purified," or "biologically pure" refer to varying degrees of separation of a material from components normally associated with it in its natural state. "Isolated" refers to a degree of separation from the original source or surrounding environment. "Purified" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. In other words, a nucleic acid or peptide of the invention is considered purified if it is produced by recombinant DNA techniques and is substantially free of cellular material, viral material, or culture media, or if it is chemically synthesized and is substantially free of chemical precursors or other chemicals. Purity and homogeneity are typically determined by analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can mean that a nucleic acid or protein produces essentially a single band in an electrophoretic gel. For proteins that can be modified, for example, for phosphorylation or glycosylation, different modifications may result in different isolated proteins, which can be purified separately.

[0418] "Isolated nucleic acid" means a nucleic acid (e.g., DNA) that is free of the genes that flank the nucleic acid in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. The term thus encompasses, for example, recombinant DNA incorporated into a vector; incorporated into an autonomously replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryotic or eukaryotic organism; or existing as a separate molecule (e.g., a cDNA or genomic or cDNA fragment generated by PCR or restriction endonuclease digestion) independent of other sequences. Additionally, the term encompasses RNA molecules transcribed from a DNA molecule, as well as recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0419] "Isolated polypeptide" means a polypeptide of the present invention that has been separated from components with which it is naturally associated. Generally, a polypeptide is considered isolated when it is at least 60% free, by weight, from the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, of the polypeptide of the present invention. Isolated polypeptides of the present invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding the polypeptide, or by chemically synthesizing the protein. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or by HPLC (high performance liquid chromatography) analysis.

[0420] The term "linker", as used herein, may refer to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group, or a molecule that links two molecules or moieties, for example, two components of a protein complex or a ribonucleic acid complex, or two domains of a fusion protein, such as, for example, a polynucleotide-programmable DNA binding domain (e.g., dCas9) and one or more deaminase domains (e.g., adenosine deaminase and / or cytidine deaminase). The linker can connect different components or different parts of a base editor system. For example, in some embodiments, the linker can connect the guide polynucleotide binding domain of the polynucleotide-programmable nucleotide binding domain and the catalytic domain of the deaminase. In some embodiments, the linker can connect the CRISPR polypeptide and the deaminase. In some embodiments, the linker can connect Cas9 and the deaminase. In some embodiments, the linker can connect dCas9 and the deaminase. In some embodiments, the linker can connect nCas9 and the deaminase. In some embodiments, the linker can connect the guide polynucleotide and the deaminase. In some embodiments, the linker can connect the deamination component of the base editor system and the polynucleotide programmable nucleotide binding component. In some embodiments, the linker can connect the RNA-binding portion of the deamination component of the base editor system and the polynucleotide programmable nucleotide binding component. In some embodiments, the linker can connect the RNA-binding portion of the deamination component of the base editor system and the RNA-binding portion of the polynucleotide programmable nucleotide binding component. The linker can be located between or on both sides of two groups, molecules or other moieties and is linked to each of the two via a covalent bond or non-covalent interaction, thereby linking the two together. In some embodiments, the linker can be an organic molecule, group, polymer or chemical moiety. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, the linker can include 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 include an aptamer that can be derived from a riboswitch. The riboswitch from which the aptamer is derived can be selected from the group consisting of a theophylline riboswitch, a thiamine pyrophosphate (TPP) riboswitch, an adenosylcobalamin riboswitch, an S-adenosylmethionine (SAM) riboswitch, a SAH riboswitch, a flavin mononucleotide (FMN) riboswitch, a tetrahydrofolate riboswitch, a lysine riboswitch, a glycine riboswitch, a purine riboswitch, a GlmS riboswitch, or a pre-queosine 1 (PreQ1) riboswitch. In some embodiments, a linker can include an aptamer that is bound to a polypeptide or protein domain, such as a polypeptide ligand.In some embodiments, the polypeptide ligand may 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 may be part of a base editor system component. For example, a nucleobase editing component may include one or more deaminase domains and an RNA recognition motif.

[0421] In some embodiments, the linker can be an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker can be a length of about 5-100 amino acids, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids in length. In some embodiments, the linker can be a length of about 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 amino acids in length. Longer or shorter linkers can also be expected.

[0422] In some embodiments, the linker connects the gRNA binding domain of an RNA-programmable nuclease (which comprises a Cas9 nuclease domain) and the catalytic domain of a nucleic acid editing protein (e.g., cytidine and / or adenosine deaminase). In some embodiments, the linker connects dCas9 and a nucleic acid editing protein. For example, the linker is located between or on both sides of two groups, molecules, or other moieties and is linked to each of the two via a covalent bond, thereby linking the two. In some embodiments, the linker is an amino acid or a plurality of 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, for example, 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.

[0423] In some embodiments, the domains of a nucleobase editor (e.g., a multi-effector nucleobase editor) are fused via a linker comprising the following amino acid sequence: SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS. In some embodiments, the domains of a nucleobase editor (e.g., a multi-effector nucleobase editor) are fused via a linker comprising the following amino acid sequence: SGSETPGTSESATPES, which may also be referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker comprises (SGGS) n 、(GGGS) n 、(GGGGS) n 、(G) n 、(EAAAK) n 、(GGS) n , SGSETPGTSESATPES, or (XP) n motif, or any combination thereof, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0424] In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS. In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0425] "Marker" means any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or condition.

[0426] The term "mutation", as used herein, refers to the replacement of a residue in a sequence (e.g., a nucleic acid or amino acid sequence) by another residue, or the deletion or insertion of one or more residues in the sequence. In this article, the description of the mutation is usually by first indicating the original residue, then indicating the position of the residue in the sequence, and then indicating the identity of the newly replaced residue. Various methods for producing amino acid substitutions (mutations) provided herein are well known in the art, such as provided by Green and Sambrook, "Molecular Cloning: A Laboratory Manual" (4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). In some embodiments, the base editor disclosed herein can effectively generate "expected mutations" (such as point mutations) in nucleic acids (e.g., nucleic acids in the genome of a subject) without generating a significant number of unexpected mutations (such as unexpected point mutations). In some embodiments, the expected mutation is a mutation generated by a specific base editor bound to a guide polynucleotide (e.g., gRNA), and the guide polynucleotide is specifically designed to produce the expected mutation.

[0427] Typically, mutations created or identified in a sequence (e.g., an amino acid sequence as described herein) are numbered relative to a reference (or wild-type) sequence (i.e., a sequence that does not contain the mutation). Those skilled in the art will readily understand how to determine the position of mutations in amino acid and nucleic acid sequences relative to a reference sequence.

[0428] The term "non-conservative mutation" refers to amino acid substitutions between different groups, for example, tryptophan for lysine, serine for phenylalanine, etc. 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.

[0429] The terms "nuclear localization sequence," "nuclear localization signal," or "NLS" refer to an amino acid sequence that facilitates the import of a protein into the cell nucleus. Nuclear localization sequences are known in the art and are described, for example, in Plank et al., International PCT Application No. PCT / EP2000 / 011690, filed November 23, 2000, and published as WO / 2001 / 038547 on May 31, 2001, for their disclosure of exemplary nuclear localization sequences, the contents of which are incorporated herein by reference. In other embodiments, the NLS is an optimized NLS, as described, for example, in 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.

[0430] The terms "nucleic acid" and "nucleic acid molecule", as used herein, refer to a compound comprising a core base and an acidic moiety, such as a polymer of a nucleoside, a nucleotide, or a nucleotide. Typically, a polymeric nucleic acid, such as a nucleic acid molecule comprising three or more nucleotides, is a linear molecule in which adjacent nucleotides are linked to each other via phosphodiester bonds. In some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., a nucleotide and / or a nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more single nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA and single-stranded and / or double-stranded DNA. Nucleic acids can be naturally occurring, such as in the following contexts: genomes, transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plasmids, cosmids, chromosomes, chromatids, or other naturally occurring nucleic acid molecules. On the other hand, nucleic acid molecules can be non-natural molecules, for example, recombinant DNA or RNA, artificial chromosomes, engineered genomes, or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids, or comprise non-natural nucleotides or nucleosides. In addition, terms " nucleic acid ", " DNA ", " RNA " and / or similar terms comprise nucleic acid analogs, for example, analogs with non-phosphodiester backbones. Nucleic acid can be purified from natural sources, produced using a recombinant expression system, and purified, chemically synthesized, etc., alternatively. In appropriate cases, for example, in the case of chemically synthesized molecules, nucleic acid can comprise nucleoside analogs, such as bases or sugars with chemical modification, and backbone modifications. Unless otherwise indicated, nucleotide sequences are presented in 5' to 3' directions. In some embodiments, the nucleic acid is or includes natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrole-pyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine); guanosine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated 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)

[0431] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" is used interchangeably with "polynucleotide programmable nucleotide binding domain" to refer to a protein associated with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid that directs the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. The Cas9 protein can be associated with a guide RNA that directs the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactivated 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 known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, type II Cas effector protein, type V Cas effector protein, type VI Cas effector protein, 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, although they may not be explicitly listed in this disclosure. See, for example, Makarova et al., "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR J. 2018 Oct; 1: 325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., "Functionally diverse type V CRISPR-Cas systems" Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, the entire contents of each of which are hereby incorporated by reference.

[0432] The terms "nucleobase," "nitrogenous base," or "base" are used interchangeably herein to refer to nitrogen-containing biological compounds that form nucleosides, which are components of nucleotides. The ability of nucleobases to form base pairs and stack with each other directly leads to long 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 the 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 bases may 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, both by deamination (substituting an amino 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 (either ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides with modified nucleobases include inosine (I), xanthine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). A "nucleotide" consists of a nucleobase, a five-carbon sugar (either ribose or deoxyribose), and at least one phosphate group.

[0433] The term "nucleobase editing domain" or "nucleobase editing protein", as used herein, refers to a protein or enzyme that can catalyze nucleobase modification reactions in RNA or DNA, such as the deamination reaction of cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine), and the deamination reaction of adenine (or adenosine) to hypoxanthine (or inosine), as well as non-templated nucleotide additions and insertions. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., an adenine deaminase or adenosine deaminase; or a cytidine deaminase or a cytosine deaminase). In some embodiments, the nucleobase editing domain is more than one deaminase domain (e.g., an adenine deaminase or adenosine deaminase and a cytidine or cytosine deaminase). In some embodiments, the nucleobase editing domain can be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase editing domain can be an engineered or evolved version of a naturally occurring nucleobase editing domain. The nucleobase editing domain can be from any organism, such as bacteria, humans, chimpanzees, gorillas, monkeys, cows, dogs, rats, or mice.

[0434] As used herein, "obtaining," as in "obtaining a dose," includes synthesizing, purchasing, or otherwise acquiring the dose.

[0435] "Patient" or "subject," as used herein, refers to a mammalian subject or individual who has been diagnosed with, is at risk of having or developing, or is suspected of having or developing a disease or condition. In some embodiments, the term "patient" refers to a mammalian subject who has an above-average likelihood of developing a disease or condition. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cows, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), and other mammals that can benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female.

[0436] A "patient in need thereof" or "subject in need thereof" as used herein refers to a patient diagnosed with, at risk of, or already suffering from, prone to, or suspected of having a disease or condition.

[0437] The terms "pathogenic mutation," "pathogenic variant," "disease-causing mutation," "pathogenic variant," "deleterious mutation," or "susceptibility mutation" refer to a genetic alteration or mutation that increases an individual's susceptibility or predisposition to a disease or condition. In some embodiments, the pathogenic mutation comprises a substitution of at least one pathogenic amino acid for at least one wild-type amino acid in a protein encoded by a gene.

[0438] 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 stearate or zinc stearate, or stearic acid), or a solvent encapsulating material that is involved in carrying or transporting the compound from one location in the body (e.g., delivery site) to another location (e.g., an organ, tissue, or body part of the human body). A pharmaceutically acceptable carrier is "acceptable" in the sense that it is compatible with the other ingredients of the formulation and is not harmful to the tissues of the subject (e.g., physiological compatibility, sterility, physiological pH, etc.). Terms such as "excipient," "carrier," "pharmaceutically acceptable carrier," "vehicle," etc. are used interchangeably herein.

[0439] The term "pharmaceutical composition" means a formulated composition for pharmaceutical use.

[0440] 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. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids in length. A protein, peptide, or polypeptide may refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for coupling, functionalization, or other modifications, and the like. A protein, peptide, or polypeptide may also be a single molecule or may be a multimolecular complex. A protein, peptide, or polypeptide may be simply a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. The term "fusion protein," as used herein, refers to a hybrid polypeptide comprising protein domains from at least two different proteins. A protein can be located at the amino-terminal (N-terminal) part or the carboxyl-terminal (C-terminal) of the fusion protein, thereby forming an amino-terminal fusion protein or a carboxyl-terminal fusion protein respectively. Protein can include different domains, for example, a nucleic acid binding domain (for example, the gRNA binding domain of Cas9, which guides the combination of the protein and the target site) and a nucleic acid cutting domain, or the catalytic domain of nucleic acid editing protein. In some embodiments, the protein includes a part of the protein, for example, an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, for example, a compound that can serve as a nucleic acid cutting agent. In some embodiments, the protein forms a complex or is associated with nucleic acid (for example, RNA or DNA). Any protein provided herein can be produced by methods known in the art. For example, the protein provided herein can be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins including peptide linkers. Methods of recombinant protein expression and purification are well known and include those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), the entire contents of which are incorporated herein by reference.

[0441] The polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) may include synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline- 2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. The 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 bonds, sulfation, myristoylation, palmitoylation, prenylation, farnesylation, geranylation, glypiation, lipoylation, and iodination.

[0442] The term "recombinant," as used herein in the context of 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 comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any naturally occurring sequence.

[0443] "Decrease" means a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0444] "Reference" means a standard or control condition. In one embodiment, the reference is a wild-type or healthy cell. In other embodiments and without limitation, the reference is an untreated cell that has not been subjected to the test condition, or has been subjected to a placebo or regular saline, culture medium, buffer, and / or a control vector without the polynucleotide of interest.

[0445] A "reference sequence" is a defined sequence used as a benchmark for sequence alignment. A reference sequence can be a subset or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides, or any integer approximately equal thereto or therebetween. In some embodiments, the reference sequence is the wild-type sequence of the protein of interest. In other embodiments, the reference sequence is a polynucleotide sequence encoding the wild-type protein.

[0446] The terms "RNA-programmable nuclease" and "RNA-guided nuclease" are used in conjunction with (e.g., bound to or associated with) one or more RNAs that are not cleavage targets. In some embodiments, when an RNA-programmable nuclease forms a complex 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).

[0447] In some embodiments, the RNA-programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 from Streptococcus pyogenes (Csnl) (see, e.g., “Complete genome sequence of an Mlstrain of Streptococcus pyogenes.” Ferretti JJ et al., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and hostfactor RNase III.” Deltcheva E. et al., Nature 471:602-607 (2011)).

[0448] Because RNA-programmable nucleases (such as Cas9) use RNA:DNA hybrids to target DNA cleavage sites, these proteins have the ability, in principle, to be targeted to any sequence specified by the guide RNA. Methods of using RNA-programmable nucleases (such as Cas9) for site-specific cleavage (e.g., to modify the genome) are known in the art (see, e.g., Cong, L. et al., Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, WY et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference).

[0449] The term "single nucleotide polymorphism (SNP)" refers to a variation in a single nucleotide at a specific location in the genome, where each variation is present in a population to a significant degree (e.g., >1%). For example, at a specific base position in the human genome, a C nucleotide may occur in most individuals, but in a minority of individuals, that position is occupied by an A. This means that a SNP exists at this specific position, and the two possible nucleotide variations, C or A, are considered alleles at that position. SNPs underlie differences in disease susceptibility. The severity of disease and how the body responds to treatment are also clinical manifestations of genetic variation. SNPs can fall within the coding region of a gene, the non-coding region of a gene, or be located in intergenic regions (the region between genes). In some embodiments, due to the degeneracy of the genetic code, SNPs within coding sequences do not necessarily change the amino acid sequence of the produced protein. There are two types of SNPs within coding regions: synonymous and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, while non-synonymous SNPs alter the protein's amino acid sequence. There are two types of non-synonymous SNPs: missense and nonsense. SNPs that are not in protein-coding regions may still affect gene splicing, transcription factor binding, messenger RNA degradation or the sequence of non-coding RNA. The gene expression affected by this type of SNP is called eSNP (expression SNP) and can be upstream or downstream of the gene. Single nucleotide variant (SNV) is the variation in a single nucleotide, which is not limited by frequency and can occur in somatic cells. Somatic single nucleotide variation can also be called single-nucleotide change.

[0450] "Specific binding" means 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 present invention, but does not substantially recognize and bind to other molecules in a sample (e.g., a biological sample).

[0451] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide or polypeptide fragment of the present invention. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide or polypeptide fragment of the present invention. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" means the pairing and formation of a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene as described herein) or portions thereof under various stringent conditions (see, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0452] For example, stringent salt concentration will generally be less than about 750mM sodium chloride and 75mM sodium citrate, preferably less than about 500mM sodium chloride and 50mM sodium citrate, more preferably less than about 250mM sodium chloride and 25mM sodium citrate. Low stringency hybridization can be obtained in the absence of an organic solvent (e.g., formamide), while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will generally comprise a temperature of at least about 30°C, more preferably at least about 37°C, most preferably at least about 42°C. Varying other parameters, such as the inclusion or exclusion of hybridization time, detergent (e.g., sodium dodecyl sulfate (SDS)) concentration, and carrier DNA, is well known to those skilled in the art. Various stringency levels can be achieved by merging these different conditions as needed. In one embodiment, hybridization will occur at 30°C, in 750mM sodium chloride, 75mM sodium citrate, and 1% SDS. In another embodiment, hybridization will occur in 500 mM sodium chloride, 50 mM sodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C. In another embodiment, hybridization will occur in 250 mM sodium chloride, 25 mM sodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations on these conditions will be apparent to those skilled in the art.

[0453] For most applications, the post-hybridization wash step will also vary in stringency. Wash stringency can be defined both by salt concentration and by temperature. As described above, wash stringency can be increased by lowering the salt concentration or by increasing the temperature. For example, the stringent salt concentration for the wash step will preferably be less than about 30 mM sodium chloride and 3 mM sodium citrate, and most preferably less than about 15 mM sodium chloride and 1.5 mM sodium citrate. Stringent temperature conditions for the wash step will generally include a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In one embodiment, the wash step will occur at 25°C in 30 mM sodium chloride, 3 mM sodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash step will occur at 42°C in 15 mM sodium chloride, 1.5 mM sodium citrate, and 0.1% SDS. In an even more preferred embodiment, the wash step will occur at 68°C in 15 mM sodium chloride, 1.5 mM sodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, by Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., U.S. 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.

[0454] "Fragmentation" means separation into two or more fragments.

[0455] "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. The polypeptides corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein can be spliced ​​to form a "reconstructed" Cas9 protein. In a specific embodiment, the Cas9 protein is divided into two fragments within the disordered region of the protein, for example, as described in Nishimasu et al., Cell, Vol. 156, No. 5, pp. 935-949, 2014, or as described in Jiang et al. (2016) Science 351: 867-871. PDB file: 5F9R, the entire contents of each of which are incorporated herein by reference. In some embodiments, the protein is split into two fragments at any C, T, A, or S within a region of SpCas9 between approximately amino acids A292-G364, F445-K483, or E565-T637, or at corresponding positions in any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other napDNAbp. In some embodiments, the protein is split into two fragments at T310, T313, A456, S469, or C574 of SpCas9. In some embodiments, the process of splitting a protein into two fragments is referred to as the "cleavage process" of the protein.

[0456] In other embodiments, the N-terminal portion of the Cas9 protein includes amino acids 1-573 or 1-637 of Streptococcus pyogenes Cas9 wild type (SpCas9) (NCBI Reference Sequence: NC_002737.2, Uniprot Reference Sequence: Q99ZW2) and the C-terminal portion of the Cas9 protein includes amino acids 574-1368 or a portion of 638-1368 of SpCas9 wild type.

[0457] The C-terminal portion of the split Cas9 can be connected to the N-terminal portion of the split Cas9 to form a complete Cas9 protein. In some embodiments, the C-terminal portion of the Cas9 protein starts where the N-terminal portion of the Cas9 protein ends. Therefore, in some embodiments, the C-terminal portion of the split Cas9 includes a portion of amino acids (551-651)-1368 of spCas9. "(551-651)-1368" means starting at an amino acid between amino acids 551-651 (inclusive) and ending at amino acid 1368.For example, the C-terminal portion of the split Cas9 may include a portion of any of the following amino acids: 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, or 573-1368 of spCas9. 8, 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 9-1368, 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 68, 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 includes a portion of amino acids 574-1368 or 638-1368 of SpCas9.

[0458] "Subject" means a mammal, including but not limited to a human or non-human mammal, such as a cow, horse, dog, sheep, or cat. Subjects include livestock, i.e., domesticated animals raised to produce labor and provide commodities (such as food), including but not limited to cattle, goats, chickens, horses, pigs, rabbits, and sheep.

[0459] "Substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). In one embodiment, such a sequence is at least 60%, 80%, 85%, 90%, 95%, or even 99% identical to the sequence used for comparison at the amino acid level or nucleic acid level.

[0460] Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). 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; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, where e -3 and e -100 Probability scores between and indicate closely related sequences.

[0461] For example, COBALT is used with the following parameters:

[0462] a) alignment parameters: Gap penalties -11, -1 and End - Gap penalties -5, -1,

[0463] b) CDD parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved columns and Recompute on, and

[0464] c) Query Clustering parameters: Use query clusters on; Word Size: 4; Max cluster distance: 0.8; Alphabet Regular.

[0465] For example, EMBOSS Needle is used with the following parameters:

[0466] a) Matrix: BLOSUM62;

[0467] b) GAP OPEN (slot open): 10;

[0468] c) GAP EXTEND (gap extension): 0.5;

[0469] d) OUTPUT FORMAT (output format): pair (pair);

[0470] e) END GAP PENALTY (end gap penalty): false;

[0471] f) END GAP OPEN (end gap open): 10; and

[0472] g) END GAP EXTEND (terminal gap extension): 0.5.

[0473] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a base editor. In one embodiment, the target site is deaminated by a deaminase or a fusion protein comprising a deaminase (e.g., a cytidine or adenine deaminase).

[0474] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disease and / or symptoms associated therewith or obtaining a desired pharmacological and / or physiological effect. It should be understood that, although not excluded, treatment of a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated. In some embodiments, the aforementioned effect is therapeutic, that is, but not limited to, the effect partially or completely reduces, reduces, suppresses, alleviates, alleviates, reduces the intensity of the disease and / or the harmful symptoms attributable to the disease, or cures the disease. In some embodiments, the effect is prophylactic, that is, the effect protects or prevents the occurrence or recurrence of a disease or condition. To this end, the methods disclosed herein comprise administering a therapeutically effective amount of a composition as described herein.

[0475] "Uracil glycosylase inhibitor" or "UGI" means an agent that inhibits the uracil-excision repair system. In one embodiment, the agent is a protein or fragment thereof that binds to the host uracil-DNA glycosylase and prevents uracil from being removed from DNA. In one embodiment, UGI is a protein, fragment or domain thereof that is capable of inhibiting the uracil-DNA glycosylase base-excision repair enzyme. In some embodiments, the UGI domain comprises a wild-type UGI or a modified version thereof. In some embodiments, the UGI domain comprises a fragment of the exemplary amino acid sequence listed below. In some embodiments, the amino acid sequence included in the UGI fragment includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the exemplary UGI sequence provided below. In some embodiments, UGI comprises an amino acid sequence homologous to the exemplary UGI amino acid sequence or a fragment thereof (as listed below). In some embodiments, the UGI, or a portion thereof, is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%, or 100% identical to wild-type UGI or a UGI sequence as listed below, or a portion thereof. Exemplary UGIs include the following amino acid sequences:

[0476] MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML.

[0477] The term "vector" refers to a means for introducing a nucleic acid sequence into a cell to transform the cell. Vectors include plasmids, transposons, phages, viruses, liposomes, and episomes. An "expression vector" is a nucleic acid sequence that contains a nucleotide sequence to be expressed in a recipient cell. An expression vector may contain additional nucleic acid sequences to enhance and / or promote expression of the introduced sequence, such as initiation, termination, enhancer, promoter, and secretion sequences.

[0478] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0479] DNA editing has emerged as a viable means of modulating disease states by correcting pathogenic mutations at the genetic level. Until recently, all DNA editing platforms functioned by inducing DNA double-strand breaks (DSBs) at specific genomic loci and relying on endogenous DNA repair pathways to determine product outcomes in a semi-stochastic manner, resulting in a complex population of gene products. While precise, user-defined repair outcomes can be achieved through the homology-directed repair (HDR) pathway, several challenges have hindered the efficient use of HDR for repair in therapeutically relevant cell types. In practice, this pathway is inefficient relative to its competitor, the error-prone non-homologous end-joining pathway. Furthermore, HDR is strictly restricted to the G1 and S phases of the cell cycle, hindering the precise repair of DSBs in post-mitotic cells. Consequently, efficiently altering genomic sequence in a user-defined, programmable manner in these populations has proven difficult or impossible. BRIEF DESCRIPTION OF THE DRAWINGS

[0480] Figures 1A-1C The cis-trans activity of the free deaminase is depicted. Figure 1A Schematic diagram depicting the experimental design for a cis-trans assay with SpCas9 and a deaminase, either in the form of a base editor complex or in an untethered form. Figure 1B The cis-trans activity of rAPOBEC is depicted. Figure 1C The cis-trans activities of TadA7.10 and TadA-TadA7.10 are depicted.

[0481] Figures 2A-2F Depicted are the cis-trans assay for base editors, a diagram of the deaminase similarity network, and a screen of 153 deaminases. Figure 2A Schematic diagram depicting the experimental design of the cis-trans assay. HEK293T cells were transfected with separate plasmids encoding SaCas9, gRNA for SaCas9, and target base editors. Figure 2BSchematic diagram depicting the similarity network of APOBEC-like deaminases. Each dot represents a cytidine deaminase that was screened for next-generation CBEs, and the core next-generation CBEs are indicated. The shading of the dot represents the average trans / cis ratio; the size of the dot represents the average cis activity. Figure 2B The method for the cytidine deaminase similarity network shown is as follows: In order to focus the search space within the APOBEC1-like protein family, human APOBEC1 was used as the query sequence for a protein BLAST search against the NCBI non-redundant protein sequence database (nr_v5). The first 1000 sequences were used to generate a sequence similarity network (SSN) with 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. In order to identify deaminases from other families that can act as base-editing enzymes, 80 sequences were sampled from the SSN constructed with all deaminases and the following InterPro annotations were used: IPR002125 (cytidine and deoxycytidylic acid deaminase domain), IPR016192 (APOBEC / CMP deaminase, zinc-binding) and IPR016193 (cytidine deaminase-like). The 82,043 sequences in this collection were first analyzed using Cd-HIT 3 Clustering at 55% identity was followed by generation of SSN networks by protein BLAST with a -log(E-value) edge threshold of 50. Sequences were selected based on their centrality within the cluster. Figure 2C is an S-diagram depicting the cis-trans activities of ppBE4 and its mutants. Figure 2D is a graph depicting the cis-trans activity of the selected editors. Separately, data on cis-trans-activity were generated based on cis / trans assays at three target sites, site 1, site 4, and site 6, as shown in Figure 2E and Figure 2F shown. Figure 2EA bar graph showing the cis- and trans-editing activities of the identified CBEs is presented. Shown is a comparison of the cis- and trans-editing frequencies of mammalian cells treated with candidate CBEs. Editors numbered 1-36 are 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, pYY The reported base editing efficiencies are for the most edited base in the target site. Figure 2FA bar graph is presented showing the cis- and trans-editing activities of the identified CBEs. Shown is a comparison of the cis- and trans-editing frequencies of mammalian cells treated with candidate CBEs. Editors numbered 1-37 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, hAPOBEC-3D, and hAPOBEC-3A. 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, pYY-BEM3.7. Base editing efficiencies reported are for the most edited base within the target site.

[0482] Figure 3A and 3B Cis-trans activity is depicted. Figure 3A is a graph depicting the cis-trans activity of ABE7.10. Figure 3B is a graph depicting the cis-trans activity of BE4max.

[0483] Figure 4A and 4B Depicted is the rAPOBEC1 homology model generated by SWISSMODEL using the hAPOBEC3C structure (PDB ID 3VM8). The ssDNA from the hAPOBEC3A structure (PDB ID 5SWW) was manually docked. Figure 4A is a schematic diagram depicting mutations with the potential to affect ssDNA binding. Figure 4B is a schematic diagram depicting mutations with the potential to affect catalytic activity.

[0484] Figures 5A-5C The cis-trans activities of rAPOBEC1 mutants are depicted.

[0485] Figures 6A-6E The cis-trans activities of rAPOBEC1 double mutants are depicted. Figure 6A is a graph depicting the cis and trans activities of rAPOBEC1 double mutants. Figure 6Bis a graph depicting cis-activity at six sites. Figure 6C is a diagram depicting cis / trans activity. Figure 6D is a graph depicting cis-activity at five sites. Figure 6E is a diagram depicting cis / trans activity.

[0486] Figure 7A and 7B The cis-trans activities of the deaminases in the first round of screening are depicted.

[0487] Figures 8A-8C is a graph depicting the on-target activity of ppAPOBEC1 relative to rAPOBEC1.

[0488] Figure 9 is a schematic diagram depicting the similarity network of APOBEC-like proteins.

[0489] Figure 10A and 10B are graphs depicting dose-dependency studies of cis-activity and trans-activity in TadA-TadA7.10 and rAPOBEC1, respectively.

[0490] Figure 11 Figure 2 is a graph depicting off-target editing of selected CBEs. SNVs were identified by exome sequencing.

[0491] Figure 12A and 12B are graphs depicting the quantification of base editor mRNA and protein, respectively, from HEK293T cells transfected with base editor plasmids.

[0492] Figure 13 Figure 2 is a diagram depicting targeted RNA sequencing of selected editors. Three 200-300 bp regions were sequenced.

[0493] Figure 14 is a diagram depicting directed off-target editing of selected CBEs.

[0494] Figures 15A-15E Depicts the editing window of the selected editor.

[0495] Figure 16 is a graph depicting the indel rates of selected CBEs at 10 target sites.

[0496] Figures 17A-17D Shown are pictorial diagrams and graphs related to non-guided ssDNA deamination reactions and cis / trans assays. Figure 17A The potential formation of ssDNA in the genome during transcription or translation is illustrated. Figure 17BThe experimental design of the cis / trans assay is described. HEK293T cells were transfected with separate constructs encoding SaCas9, gRNA for SaCas9, and base editors. Cis- and trans-activity at the target site (with the NGGRRT PAM sequence) was measured in different transfections. Figure 17C Cis / trans activity of BE4 with rAPOBEC1 is shown. Figure 17D ABE7.10 variants at 34 genomic sites are shown. The leftmost bar on each genomic site on the x-axis represents cis-on-target editing. The rightmost bar on each genomic site on the x-axis represents trans-editing. The reported base editing efficiency is for the most edited base in the target site. Values ​​and error bars reflect the mean and standard deviation (SD) of independent biological replicates.

[0497] Figure 18 The bar graph shows the identification of next-generation CBEs with high cis-activity and reduced trans-activity compared to BE4 with rAPOBEC1. Shown is a comparison of cis- and trans-editing frequencies at 10 genomic loci in mammalian cells treated with these 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 within the target site. Values ​​and error bars reflect the mean and standard deviation of four independent biological replicates.

[0498] Figures 19A-19E Shown are allele frequencies and graphs associated with next-generation CBEs with reduced off-target DNA and RNA editing in mammalian cells relative to BE4. Figure 19A Whole transcriptome sequencing and target RNA sequencing of Hek293T cells expressing cytosine base editors with minimized spurious deaminations are shown ( Figure 19B ). Figure 19C The percentage of C-to-T editing at known off-target sites is shown. Figure 19D The percentage of C-to-T editing in an in vitro enzymatic assay on a single-stranded DNA substrate is shown. Core next-generation CBEs were assayed for C-to-U editing on a ssDNA substrate. Each dot represents the number of N edited. C Native sequence context. The black line indicates the average editing efficiency of the target cytosine in the substrate. Figure 19E Presented are the time courses of product formation in in vitro enzymatic assays performed with cell lysates containing selected CBEs. Figure 19D and 19EThe oligonucleotide sequences used in the present invention are listed in the table below in Example 5. The values ​​and error bars reflect the results of three independent biological replicates ( Figure 19A , B, C) and two independent biological replicates ( Figure 19D , E) mean and standard deviation.

[0499] Figure 20 Diagrammatically depicts the Figure 4A and 4B Cis- and trans-editing activity of BE4 at target site 1 for the indicated rAPOBEC1 mutants. Base editing efficiencies are reported for the most edited base within the target site. Trans-editing efficiency is represented by the leftmost bar on the x-axis for each target site; cis-editing efficiency is represented by the rightmost bar on the x-axis for each target site. Values ​​and error bars reflect the means and standard deviations of independent biological replicates.

[0500] Figure 21 Depicted are the cis / trans editing activities of BE4-rAPOBEC1 harboring HiFi mutations at 10 target sites. Values ​​and error bars reflect the mean and standard deviation of four independent biological replicates.

[0501] Figure 22A and 22B Figures and sequence alignments related to the cis / trans editing activities and sequence alignments of the CBEs tested in the aforementioned first round of screening are shown. Figure 22A ) and sequence alignment ( Figure 22B The amino acid residues that mapped to the HiFi mutation in rAPOBEC1 are highlighted. Values ​​and error bars reflect the means and standard deviations of independent biological replicates.

[0502] Figure 23 Shown are the cis / trans activities of BE4-PpAPOBEC1 and BE4-PpAPOBEC with HiFi mutations at 10 target sites. Base editing efficiencies are reported for the most edited base within the target site. Values ​​and error bars reflect the mean and standard deviation of four independent biological replicates.

[0503] Figure 24 A heat map is shown, which shows Figure 18 Previous base preferences of the CBEs shown in . The values ​​used to generate this heat map reflect the average of four independent biological replicates.

[0504] Figure 25 The results of 10 target sites are presented. Figure 18Figure 4. Editing windows of the CBEs shown in Figure 4. Values ​​reflect the average of four independent biological replicates. Cis- and trans-editing are presented in the heatmaps in the left- and right-most panels, respectively.

[0505] Figure 26 A table showing the expression of Figure 18 Indel rates for the CBEs shown in . The values ​​used to generate this heat map reflect the average of four independent biological replicates.

[0506] Figures 27A-27D Homology models of four selected cytidine deaminases based on available crystal structures are depicted. Figure 27A : Homology model of PpAPOBEC1 based on the putative 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).

[0507] Figures 28A-28D Presented are diagrams illustrating guided off-target editing of selected next-generation CBEs. Figure 28A : Editing efficiency of next-generation CBEs at HEK2, HEK3, and HEK4 loci, and Figure 28B and 28C :The reported off-target sites of HEK2 sgRNA and HEK3 sgRNA and Figure 28D HEK4 sgRNA. Base editing efficiencies reported are based on the most edited base within the target site. Values ​​and error bars reflect the mean and standard deviation of three independent biological replicates.

[0508] Figure 29 The graphs presented show the C-to-T editing efficiency of selected CBEs on ssDNA substrates in an in vitro enzymatic assay. Editing efficiency was measured at all 25 cytidines on two DNA substrates and was expressed as N C Sequence context grouping. The sequences of the two substrates used are listed herein in Table 18. Values ​​and error bars reflect the mean and standard deviation of data from independent biological replicates.

[0509] Figure 30The presented graph shows the quantitative analysis of CBE protein concentration in HEK293T cells transfected with a base editor expression plasmid. Base editor protein concentration was quantified by measuring total Cas9 protein concentration and total protein amount in cell lysate. BE protein concentration was normalized to BE4-rAPOBEC1. Values ​​and error bars reflect the mean and standard deviation from two or more independent biological replicates.

[0510] Figure 31 The presented graph shows the pseudodeamination activity of CBEs examined by whole-genome sequencing (WGS). The relative mutation rates are shown as odds ratios. DETAILED DESCRIPTION

[0511] The present invention provides nucleobase editors and multi-effector nucleobase editors with improved editing settings (i.e., minimal off-target deamination reactions), compositions including such editors, and methods of using them to generate modifications in target nucleobase sequences.

[0512] Nucleobase editors

[0513] Disclosed herein are base editors or nuclear base editors or multi-effector nuclear base editors for editing, modifying or changing the target nucleotide sequence of a polynucleotide. A nuclear base editor or base editor or multi-effector nuclear base editor is described herein, comprising a polynucleotide programmable nucleotide binding domain (e.g., Cas9) and at least one nuclear base editing domain (e.g., adenosine deaminase and / or cytidine deaminase). A polynucleotide programmable nucleotide binding domain (e.g., Cas9), when connected to a bound guide polynucleotide (e.g., gRNA), can specifically bind to a target nucleotide sequence (i.e., via complementary base pairing between the bound guide nucleic acid and the target nucleotide sequence base), thereby positioning the base editor to the target nucleic acid sequence to be edited.

[0514] Polynucleotide-programmable nucleotide-binding domain

[0515] It should be understood that a polynucleotide programmable nucleotide binding domain can also comprise a nucleic acid programmable protein that binds to RNA. For example, the polynucleotide programmable nucleotide binding domain can be associated with a nucleic acid that directs the polynucleotide programmable nucleotide binding domain to RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, although they are not specifically listed in this disclosure.

[0516] The polynucleotide programmable nucleotide binding domain of the base editor itself may include one or more domains. For example, the polynucleotide programmable nucleotide binding domain may include one or more nuclease domains. In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain may include an endonuclease or an exonuclease. The term "exonuclease" in this article refers to a protein or polypeptide that can digest a nucleic acid (such as RNA or DNA) from the end, while the term "endonuclease" refers to a protein or polypeptide that can catalyze (such as cut) an internal region in a nucleic acid (such as DNA or RNA). In some embodiments, an endonuclease can cut a single strand of a double-stranded nucleic acid. In some embodiments, an endonuclease can cut two strands of a double-stranded nucleic acid molecule. In some embodiments, the polynucleotide programmable nucleotide binding domain may be a deoxyribonuclease. In some embodiments, the polynucleotide programmable nucleotide binding domain may be a ribonuclease.

[0517] In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain can cut zero, one or two strands of the target polynucleotide. In some embodiments, the polynucleotide programmable nucleotide binding domain can include a nickase domain. The term "nickase" refers to a polynucleotide programmable nucleotide binding domain including a nuclease domain in this article, which can only cut one of the two strands in a double-stranded helix nucleic acid molecule (such as DNA). In some embodiments, a nickase can be derived from a polynucleotide programmable nucleotide binding domain of a (such as natural) form of complete catalytic activity, which is a polynucleotide programmable nucleotide binding domain by introducing one or more mutations into an active polynucleotide programmable nucleotide binding domain. For example, when a polynucleotide programmable nucleotide binding domain includes a nickase domain derived from Cas9, the Cas9-derived nickase domain can include a D10A mutation and a histidine at position 840. In this embodiment, the residue H840 retains catalytic activity and can therefore cut a single strand of the nucleic acid double helix. In another example, the Cas9-derived nickase domain can include an H840A mutation, while the amino acid residue at position 10 remains D. In some embodiments, the nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide-programmable nucleotide binding domain by removing all or part of a nuclease domain that is not essential for nickase activity. For example, when the polynucleotide-programmable nucleotide binding domain includes a nickase domain derived from Cas9, the Cas9-derived nickase domain can include a deletion of all or part of a RuvC domain or a HNH domain.

[0518] The amino acid sequence of an exemplary catalytically active Cas9 is as follows:

[0519] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLNLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKIKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT

[0520] .

[0521] A base editor comprising a polynucleotide programmable nucleotide binding domain (which includes a nickase domain) is therefore capable of generating a single-stranded DNA break (nick) at a specific polynucleotide target sequence (e.g., determined by the complementary sequence of the bound guide nucleic acid). In some embodiments, the strand of the nucleic acid double-stranded helix target polynucleotide sequence that is cut by a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain) is the strand that is not edited by the base editor (i.e., the strand that is cut by the base editor is on the opposite side of the strand that includes the base to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain) can cut the strand of DNA that is to be targeted for editing. In such embodiments, the non-targeted strand is not cut.

[0522] Also provided herein is a base editor comprising a catalytically inactivated (i.e., a target polynucleotide sequence that cannot be cut) polynucleotide programmable nucleotide binding domain. The terms "catalytically inactivated" and "nuclease-inactivated" are used interchangeably herein to refer to a polynucleotide programmable nucleotide binding domain having one or more mutations and / or deletions that cause it to be unable to cut a nucleic acid strand. In some embodiments, a catalytically inactivated polynucleotide programmable nucleotide binding domain base editor may lack nuclease activity due to specific point mutations in one or more nuclease domains. For example, in the case of a base editor comprising a Cas9 domain, the Cas9 may include two mutations, i.e., a D10A mutation and an H840A mutation. Such mutations deactivate two nuclease domains, resulting in the loss of nuclease activity. In other embodiments, a catalytically inactivated polynucleotide programmable nucleotide binding domain may include one or more deletions of all or part of a catalytic domain (e.g., RuvC1 and / or HNH domain). In further embodiments, the catalytically inactive polynucleotide-programmable nucleotide binding domain comprises a point mutation (eg, D10A or H840A) and a deletion of all or part of the nuclease domain.

[0523] Mutations that can generate catalytically inactive polynucleotide programmable nucleotide binding domains from a previous functional version of the polynucleotide programmable nucleotide binding domain are also examined herein. For example, in the case of catalytically inactive Cas9 ("dCas9"), variants are provided having mutations other than D10A and H840A (which result in inactivated Cas9). Such mutations, for example, include other amino acid replacements at D10 and H840, or other replacements within the nuclease domain of Cas9 (e.g., replacements within the HNH nuclease subdomain and / or RuvC1 subdomain). Based on the present disclosure and knowledge in the art, additional suitable nuclease-inactivated dCas9 domains will be apparent to those skilled in the art and are within the scope of the present disclosure. Such additional exemplary suitable nuclease-inactivated 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).

[0524] Non-limiting examples of polynucleotide programmable nucleotide binding domains that can be incorporated into base editors include CRISPR protein-derived domains, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some embodiments, the base editor includes a polynucleotide programmable nucleotide binding domain, which includes a natural or modified protein or portion thereof, which protein or portion thereof can bind to a nucleic acid sequence during CRISPR (i.e., clustered regularly interspaced short palindromic repeats)-mediated nucleic acid modification via a bound guide nucleic acid. This protein is referred to herein as a "CRISPR protein." Therefore, disclosed herein is a base editor comprising a polynucleotide programmable nucleotide binding domain, which includes a polynucleotide programmable nucleotide binding domain that includes all or part of a CRISPR protein (i.e., a base editor comprising all or part of a CRISPR protein as a domain, the domain is also referred to as a "CRISPR protein-derived domain" of the base editor). The CRISPR protein-derived domain incorporated into the base editor can be modified compared to the wild-type or natural version of the CRISPR protein. For example, as described below, a CRISPR protein-derived domain can include one or more mutations, insertions, deletions, rearrangements, and / or recombinations relative to a wild-type or naturally occurring version of a CRISPR protein.

[0525] CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposons, and conjugative plasmids). The CRISPR cluster contains a spacer sequence, a sequence that is complementary to the previous mobile element and targets invading nucleic acids. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In the type II CRISPR system, the correct processing of pre-crRNA requires trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. The tracrRNA acts as a guide for the ribonuclease 3-assisted pre-crRNA processing process. Subsequently, Cas9 / crRNA / tracrRNA cuts the linear or circular dsDNA target complementary to the spacer sequence in a nucleic acid cleavage manner. The target strand that is not complementary to the crRNA is first cut in a nucleic acid cleavage manner and then trimmed 3′-5′ in a nucleic acid cleavage manner. In nature, DNA-binding and cutting usually require protein and two types of RNA. However, single guide RNAs ("sgRNAs," or "gRNAs" for short) can be engineered to incorporate aspects of both crRNA and tracrRNA into a single RNA species. See, for example, Jinek M. et al., Science 337:816-821 (2012), which is incorporated herein by reference in its entirety. Cas9 recognizes a short motif (a PAM or protospacer-adjacent motif) within the CRISPR repeat sequence to help distinguish self from non-self.

[0526] In some embodiments, the methods described herein can utilize engineered Cas proteins. A guide RNA (gRNA) is a short synthetic RNA that consists of a scaffold sequence required for Cas-binding and a user-defined spacer sequence of approximately 20 nucleotides that defines the genomic target to be modified. Thus, one skilled in the art can alter the genomic target specificity of the Cas protein, which depends in part on the specificity of the gRNA targeting sequence for the genomic target compared to the rest of the genome.

[0527] In some embodiments, the gRNA scaffold sequence is as follows:

[0528] In some embodiments, the gRNA scaffold sequence is as follows: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAUAAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU.

[0529] In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is an endonuclease (e.g., a deoxyribonuclease or ribonuclease) that is capable of binding to a target polynucleotide sequence when attached to a bound guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is a nickase that is capable of binding to a target polynucleotide sequence when attached to a bound guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is a catalytically inactive domain that is capable of binding to a target polynucleotide sequence when attached to a bound guide nucleic acid. In some embodiments, the target polynucleotide bound by the CRISPR protein-derived domain of the base editor is DNA. In some embodiments, the target polynucleotide bound by the CRISPR protein-derived domain of the base editor is RNA.

[0530] Cas proteins useful herein include class 1 and class 2. Non-limiting examples of Cas proteins include: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, C Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i, CARF, DinG, homologs thereof, or modified versions thereof. Unmodified CRISPR enzymes can have DNA cleavage activity, such as Cas9, which has two functional endonuclease domains: RuvC and HNH. The CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within the target sequence and / or within the complement of the target sequence. For example, the CRISPR enzyme can direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence.

[0531] Vectors encoding a CRISPR enzyme that is mutated (relative to a corresponding wild-type enzyme) such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide comprising a target sequence can be used. Cas9 can refer to a polypeptide having at least or about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology relative to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from Streptococcus pyogenes). Cas9 can refer to a polypeptide having at most or at most about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology relative to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from Streptococcus pyogenes). Cas9 can refer to a wild-type or modified form of a Cas9 protein, including amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0532] In some embodiments, the CRISPR protein-derived domain of the base editor can include all or part of Cas9, which 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, China (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021847.1); Ref: NC_021314.1; Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningitidis (NCBI Ref: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus aureus).

[0533] Cas9 domains of nucleobase editors

[0534] The Cas9 nuclease sequence and structure are well known to those skilled in the art (see, for example, "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E. et al., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M. et al., Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference). Heterologous homologs of Cas9 have been described in various species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on the present disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0535] 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 may be a nuclease activity Cas9 domain, a nuclease inactivation Cas9 domain (dCas9) or a Cas9 nickase (nCas9). In some embodiments, the Cas9 domain is a nuclease activity domain. For example, the Cas9 domain may be a Cas9 domain that cuts two strands of a double-stranded helical nucleic acid (e.g., two strands of a double-stranded helical DNA molecule). In some embodiments, the Cas9 domain includes any one of the amino acid sequences listed herein. In some embodiments, the amino acid sequence included in the Cas9 domain 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 compared to any one of the amino acid sequences listed 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 listed 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 listed herein.

[0536] In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, the protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or a fragment thereof are referred to as "Cas9 variants." Cas9 variants share homology with Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 variant 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 includes a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding wild-type Cas9 fragment. In some embodiments, the fragment is at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% of the amino acid length of the corresponding wild-type Cas9. In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0537] In some embodiments, the Cas9 fusion protein provided herein includes the full-length amino acid sequence of the Cas9 protein, such as one of the Cas9 sequences provided herein. However, in other embodiments, the fusion protein provided herein does not include the full-length Cas9 sequence, but only includes 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.

[0538] The Cas9 protein can be associated with a guide RNA that directs the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the polynucleotide-programmable nucleotide binding domain is a Cas9 domain, for example, a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Examples of nucleic acid-programmable DNA-binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b / C2C1, and Cas12c / C2C3.

[0539] 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).

[0540]

[0541]

[0542]

[0543] (Single underline: HNH domain; double underline: RuvC domain)

[0544] In some embodiments, the wild-type Cas9 corresponds to or includes the following nucleotide and / or amino acid sequence:

[0545]

[0546]

[0547]

[0548] (Single underline: HNH domain; double underline: RuvC domain).

[0549] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI reference sequence: NC_002737.2) (nucleotide sequence below); and Uniprot reference sequence: Q99ZW2 (amino acid sequence below)

[0550]

[0551]

[0552]

[0553]

[0554] (Single underline: HNH domain; double underline: RuvC domain)

[0555] In some embodiments, Cas9 refers to Cas9 from the following: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheriae (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma aphididae (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanensis (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella balticola (NCBI Ref: NC_018010.1); Campylobacter contortus I (NCBI Ref: NC_018010.1). 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.

[0556] It will be understood that additional Cas9 proteins (e.g., nuclease-inactivated Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including variants and homologs thereof, are within the scope of the present disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is a nuclease-inactivated Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease-active Cas9.

[0557] In some embodiments, the Cas9 domain is a nuclease-inactivated Cas9 domain (dCas9). For example, the dCas9 domain can be bound to a double-stranded nucleic acid molecule (e.g., via a gRNA molecule) without cutting any of the double-stranded nucleic acid molecules. In some embodiments, the nuclease-inactivated dCas9 domain includes a D10X mutation and an H840X mutation of the amino acid sequence listed herein, or a corresponding mutation in any amino acid sequence provided herein, wherein X is any amino acid change. In some embodiments, the nuclease-inactivated dCas9 domain includes a D10A mutation and an H840A mutation of the amino acid sequence listed herein, or a corresponding mutation in any amino acid sequence provided herein. As an example, the nuclease-inactivated Cas9 domain includes the amino acid sequence listed in the cloning vector pPlatTET-gRNA2 (accession number BAV54124).

[0558] The amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows:

[0559]

[0560] GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK RMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0561] 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).

[0562] Based on the present disclosure and the knowledge in the art, additional suitable nuclease-inactivated dCas9 domains will be apparent to those skilled in the art and are within the scope of the present disclosure. Such additional exemplary suitable nuclease-inactivated 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)

[0563] In some embodiments, the Cas9 nuclease has an inactivated (e.g., deactivated) DNA cleavage domain, that is, the Cas9 is a nickase and is referred to as a "nCas9" protein (from "nickase" Cas9). Nuclease-inactivated Cas9 proteins are interchangeably referred to as "dCas9" proteins (from nuclease-"dead" Cas9) or catalytically inactive Cas9. Methods for generating Cas9 proteins (or fragments thereof) with inactivated DNA cleavage domains are known (see, e.g., Jinek et al., Science. 337:816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, it is known that the DNA cleavage domain of Cas9 contains two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cuts the strand complementary to the gRNA, while the RuvC1 subdomain cuts the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337: 816-821 (2012); Qi et al., Cell. 28; 152 (5): 1173-83 (2013)).

[0564] In some embodiments, the dCas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any dCas9 domain 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 listed 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 listed herein.

[0565] In some embodiments, the dCas9 corresponds to or includes a portion or all of a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain includes the D10A and H840A mutations, or corresponding mutations in another Cas9.

[0566] In some embodiments, the dCas9 comprises the amino acid sequence of dCas9(D10A and H840A):

[0567]

[0568] (Single underline: HNH domain; double underline: RuvC domain).

[0569] In some embodiments, the Cas9 domain includes a D10A mutation, and 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.

[0570] In other embodiments, dCas9 variants are provided having mutations other than D10A and H840A, which, for example, result in nuclease-inactivated Cas9 (dCas9). Such mutations, for example, include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions within the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs of dCas9 are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants of dCas9 are provided that have an amino acid sequence that is shorter or longer by about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or more.

[0571] In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase can be a Cas9 protein that can cut only one strand of a double-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, the Cas9 nickase cuts the target strand of the double-stranded nucleic acid molecule, which means that the Cas9 nickase cuts the strand that is base-paired with (complementary to) the gRNA (e.g., sgRNA), and the gRNA is bound to the Cas9. In some embodiments, the Cas9 nickase includes a D10A mutation and has a histidine at position 840. In some embodiments, the Cas9 nickase cuts the non-target, non-base-edited strand of the double-stranded nucleic acid molecule, which means that the Cas9 nickase cuts the strand that is not base-paired with the gRNA (e.g., sgRNA), and the gRNA is bound to Cas9. In some embodiments, the Cas9 nickase includes an H840A mutation and has an aspartic acid residue at position 10, or a corresponding mutation. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the Cas9 nickases provided herein. Additional suitable Cas9 nickases will be apparent to those skilled in the art based on this disclosure and knowledge in the art and are within the scope of this disclosure.

[0572] The amino acid sequence of an exemplary catalytic Cas9 nickase (nCas9) is as follows:

[0573]

[0574] In some embodiments, Cas9 refers to Cas9 from an archaeon (e.g., Nanoarchaeota), which constitutes a domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, the programmable nucleotide-binding protein refers to a CasX or CasY protein, which has been 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 hereby incorporated by reference. Using genome-resolved metagenomics, several CRISPR-Cas systems have been identified, including Cas9, which was first reported in the Archaeal domain of life. This distinct Cas9 protein was discovered as part of an active CRISPR-Cas system in the little-studied genus Nanoarchaeota. 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 may be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0575] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein may 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 amino acid sequence included in the napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp is a naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide binding protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any CasX or CasY protein described herein. It should be understood that CasX and CasY from other bacterial species can also be used according to the present disclosure.

[0576] An exemplary CasX ((uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53)tr|F0NN87|F0NN87_SULIHCRISPR-associated Casx protein OS=Sulfolobus icelandica (strain HVE10 / 4) GN=SiH_0402PE=4SV=1) amino acid sequence is as follows:

[0577] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0578] An exemplary CasX (>tr|F0NH53|F0NH53_SULIR CRISPR-associated protein, Casx OS=Sulfolobus icelandica (strain REY15A) GN=SiRe_0771 PE=4SV=1) amino acid sequence is as follows:

[0579] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKV SEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0580] Delta-Proteobacteria CasX

[0581] MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQ KWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0582] The amino acid sequence of an exemplary CasY ((ncbi.nlm.nih.gov / protein / APG80656.1)>APG80656.1 CRISPR-associated protein CasY (uncultured Gemmatimonadetes bacterium)) is as follows:

[0583] MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPREIVSAINDDYVGLYGLSNFDDLYNAEKRNEEKVYSVLDFWYDCVQYGAVFSYTAPGLLKNVAEVRGGSYELTKTLKGSHLYDELQIDKVIKFLNKKEISRANGSLDKLKKDIIDCFKAEYRERHKDQCNKLADDIKNAKKDAGASLGERQKKLFRDFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNRNRGEVLFNKLKEYAQKLDKNEGSLEMWEYIGIGNSGTAFSNFLGEGFLGRLRENKITELKKAMMDITDAWRGQEQEEELEKRLRILAALTIKLREPKFDNHWGGYRSDINGKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEMINRFGESDTKEEAVVSSLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDVQEALIKERLEAEKKKKPKKRKKKSDAEDEKETIDFKELFPHLAKPLKL

[0584] .

[0585] The Cas9 nuclease has two functional nuclease domains: RuvC and HNH. Upon binding to the target, Cas9 undergoes a conformational change that positions the nuclease domains to cleave opposite strands of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (approximately 3-4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired by one of two 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.

[0586] The "efficiency" of non-homologous end joining (NHEJ) and / or homology directed repair (HDR) can be calculated by any convenient method. For example, in some embodiments, efficiency can be expressed as a percentage of successful HDR. For example, a surveyor nuclease assay can be used to generate cleavage products, and the ratio of product to substrate can be used to calculate the percentage. For example, a surveyor nuclease can be used to directly cleave DNA containing newly integrated restriction endonuclease recognition sequences (as a result of successful HDR). The more substrate that is cleaved, the higher the HDR percentage (the higher the efficiency of HDR). As an illustrative example, the following equation [(cleavage product) / (substrate plus cleavage product)] can be used (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).

[0587] In some embodiments, efficiency can be expressed as a percentage of successful NHEJ. For example, the 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 cuts mismatched heteroduplex DNA, which occurs when wild and mutant DNA strands are hybridized (NHEJ generates small random insertions or deletions at the original break site). The more cuts, the higher the percentage of NHEJ (the higher the efficiency of NHEJ). As an illustrative example, the fraction (percentage) of NHEJ can be calculated using the following equation: (1-(1-(b+c) / (a+b+c)) 1 / 2 )×100, where “a” is the band intensity of the DNA substrate, and “b” and “c” are the cleavage products (Ran et al., Cell. 2013 Sep. 12; 154(6): 1380-9; and Ran et al., Nat Protoc. 2013 Nov.; 8(11): 2281–2308).

[0588] The NHEJ repair pathway is the most active repair mechanism, and it often causes small nucleotide insertions or deletions at DSB sites. The randomness of NHEJ-mediated DSB repair has important practical significance because cell populations expressing Cas9 and gRNA or guide polynucleotides can lead to a variety of mutations. In most embodiments, NHEJ causes small insertions / deletions in the target DNA, which lead to amino acid deletions, insertions, or frameshift mutations, and these mutations lead to premature stop codons within the open reading frame (ORF) of the targeted gene. The ideal end result is a loss-of-function mutation within the targeted gene.

[0589] NHEJ-mediated DSB repair often interrupts the open reading frame of a gene, while homology-directed repair (HDR) can be used to generate specific nucleotide changes, ranging from single nucleotide changes to large insertions, such as the addition of fluorophores or tags. To use HDR for gene editing, a DNA repair template containing the desired sequence can be delivered into the target cell type along with (single or multiple) gRNAs and Cas9 or Cas9 nickase. The repair template can contain the desired arrangement, as well as additional homologous sequences immediately upstream and downstream of the target (called left & right homology arms). The length of each homology arm can depend on the size of the change to be introduced, with larger insertions requiring longer homology arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. Even in cells expressing Cas9, gRNA, and exogenous repair templates, the efficiency of HDR is typically low (<10% of modified alleles). Since HDR occurs during the S and G2 phases of the cell cycle, the efficiency of HDR can be improved by synchronizing the cells. Chemical or genetic suppressors of genes involved in NHEJ can also increase the frequency of HDR.

[0590] In some embodiments, Cas9 is a modified Cas9. A given gRNA targeting sequence can have additional sites with partial homology throughout the genome. These sites are called off-target sites and need to be taken into account when designing the 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. The D10A mutant of Cas9 nickase, SpCas9, retains one nuclease domain and generates DNA nicks instead of DSBs. This nickase system can also be combined with HDR-mediated gene editing for specific gene editing.

[0591] In some embodiments, Cas9 is a variant Cas9 protein. When compared to the amino acid sequence of the wild-type Cas9 protein, the amino acid sequence of the variant Cas9 polypeptide is different from one amino acid (e.g., having a deletion, insertion, substitution, fusion). In some instances, the variant Cas9 polypeptide has an amino acid change (e.g., a deletion, insertion, or substitution) that reduces the nuclease activity of the Cas9 polypeptide. For example, in some instances, the variant Cas9 polypeptide has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 protein. In some embodiments, the variant Cas9 protein has no substantial nuclease activity. When a test Cas9 protein is a variant Cas9 protein that does not have substantial nuclease activity, it can be referred to as "dCas9".

[0592] In some embodiments, the variant Cas9 protein has reduced nuclease activity. For example, the variant Cas9 protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the endonuclease activity of a wild-type Cas9 protein (e.g., a wild-type Cas9 protein).

[0593] In some embodiments, the variant Cas9 protein can cut the complementary strand of the guide target sequence, but its ability to cut the non-complementary strand of the double-stranded guide target sequence is reduced. For example, the variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, the Cas9 variant protein has a D10A (aspartic acid at amino acid position 10 is changed to alanine) mutation, and thus can cut the complementary strand of the double-stranded guide target sequence, but its ability to cut the non-complementary strand of the double-stranded guide target sequence is reduced (thus, when the variant Cas9 protein cuts the double-stranded target nucleic acid, a single-strand break (SSB) is obtained instead of a double-strand break (DSB)) (see, for example, Jinek et al., Science. 2012 Aug. 17; 337(6096): 816-21).

[0594] In some embodiments, the variant Cas9 protein can cut the non-complementary strand of a double-stranded guide target sequence, but its ability to cut the complementary strand of the guide target sequence is reduced. For example, the variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif). As a non-limiting example, in some embodiments, the variant Cas9 protein has an H840A (histidine at amino acid position 840 is changed to alanine) mutation, and thus can cut the non-complementary strand of the guide target sequence, but its ability to cut the complementary strand of the guide target sequence is reduced (thus, when the variant Cas9 protein cuts the double-stranded target nucleic acid, an SSB is obtained instead of a DSB). The ability of such a Cas9 protein to cut a guide target sequence (e.g., a single-stranded guide target sequence) is reduced, but the ability to bind to the guide target sequence (e.g., a single-stranded guide target sequence) is retained.

[0595] 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 carries two mutations, D10A and H840A, such that the polypeptide has a reduced ability to cleave both the complementary and non-complementary strands of double-stranded target DNA. Such a Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0596] As another non-limiting example, in some embodiments, the variant Cas9 protein carries two mutations, W476A and W1126A, such that the ability of the polypeptide to cleave target DNA is reduced. Such a Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0597] As another non-limiting example, in some embodiments, the variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, such that the ability of the polypeptide to cleave target DNA is reduced. Such a Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0598] As another non-limiting example, in some embodiments, the variant Cas9 protein carries H840A, W476A, and W1126A mutations, such that the ability of the polypeptide to cleave target DNA is reduced. The ability of such a Cas9 protein to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to target DNA (e.g., single-stranded target DNA) is retained. As another non-limiting example, in some embodiments, the variant Cas9 protein carries H840A, D10A, W476A, and W1126A mutations, such that the ability of the polypeptide to cleave target DNA is reduced. The ability of such a Cas9 protein to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to target DNA (e.g., single-stranded target DNA) is retained. In some embodiments, the variant Cas9 has restored the catalytic His residue (A840H) at position 840 of the Cas9 HNH domain.

[0599] As another non-limiting example, in some embodiments, the variant Cas9 protein carries H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, such that the ability of the polypeptide to cleave target DNA is reduced. The ability of such a Cas9 protein to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to target DNA (e.g., single-stranded target DNA) is retained. As another non-limiting example, in some embodiments, the variant Cas9 protein carries D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, such that the ability of the polypeptide to cleave target DNA is reduced. The ability of such a Cas9 protein to cleave target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind to target DNA (e.g., single-stranded target DNA) is retained. In some embodiments, when the variant Cas9 protein has two mutations, W476A and W1126A, or when the variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, the variant Cas9 protein cannot effectively bind to the PAM sequence. Therefore, 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 can include a guide RNA, but the method can be performed in the absence of a PAM sequence (and the specificity of binding is therefore provided by the targeting segment of the guide RNA). Other residues can be mutated to achieve the above effects (i.e., to inactivate one or the other nuclease portion). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 can be altered (i.e., replaced). Similarly, mutations other than alanine replacements are also suitable.

[0600] In some embodiments, a variant Cas9 protein with reduced catalytic activity (e.g., when the Cas9 protein has D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 mutations, such as D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A), as long as it retains the ability to interact with the guide RNA, the variant Cas9 protein can still bind to the target DNA in a site-specific manner (because it can still be guided to the target DNA sequence by the guide RNA).

[0601] 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.

[0602] In some embodiments, a modified SpCas9 is used that comprises the amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and is specific for the altered PAM 5'-NGC-3'.

[0603] Alternatives to S. pyogenes Cas9 could include RNA-guided endonucleases from the Cpf1 family, which have demonstrated cleavage activity in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA-editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of the class II CRISPR / Cas system. This acquired immunity mechanism is found in Prevotella and Francisella bacteria. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses a guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the limitations of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, Cpf1-mediated DNA cleavage results in double-strand breaks with short 3' overhangs. The staggered cleavage pattern of Cpf1 opens up the possibility of targeted gene transfer (similar to traditional restriction endonuclease cloning), which can improve the efficiency of gene editing. Like the Cas9 variants and heterologs mentioned above, Cpf1 can also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM site preferred by SpCas9. The Cpf1 locus contains a mixed α / β domain, RuvC-I, followed by a helical region, RuvC-II, and a zinc finger-like domain. The Cpf1 protein has a RuvC-like nuclease domain that is similar to the RuvC domain of Cas9. In addition, Cpf1 does not have an HNH nuclease domain, and the N-terminus of Cpf1 does not have the α-helical recognition lobe of Cas9. The structure of the Cpf1 CRISPR-Cas domain shows that Cpf1 has unique functions and is classified as a Class 2 type V CRISPR system. The Cas1, Cas2, and Cas4 proteins encoded by the Cpf1 locus are more similar to type I and type III systems than to type II systems. Functional Cpf1 does not require trans-activating CRISPR RNA (tracrRNA), so only CRISPR (crRNA) is required. This is beneficial for genome editing, not only because Cpf1 is smaller than Cas9, but also because it has a smaller sgRNA molecule (about half the number of Cas9 nucleotides). In contrast to the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex cuts the target DNA or RNA by recognizing the protospacer adjacent motif 5'-YTN-3'. After recognizing the PAM, Cpf1 introduces a sticky-end-like DNA double-strand break with an overhang of 4 or 5 nucleotides.

[0604] Nucleic acid-programmable DNA-binding proteins

[0605] Some aspects of the present disclosure provide a fusion protein including a domain that acts as a nucleic acid programmable DNA binding protein, which can be used to guide a protein, such as a base editor, to a specific nucleic acid (e.g., DNA or RNA) sequence. In a specific embodiment, the fusion protein includes 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 known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cs e2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, C type IICas effector protein, Type V Cas effector protein, Type VI Cas effector protein, 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, although they may not be explicitly listed in this disclosure.See, e.g., Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct; 1: 325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, the entire contents of each of which are hereby incorporated by reference.

[0606] An example of a nucleic acid-programmable DNA-binding protein with a different PAM specificity than Cas9 is the clustered regularly interspaced short palindromic repeats (Cpf1) from Prevotella and Francisella 1. Similar to Cas9, Cpf1 is also a Class 2 CRISPR effector. It has been shown that Cpf1 mediates robust DNA interference, but its characteristics are different from Cas9. Cpf1 is a single RNA-guided endonuclease that lacks tracrRNA and utilizes T-rich protospacer-adjacent motifs (TTN, TTTN, or YTN). In addition, Cpf1 cuts DNA via staggered DNA double-strand breaks. Among the 16 Cpf1-family proteins, two enzymes from the genus Acidaminococcus and the family Lachnospiraceae have been shown to have efficient genome-editing activity in human cells. The Cpf1 protein is known in the art and has been described previously, for example, in Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-96; the entire contents of which are hereby incorporated by reference.

[0607] Nuclease-inactivated Cpf1 (dCpf1) variants useful in the compositions and methods of the present invention can be used as guide nucleotide sequence-programmable DNA-binding protein domains. The Cpf1 protein has a RuvC-like nuclease domain similar to the RuvC domain of Cas9, but does not have an HNH endonuclease domain, and the N-terminus of Cpf1 does not have the α-helical recognition lobe of Cas9. It has been shown in Zetsche et al., Cell, 163, 759-771, 2015 (which is incorporated herein by reference) that the RuvC-like domain of Cpf1 is responsible for cutting the two DNA strands, and deactivation of the RuvC-like domain deactivates the activity of the Cpf1 nuclease. For example, mutations corresponding to D917A, E1006A, or D1255A in Francisella novicida Cpf1 deactivate Cpf1 nuclease activity. In some embodiments, dCpf1 disclosed herein comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It should be understood that any mutation that inactivates the RuvC domain of Cpf1, such as a substitution mutation, deletion, or insertion, can be used in accordance with the present disclosure.

[0608] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein may be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In some embodiments, the Cpf1 protein is a nuclease-inactivated Cpf1 (dCpf1). In some embodiments, the amino acid sequence comprised by the Cpf1, nCpf1, or dCpf1 is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the Cpf1 sequence described herein. In some embodiments, the dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the Cpf1 sequence described herein and comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It will be understood that Cpf1 from other bacterial species can be used in accordance with the present disclosure.

[0609] Wild-type Francisella neoformans Cpf1 (D917, E1006, and D1255 are bold and underlined)

[0610]

[0611]

[0612] Francisella neoformans Cpf1 D917A (A917, E1006, and D1255 are bold and underlined)

[0613]

[0614] Francisella neoformans Cpf1 E1006A (D917, A1006, and D1255 are bold and underlined)

[0615]

[0616] Francisella neoformans Cpf1 D1255A (D917, E1006, and A1255 are bold and underlined)

[0617]

[0618]

[0619] Francisella neoformans Cpf1 D917A / E1006A (A917, A1006, and D1255 are bold and underlined)

[0620]

[0621] Francisella neoformans Cpf1 D917A / D1255A (A917, E1006, and A1255 are bold and underlined)

[0622]

[0623]

[0624] Francisella neoformans Cpf1 E1006A / D1255A (D917, A1006, and A1255 are bold and underlined)

[0625]

[0626]

[0627] Francisella neoformans Cpf1 D917A / E1006A / D1255A (A917, A1006, and A1255 are bold and underlined)

[0628]

[0629] In some embodiments, one of the Cas9 domains present in the fusion protein can be replaced by a guide nucleotide sequence-programmable DNA-binding protein domain that does not require a PAM sequence.

[0630] In some embodiments, the Cas9 domain is a Cas9 domain from Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domain is a nuclease-active SaCas9, a nuclease-inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, the SaCas9 comprises an N579A mutation, or a corresponding mutation in any of the amino acid sequences provided herein.

[0631] In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a non-positive PAM. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a NNGRRT or NNGRRT PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of E781X, N967X, and R1014X mutations, or corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of E781K, N967K, and R1014H mutations, or one or more corresponding mutations in any amino acid sequence provided herein. In some embodiments, the SaCas9 domain comprises E781K, N967K, or R1014H mutations, or corresponding mutations in any amino acid sequence provided herein.

[0632] Exemplary SaCas9 sequences

[0633]

[0634] The upper residue N579, which is underlined and shown in bold, can be mutated (e.g., to A579) to generate the SaCas9 nickase.

[0635] Exemplary SaCas9n sequences

[0636]

[0637]

[0638] The upper residue A579, which can be mutated from N579 to generate the SaCas9 nickase, is underlined and shown in bold.

[0639] Exemplary SaKKH Cas9

[0640]

[0641] The upper residue A579, which can be mutated from N579 to generate the SaCas9 nickase, is underlined and shown in bold. The upper residues K781, K967, and H1014, which can be mutated from E781, N967, and R1014 to generate the SaKKH Cas9, are underlined and shown in italics.

[0642] In some embodiments, the napDNAbp is a circular permutation array. In the following sequences, plain text represents adenosine deaminase sequences, bold sequences represent sequences derived from Cas9, italic sequences represent linker sequences, and underlined sequences represent bipartite nuclear localization sequences.

[0643] CP5 (with MSP "NGC" PID and "D10A" nickase):

[0644]

[0645]

[0646] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a single effector of a microbial CRISPR-Cas system. Single effectors of a microbial CRISPR-Cas system include, but are not limited to, Cas9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while Class 2 systems have single protein effectors. For example, Cas9 and Cpf1 are Class 2 effectors. In addition to Cas9 and Cpf1, three different 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. Two of the systems, Cas12b / C2c1 and Cas12c / C2c3, have effectors that contain a RuvC-like endonuclease domain associated with Cpf1. The third system contains an effector with two predicted HEPN RNase domains. Production of mature CRISPR RNA is independent of tracrRNA, unlike CRISPR RNA produced by Cas12b / C2c1. Cas12b / C2c1 relies on both CRISPR RNA and tracrRNA for DNA cleavage.

[0647] The crystal structure of the complex of A. acidoterrestris Cas12b / C2c1 (AacC2c1) and a chimeric single-molecule guide RNA (sgRNA) has been reported, see, for example, Liu et al., “C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism”, Mol. Cell, 2017 Jan. 19; 65(2): 310-322, the entire contents of which are hereby incorporated by reference. The crystal structure of A. acidoterrestris C2c1 bound to target DNA in the form of a ternary complex has also been reported. See, for example, 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 hereby incorporated by reference. Two catalytically competent conformations of AacC2c1 (with target and non-target DNA strands) have been independently captured, located within a single RuvC catalytic pocket, with Cas12b / C2c1-mediated cleavage resulting in staggered seven-nucleotide breaks in the target DNA. Structural comparisons of the Cas12b / C2c1 ternary complex with previously identified Cas9 and Cpf1 counterparts demonstrate the diversity of mechanisms employed by the CRISPR-Cas9 system.

[0648] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein may be Cas12b / C2c1, or Cas12c / C2c3 protein. In some embodiments, the napDNAbp is Cas12b / C2c1 protein. In some embodiments, the napDNAbp is Cas12c / C2c3 protein. In some embodiments, the amino acid sequence included in the napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp is naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the napDNAbp sequences provided herein. It should be understood that Cas12b / C2c1 or Cas12c / C2c3 from other bacterial species can also be used according to the present disclosure.

[0649] The amino acid sequence of Cas12b / C2c1 ((uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2|C2C1_ALIAGCRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acidoterrestris (strain ATCC 49025 / DSM 3922 / CIP106132 / NCIMB 13137 / GD3B) GN=c2c1 PE=1SV=1) is as follows:

[0650] MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECDKTAEECKAELLERLRARQVENGHRGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKF LSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKEKAETRKSADRTADVLRALADFGLKPLMRVYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQEYAKLVEQ

[0651] KNRFEQKNFVGQEHLVHLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSDKVFEKWGKLAPDAPFDLYDAEIKNVQRRNTRRFGSHDLFAKLAEPEYQALWREDASFLTRYAVYNSILRKLNHAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGERRHAIRFHKLLKVENGVAREVDDVTVPISMSEQLDNLLPRDPNEPIALYFRDYGAEQHFTGEFGGAKIQCRRDQLAHMHRRRGARDVYLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPFFFPIKGNDNLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPVDAANHMTPDWREAFENELQKLKSLHGICSDKEWMDAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYAKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELINQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCTQEHNPEPFPWWLNKFVVEHTLDACPLRADDLIPTGEGEIFVSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRLRCDWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYERERGKKRRKVFAQEKLSEEEAELLVEADEAREKSVVLMRDPSGIINRGNWTRQKEFWSMV NQRIEGYLVKQIRSRVPLQDSACENTGDI.

[0652] AacCas12b (Alicyclobacillus acidiphilus)-WP_067623834

[0653]

[0654]

[0655] Includes a variant called BvCas12b V4 (S893R / K846R / E837G changes relative to the wild type described above)

[0656] BhCas12b (V4) is represented as follows: 5'mRNA cap --- 5'UTR --- bhCas12b --- termination (STOP) sequence --- 3'UTR --- 120 poly A tail

[0657] 5'UTR:

[0658] GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC

[0659] 3'UTR (TriLink standard UTR)

[0660] GCTGGAGCCTCGGTGGCCATGCTTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGA

[0661] Nucleic acid sequence of bhCas12b(V4)

[0662]

[0663]

[0664] In some embodiments, the Cas12b is BvCas12B, which is a variant of BhCas12b and includes the following changes relative to BhCas12B: S893R, K846R, and E837G.

[0665] BvCas12b (Bacillus sp. V3-13) NCBI reference sequence: WP_101661451.

[0666] EGMRVMSIDMGQRTSASVSIFEVVKELPKDQEQKLFYSINDTELFAIHKRSFLLNLPGEVVTKNNKQQRQERRKKRQFVRSQIRMLANVLRLETKKTPDERKKAIHKLMEIVQSYDSWTASQKEVWEKELNLLTNMAAFNDEIWKESLVELHHRIEPYVGQIVSKWRKGLSEGRKNLAGISMWNIDELEDTRRLLISWSKRSRTPGEANRIETDEPFGSSLLQHIQNVKDDRLKQMANLIIMTALGFKYDKEEKDRYKRWKETYPACQIILFENLNRYLFNLDRSRRENSRLMKWAHRSIPRTVSMQGEMFGLQVGDVRSEYSSRFHAKTGAPGIRCHALTEEDLKAGSNTLKRLIEDGFINESELAYLKKGDIIPSQGGELFVTLSKRYKKDSDNNELTVIHADINAAQNLQKRFWQQNSEVYRVPCQLARMGEDKLYIPKSQTETIKKYFGKGSFVKNNTEQEVYKWEKSEKMKIKTDTTFDLQDLDGFEDISKTIELAQEQQKKYLTMFRDPSGYFFNNETWRPQKEYWSIVNNIIKSCLKKKILSNKVEL

[0667] Guide polynucleotide

[0668] 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 cuts linear or circular dsDNA targets complementary to the spacer sequence by endonucleolytic cleavage. Target strands that are not complementary to the crRNA are first cut by endonucleolytic cleavage and then trimmed 3'-5' by exonucleolytic cleavage. In nature, DNA-binding and cleavage typically require proteins and two RNAs. However, single guide RNAs ("sgRNAs", or "gRNAs" for short) can be engineered to incorporate aspects of crRNA and tracrRNA into a single RNA species. See, for example, Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are hereby incorporated by reference. Cas9 recognizes short motifs (PAM or protospacer adjacent motifs) in CRISPR repeat sequences to help distinguish self from non-self. The Cas9 nuclease sequence and structure are well known to those skilled in the art (see, for example, "Complete genome sequence of an M1 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); and "Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M. et al., Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference). Heterologous homologs of Cas9 have been described in various species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus.Based on the present disclosure, additional suitable Cas9 nucleases and sequences will be apparent to those skilled in the art, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 nuclease has an inactivated (e.g., deactivated) DNA cleavage domain, i.e., the Cas9 is a nickase.

[0669] In some embodiments, the guide polynucleotide is at least one single guide RNA ("sgRNA" or "gRNA"). In some embodiments, the guide polynucleotide is at least one tracrRNA. In some embodiments, the guide polynucleotide does not require a PAM sequence to direct the polynucleotide-programmable DNA-binding domain (e.g., Cas9 or Cpf1) to the target nucleotide sequence.

[0670] The polynucleotide programmable nucleotide binding domains (e.g., CRISPR-derived domains) of the base editors disclosed herein can recognize target polynucleotides by being associated with guide polynucleotides. Guide polynucleotides (e.g., gRNAs) are typically single-stranded and can be programmed to site-specifically bind (i.e., via complementary base pairing) to a target sequence of a polynucleotide, thereby guiding the base editor connected to 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 includes natural nucleotides (e.g., adenosine). In some embodiments, the guide polynucleotide includes non-natural (or unnatural) nucleotides (e.g., peptide nucleic acids or nucleotide analogs). In some embodiments, the targeting region of the guide nucleic acid sequence can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The targeting region of the guide nucleic acid can be between 10-30 nucleotides in length, or between 15-25 nucleotides in length, or between 15-20 nucleotides in length.

[0671] In some embodiments, a guide polynucleotide comprises two or more individual polynucleotides that can interact with each other via, for example, complementary base pairing (e.g., a dual guide polynucleotide). For example, a guide polynucleotide can comprise a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). For example, a guide polynucleotide can comprise one or more trans-activating CRISPR RNAs (tracrRNAs).

[0672] In a type II CRISPR system, targeting nucleic acids by a CRISPR protein (e.g., Cas9) typically requires complementary base pairing between a first RNA molecule (crRNA) that includes a sequence that recognizes the target sequence and a second RNA molecule (trRNA) that includes a repeat sequence that forms a scaffold region that stabilizes the guide RNA-CRISPR protein complex. This dual guide RNA system can be used as a guide polynucleotide to guide the base editors disclosed herein to the target polynucleotide sequence.

[0673] In some embodiments, the base editors provided herein utilize a single guide polynucleotide (e.g., gRNA). In some embodiments, the base editors provided herein utilize dual guide polynucleotides (e.g., dual gRNA). In some embodiments, the base editors provided herein utilize one or more guide polynucleotides (e.g., multiple gRNAs). In some embodiments, a single guide polynucleotide is used for different base editors described herein. For example, a single guide polynucleotide can be used for a cytidine base editor and an adenosine base editor.

[0674] In other embodiments, the guide polynucleotide can include a polynucleotide target portion of the nucleic acid and a scaffold portion of the 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, dual, or multi-molecule nucleic acid capable of interacting with a base editor and guiding it to a target polynucleotide sequence.

[0675] Typically, a guide polynucleotide (e.g., a crRNA / trRNA complex or gRNA) includes a "polynucleotide-targeting segment" that includes a sequence that can recognize and bind to a target polynucleotide sequence, and a "protein-binding segment" that stabilizes the guide polynucleotide within the polynucleotide programmable nucleotide binding domain component of the base editor. In some embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to a DNA polynucleotide, thereby facilitating the editing of bases within the DNA. In other embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to a polynucleotide, thereby facilitating the editing of bases within the RNA. As used herein, a "segment" refers to a section or region of a molecule, for example, a contiguous stretch of nucleotides in a guide polynucleotide. A segment may also refer to a region / section of a complex, such that a segment may include a region of more than one molecule. For example, when a guide polynucleotide includes multiple nucleic acid molecules, the protein-binding segment may include all or part of multiple separate molecules (e.g., hybridized along complementary regions). In some embodiments, a protein-binding segment of a DNA-targeting RNA comprising two separate molecules can include (i) base pairs 40-75 of a first RNA molecule that is 100 base pairs in length; and (ii) base pairs 10-25 of a second RNA molecule that is 50 base pairs in length. The definition of "segment," unless otherwise specifically defined in a particular context, is not limited to a specific number of total base pairs, is not limited to any specific number of base pairs from a given RNA molecule, is not limited to a specific number of separate molecules within a complex, and can encompass regions of any total length of an RNA molecule, and can include regions that are complementary to other molecules.

[0676] A guide RNA or guide polynucleotide can include two or more RNAs, such as CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). A guide RNA or guide polynucleotide can sometimes include a single-stranded RNA, or a single guide RNA (sgRNA), which is fused to a portion (e.g., a functional portion) of tracrRNA and crRNA. A guide RNA or guide polynucleotide can also be a dual RNA, comprising crRNA and tracrRNA. In addition, crRNA can hybridize with target DNA.

[0677] As described above, the guide RNA or guide polynucleotide can be an expressed product. For example, the DNA encoding the guide RNA can be a vector comprising a sequence encoding the guide RNA. The guide RNA or guide polynucleotide can be transferred into the cell by transfection with an isolated guide RNA or plasmid DNA (comprising a sequence encoding the guide RNA and a promoter). The guide RNA or guide polynucleotide can also be transferred into the cell by other means, such as using virus-mediated gene delivery.

[0678] The guide RNA or guide polynucleotide can be isolated. For example, the guide RNA can be transfected into a cell or organism in the form of isolated RNA. The guide RNA can be prepared by in vitro transcription using any in vitro transcription system known in the art. The guide RNA can be transferred into the cell in the form of isolated RNA, rather than in the form of a plasmid containing the guide RNA coding sequence.

[0679] A guide RNA or guide polynucleotide can include three regions: a first region at the 5' end that can be complementary to a target site in a chromosomal sequence, a second internal region that can form a stem-loop structure, and a third region at the 3' end that can be single-stranded. The first region of each guide RNA can also be different, allowing each guide RNA to direct the fusion protein to a specific target site. In addition, the second and third regions of each guide RNA can be identical across all guide RNAs.

[0680] The first region of the guide RNA or guide polynucleotide can be complementary to the target site in the chromosomal sequence so 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 include from or about 10 nucleotides to 25 nucleotides (i.e., from 10 nucleotides to 25 nucleotides; or from about 10 nucleotides to about 25 nucleotides; or from 10 nucleotides to about 25 nucleotides; or from 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 or can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides in length. Sometimes, the first region of the guide RNA can be or can be about 19, 20, or 21 nucleotides in length.

[0681] The guide RNA or guide polynucleotide may also include a second region that forms a secondary structure. For example, the secondary structure formed by the guide RNA may include a stem (or hairpin) and a loop. The length of the loop and the stem may vary. For example, the loop may range in length from or about 3 to 10 nucleotides, while the length may range from or about 6 to 20 base pairs. The stem may include one or more protrusions of 1 to 10 or about 10 nucleotides. The total length of the second region may be within the length range of about 16 to 60 nucleotides. For example, the loop may be or may be a length of about 4 nucleotides, while the stem may be or may be about 12 base pairs.

[0682] The guide RNA or guide polynucleotide can also include a third region at the 3' end that can be substantially single-stranded. For example, the third region can sometimes be non-complementary to any chromosomal sequence in the target cell and sometimes be non-complementary to the rest of the guide RNA. Furthermore, the length of the third region can vary. The length of the third region can be more than or equal to about 4 nucleotides. For example, the length of the third region can be in the range of about 5 to 60 nucleotides.

[0683] A guide RNA or guide polynucleotide can target any exon or intron of a gene target. In some embodiments, a guide can target exon 1 or 2 of a gene; in other embodiments, a guide can target exon 3 or 4 of a gene. A composition can include multiple guide RNAs that all target the same exon, or in some embodiments, multiple guide RNAs that target different exons. Exons and introns of a gene can be targeted.

[0684] The guide RNA or guide polynucleotide can target a nucleic acid sequence having or about 20 nucleotides. The target nucleic acid can be less than or less than about 20 nucleotides. The target nucleic acid can be at least or about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or any number between 1 and 100 nucleotides in length. The target nucleic acid can be at most or about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or any number between 1 and 100 nucleotides in length. The distance from the first nucleotide of the PAM immediately 5' to the target nucleic acid sequence can be or about 20 bases. The guide RNA can target a nucleic acid sequence. A target nucleic acid can be at least or at least about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, or 1-100 nucleotides.

[0685] A guide polynucleotide, such as a guide RNA, can refer to a nucleic acid that can hybridize to another nucleic acid, for example, a target nucleic acid or a protospacer sequence in the genome of a cell. The guide polynucleotide can be RNA. The guide polynucleotide can be DNA. The guide polynucleotide can be programmed or designed to site-specifically bind to a nucleic acid sequence. A guide polynucleotide can include a polynucleotide chain and can be referred to as a single guide polynucleotide. A guide polynucleotide can include two polynucleotide chains and can be referred to as a dual guide polynucleotide. A guide RNA can be introduced into a cell as an RNA molecule. For example, an RNA molecule can be transcribed in vitro and / or can be chemically synthesized. The RNA can be derived from a synthetic DNA molecule (e.g., The guide RNA can then be transcribed from a gene fragment (a gene fragment). The guide RNA can then be introduced into the cell as an RNA molecule. The guide RNA can also be introduced into the cell in the form of a non-RNA nucleic acid molecule (such as a DNA molecule). For example, the DNA encoding the guide RNA can be operably linked to a promoter control sequence for expression of the guide RNA in the target cell. The RNA coding sequence can be operably linked to a promoter sequence recognized by RNA polymerase subunit III (PolIII). Plasmid vectors that can be used to express guide RNA include but are not limited to px330 vectors and px333 vectors. In some embodiments, a plasmid vector (e.g., a px333 vector) may include at least two DNA sequences encoding guide RNA.

[0686] 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, in order to minimize the effect of potential substrate contamination of a deaminase domain (e.g., an AID domain) in a base editor system, the number of residues that may be unintentionally targeted for deamination reactions (e.g., off-target C residues on ssDNA that may potentially reside in the target nucleic acid locus) that reside in the target nucleic acid) can be minimized. In addition, software tools can be used to optimize gRNAs corresponding to target nucleic acid sequences, for example, to minimize total off-target activity across the genome. For example, when using Streptococcus pyogenes Cas9, for each possible selection of a targeting domain, all off-target sequences (preceding the selected PAM, such as NAG or NGG) can be identified across the genome, containing up to a specific number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base pairs. The first region of the gRNA that is complementary to the target site can be identified, and all first regions (e.g., crRNAs) can be ranked according to their total predicted off-target scores; the top-ranked target domains represent those that are likely to have the greatest on-target and minimal off-target activity. Candidate targeting gRNAs can be functionally evaluated using methods known in the art and / or as described herein.

[0687] As a non-limiting example, a DNA sequence search algorithm can be used to identify the target DNA hybridization sequence in the crRNA of the guide RNA used with Cas9. gRNA design can be performed using a custom gRNA design software based on the public tool cas-offinder, as described in the following literature, 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 first calculates the genome-wide off-target tendency of each guide and then scores each guide. Typically, matches within the range of perfect matches to 7 mismatches are considered to be guides with a length ranging from 17 to 24. Once the off-target sites are determined by computer operation, the total score is calculated for each guide and summarized in a table output using a network-interface. In addition to identifying potential target sites adjacent to the PAM sequence, the software can also identify all PAM-adjacent sequences that differ from the selected target site by 1, 2, 3, or more nucleotides. The genomic DNA sequence of the target nucleic acid sequence (e.g., the target gene) can be obtained, and publicly available tools such as the RepeatMasker program can be used to screen for repetitive elements. RepeatMasker searches the input DNA sequence for repetitive elements and low-complexity regions. The output is a detailed annotation of the repeats (elements) present in the given query sequence.

[0688] After identification, the first regions of the guide RNA (e.g., crRNA) can be ranked into different tiers based on their distance from the target site, their orthogonality, and the presence of a 5' nucleotide that is a close match to a relevant PAM sequence (e.g., 5'G, based on its close match identified in the human genome containing a relevant PAM (e.g., NGG PAM for Streptococcus pyogenes, or NNGRRT or NNGRRV PAM for Staphylococcus aureus). As used herein, orthogonality refers to the number of sequences in the human genome that contain the fewest number of mismatches with the target sequence. For example, "high level of orthogonality" or "good orthogonality" can refer to a 20-base (20-mer) targeting domain that has no identical sequence in the human genome other than the intended target, nor any sequence that contains one or two mismatches in the target sequence. Targeting domains with good orthogonality can be selected to minimize off-target DNA cleavage.

[0689] In some embodiments, a reporter system can be used to detect base-editing activity and test candidate guide polynucleotides. In some embodiments, the reporter system may include a reporter gene-based assay, wherein the base editing activity results in the expression of a reporter gene. For example, the reporter system may include a reporter gene that includes a deactivated start codon, for example, a mutation from 3'-TAC-5' to 3'-CAC-5 on the template strand. Once the deamination reaction of target C is successful, the corresponding mRNA will be transcribed as 5'-AUG-3' instead of 5'-GUG-3', thereby allowing translation of the reporter gene. Suitable reporter genes are obvious to those skilled in the art. Non-limiting examples of reporter genes include: genes encoding green fluorescent protein (GFP), red fluorescent protein (RFP), luciferase, secreted alkaline phosphatase (SEAP), or any other gene whose expression is detectable and obvious to those skilled in the art. The reporter system can be used to test many different gRNAs, for example, to determine which residues of the target DNA sequence will be targeted by the corresponding deaminase. sgRNAs targeting non-template strands 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 polynucleotides can include standard ribonucleotides, modified ribonucleotides (e.g., pseudouridine), ribonucleotide isomers, and / or ribonucleotide analogs. In some embodiments, the guide polynucleotides can include at least one detectable label. The detectable label can be a fluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluors, Halo tags, or suitable fluorescent dyes), a detection label (e.g., biotin, digoxin, etc.), a labeling agent, a quantum dot, or gold particles.

[0690] The guide polynucleotide can be synthesized by chemical synthesis, enzymatic synthesis or a combination thereof. For example, the guide RNA can be synthesized using a standard phosphoramidite-based solid-phase synthesis method. Alternatively, the guide RNA can be synthesized in vitro by operably linking the DNA encoding the guide RNA to a promoter sequence recognized by a phage RNA polymerase. Examples of suitable phage promoter sequences include T7, T3, SP6 promoter sequences or variants thereof. In embodiments where the guide RNA includes two separate molecules (e.g., crRNA and tracrRNA), the crRNA can be chemically synthesized, and the tracrRNA can be enzymatically synthesized.

[0691] In some embodiments, the base editor system may include multiple guide polynucleotides, such as multiple gRNAs. For example, the multiple gRNAs may target one or more target loci included in the base editor system (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 g RNAs, at least 50 gRNAs). The multiple gRNA sequences can be arranged in tandem and are preferably separated by direct repeat sequences.

[0692] The DNA sequence encoding the guide RNA or guide polynucleotide can also be part of the vector. In addition, the vector can include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), optional marker sequences (e.g., GFP or antibiotic resistance genes such as puromycin), replication origins, etc. The DNA molecule encoding the guide RNA can also be linear. The DNA molecule encoding the guide RNA or guide polynucleotide can also be circular.

[0693] In some embodiments, one or more components of the base editor system may be encoded by several DNA sequences.Such several DNA sequences can be introduced into an expression system, such as a cell, together or separately.For example, two DNA sequences encoding polynucleotide programmable nucleotide binding domains and a guide RNA can be introduced into a cell, wherein each DNA sequence can be part of a separate molecule (e.g., a vector containing the polynucleotide programmable nucleotide binding domain encoding sequence and a second vector containing the guide RNA encoding sequence) or the two can be part of the same molecule (e.g., a vector containing the coding (and regulatory) sequences of both the polynucleotide programmable nucleotide binding domain and the guide RNA).

[0694] A guide polynucleotide can include one or more modifications to provide a nucleic acid with new or enhanced properties. A guide polynucleotide can include a nucleic acid affinity tag. A guide polynucleotide can include synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides.

[0695] In some embodiments, a gRNA or guide polynucleotide can include modifications. Modifications can be made at any location on the gRNA or guide polynucleotide. More than one modification can be made to a single gRNA or guide polynucleotide. The gRNA or guide polynucleotide can be subjected to quality control after modification. In some embodiments, quality control can include PAGE, HPLC, MS, or any combination thereof.

[0696] Modifications of the gRNA or guide polynucleotide can be substitutions, insertions, deletions, chemical modifications, physical modifications, stabilization, purification, or any combination thereof.

[0697] The gRNA or guide polynucleotide can also be modified with the following: 5' adenosine, 5' guanosine-triphosphate cap, 5' N7-methylguanosine-triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' phosphorothioate, 5' phosphate e, 5' phosphorothioate, cis-ipsilateral (Cis-Syn) thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer r, rSpacer, Spacer 18, Spacer 9, 3'-3' modification, 5'-5' modification, abasic / absic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesterol TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, dual biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3'DABCYL, Black Hole Quencher 1, Black Hole Quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linkers, thiol linkers, 2'-deoxyribonucleoside analogs purines, 2'-deoxyribonucleoside analogs pyrimidines, ribonucleoside analogs, 2'-O-methyl ribonucleoside analogs, sugar-modified analogs, wobble / universal bases, fluorescent dye labels, 2'-fluoro RNA, 2'-O-methyl RNA, methylphosphonates, phosphodiester DNA, phosphodiester RNA, phosphorothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate, 5'-methylcytidine-5'-triphosphate, or any combination thereof.

[0698] In some embodiments, the modification is permanent. In other embodiments, the modification is temporary. In some embodiments, multiple / various modifications are made to the gRNA or guide polynucleotide. Modifications to the gRNA or guide polynucleotide can alter the physicochemical properties of the nucleotides, such as their conformation, polarity, hydrophobicity, chemical reactivity, base-pairing interactions, or any combination thereof.

[0699] The PAM sequence can be any PAM sequence known in the art. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NGN, NGT, NGCG, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNNGATT, NNAGAAW, or NAAAC. Y is a pyrimidine; N is any nucleotide base; and W is A or T.

[0700] The modification can also be a thiophosphate substitute. In some embodiments, the natural phosphodiester bond can be easily and rapidly degraded by the cell's nucleases; the modification of the internucleotide link using a thiophosphate (PS) bond substitute can be more stable to hydrolysis (by cell degradation). The modification can increase the stability of the gRNA or guide polynucleotide. The modification can also enhance biological activity. In some embodiments, the gRNA whose RNA is enhanced by thiophosphate can inhibit RNase A, RNaseT1, calf serum nuclease, or any combination thereof. These features allow PS-RNA gRNA to be used in applications where there is a high possibility of exposure to nucleases in vivo or in vitro. For example, a thiophosphate (PS) bond can be introduced between the last 3 to 5 nucleotides of the 5'- or 3'-end of the gRNA, which can inhibit exonuclease degradation. In some embodiments, thiophosphate bonds can be added throughout the gRNA to reduce endonuclease attack.

[0701] protospacer adjacent motif

[0702] The term "protospacer adjacent motif (PAM)" or PAM-like motif refers to a 2 to 6 base pair DNA sequence that immediately follows the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. In some embodiments, the PAM can be a 5' PAM (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM (i.e., located downstream of the 5' end of the protospacer).

[0703] This PAM sequence is necessary for target binding, but the exact sequence depends on the type of Cas protein.

[0704] The base editors provided herein may include a CRISPR protein-derived domain that is capable of binding to a nucleotide sequence containing an aligned or non-aligned protospacer adjacent motif (PAM) sequence. A PAM site is a nucleotide sequence adjacent to a target polynucleotide sequence. Some aspects disclosed herein provide base editors that include all or part of a CRISPR protein with different PAM specificities. For example, Cas9 proteins typically, such as Cas9 (spCas9) of Streptococcus pyogenes, require an aligned NGG PAM sequence to bind to a specific nucleic acid region, where the “N” in “NGG” is adenine (A), thymine (T), guanine (G), or cytosine (C), and G is guanine. PAM can be CRISPR protein-specific and can be different between different base editors (including different CRISPR protein-derived domains). PAM can be at 5' or 3' of the target sequence. PAM can be upstream or downstream of the target sequence. The length of a PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. Typically, the length of a PAM is between 2 and 6 nucleotides. Several PAM variants are described in Table 1 below.

[0705] Table 1. Cas9 proteins and corresponding PAM sequences

[0706] Variants PAM spCas9 NGG spCas9-VRQR NGA spCas9-VRER NGCG xCas9(sp) NGN saCas9 NNGRRT saCas9-KKH NNNRRT spCas9-MQKSER NGCG spCas9-MQKSER NGCN spCas9-LRKIQK NGTN spCas9-LRVSQK NGTN spCas9-LRVSQL NGTN spCas9-MQKFRAER NGC Cpf1 5'(TTTV) SpyMac 5'-NAA-3'

[0707] In some embodiments, the PAM is NGC. In some embodiments, the NGC PAM is recognized by a Cas9 variant. In some embodiments, the NGC PAM variant comprises one or more amino acid substitutions selected from the group consisting of: D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (collectively referred to as "MQKFRAER").

[0708] In some embodiments, the PAM is NGT. In some embodiments, the NGT PAM is recognized by a Cas9 variant. In some embodiments, the NGT PAM variant is generated by targeted mutations at one or more 1335, 1337, 1135, 1136, 1218, and / or 1219 residues. In some embodiments, the NGT PAM variant is generated by targeted mutations at one or more 1219, 1335, 1337, 1218 residues. In some embodiments, the NGT PAM variant is generated by targeted mutations at one or more 1135, 1136, 1218, 1219, and 1335 residues. In some embodiments, the NGT PAM variant is selected from the set of targeted mutations provided in Tables 2 and 3 below.

[0709] Table 2: NGT PAM variant mutations at residues 1219, 1335, 1337, 1218

[0710] Variants E1219V R1335Q T1337 G1218 1 F V T 2 F V R 3 F V Q 4 F V L 5 F V T R 6 F V R R 7 F V Q R 8 F V L R 9 L L T 10 L L R 11 L L Q 12 L L L 13 F I T 14 F I R 15 F I Q 16 F I L 17 F G C 18 H L N 19 F G C A 20 H L N V 21 L A W 22 L A F 23 L A Y 24 I A W 25 I A F 26 I A Y

[0711] Table 3: NGT PAM variant mutations at residues 1135, 1136, 1218, 1219, and 1335

[0712]

[0713]

[0714] In some embodiments, the NGT PAM variant is selected from variants 5, 7, 28, 31, or 36 in Tables 2 and 3. In some embodiments, the variants have improved NGT PAM recognition ability.

[0715] In some embodiments, the NGT PAM variants have mutations at residues 1219, 1335, 1337, and / or 1218. In some embodiments, the NGT PAM variants are selected from the variants provided in Table 4 below to obtain mutations with improved recognition capabilities.

[0716] Table 4: NGT PAM variant mutations at residues 1219, 1335, 1337, and 1218

[0717] Variants E1219V R1335Q T1337 G1218 1 F V T 2 F V R 3 F V Q 4 F V L 5 F V T R 6 F V R R 7 F V Q R 8 F V L R

[0718] In some embodiments, base editors specific for NGT PAMs can be generated, as provided in Table 5 below.

[0719] Table 5. NGT PAM variants

[0720]

[0721] In some embodiments, the NGTN variant is variant 1. In some embodiments, the NGTN variant is variant 2. In some embodiments, the NGTN variant is variant 3. In some embodiments, the NGTN variant is variant 4. In some embodiments, the NGTN variant is variant 5. In some embodiments, the NGTN variant is variant 6.

[0722] In some embodiments, the Cas9 domain is the Cas9 domain (SpCas9) of Streptococcus pyogenes. In some embodiments, the SpCas9 domain is the SpCas9 of nuclease activity, the SpCas9 (SpCas9d) of nuclease inactivation, or the SpCas9 nickase (SpCas9n). In some embodiments, the SpCas9 includes a D9X mutation, or a corresponding mutation in any amino acid sequence provided herein, wherein X is any amino acid except D. In some embodiments, the SpCas9 includes a D9A mutation, or a corresponding mutation in any amino acid sequence provided herein. In some embodiments, the SpCas9 domain, the SpCas9d domain or the SpCas9n domain can be combined with a nucleotide sequence having a non-positive PAM. In some embodiments, the SpCas9 domain, the SpCas9d domain or the SpCas9n domain can be combined with a nucleotide sequence domain having an NGG, NGA or NGCG PAM sequence. In some embodiments, the SpCas9 domain comprises one or more of D1134X, R1334X, and T1336X mutations, or corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1134E, R1334Q, and T1336R mutations, or corresponding mutations in any amino acid sequence provided herein. In some embodiments, the SpCas9 domain comprises D1134E, R1334Q, and T1336R mutations, or corresponding mutations in any amino acid sequence provided herein. In some embodiments, the SpCas9 domain comprises one or more of D1134X, R1334X, and T1336X mutations, or corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1134V, R1334Q, and T1336R mutations, or corresponding mutations in any amino acid sequence provided herein. In some embodiments, the SpCas9 domain comprises D1134V, R1334Q, and T1336R mutations, or corresponding mutations in any amino acid sequence provided herein. In some embodiments, the SpCas9 domain comprises one or more of D1134X, G1217X, R1334X, and T1336X mutations, or corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1134V, G1217R, R1334Q, and T1336R mutations, or corresponding mutations in any amino acid sequence provided herein.In some embodiments, the SpCas9 domain comprises D1134V, G1217R, R1334Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein.

[0723] In some embodiments, the Cas9 domain of any fusion protein provided herein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the Cas9 polypeptide described herein. In some embodiments, the Cas9 domain of any fusion protein provided herein comprises the amino acid sequence of any Cas9 polypeptide described herein. In some embodiments, the Cas9 domain of any fusion protein provided herein consists of the amino acid sequence of any Cas9 polypeptide described herein.

[0724] In some examples, a PAM recognized by a CRISPR protein-derived domain of a base editor disclosed herein can be provided to the cell on a separate oligonucleotide relative to an insert encoding the base editor (e.g., an AAV insert). In such embodiments, providing the PAM on a separate oligonucleotide can allow for cleavage of a target sequence that would otherwise not be cleaved due to the absence of a neighboring PAM on the same polynucleotide as the target sequence.

[0725] In one embodiment, Streptococcus pyogenes Cas9 (SpCas9) can be used as a CRISPR nuclease suitable for genome engineering. However, other proteins can be used. In some embodiments, different nucleases can be used to target certain genomic targets. In some embodiments, synthetic SpCas9-derived variants with non-NGG PAM sequences can be used. In addition, other Cas9 heterologs from various species have been identified, and these "non-SpCas9" can be combined with a variety of PAM sequences that can also be used in the present disclosure. For example, the relatively large size of SpCas9 (approximately 4 kilobases (kb) coding sequence) may result in the plasmid carrying the SpCas9 cDNA being unable to be efficiently expressed in cells. Conversely, the coding sequence of Staphylococcus aureus Cas9 (SaCas9) is approximately 1 kilobase shorter than SpCas9, which may allow it to be efficiently expressed in cells. Similar to SpCas9, the SaCas9 endonuclease is able to modify target genes in vitro (in mammalian cells) and in vivo (in mice). In some embodiments, Cas proteins can target different PAM sequences. In some embodiments, for example, the target gene can be located adjacent to the Cas9 PAM (5'-NGG, for example). In other embodiments, other Cas9 heterologs may have different PAM requirements. For example, other PAMs, such as those of Streptococcus thermophilus (5'-NNAGAA for CRISPR1 and 5'-NGGNG for CRISPR3) and Neisseria meningitidis (5'-NNNNGATT), can also be found adjacent to the target gene.

[0726] In some embodiments, for the Streptococcus pyogenes system, the target gene sequence can be preceded by (i.e., 5' to) 5'-NGG PAM, and the guide RNA sequence of 20-nt can be paired with the relative strand base to mediate the Cas9 cutting near the ...

Claims

1. A cytidine base editor fusion protein, comprising: (i) Staphylococcus aureus Cas9 nickase polynucleotide programmable DNA binding domain; (ii) a cytidine deaminase having one of the following amino acid sequences: composition: a) MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVARLFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR, or said amino acid sequence having amino acid changes, wherein said amino acid changes consist of a single amino acid change selected from the group consisting of R33A, K34A, W90F and H122A, or wherein said amino acid changes consist of a single set of amino acid changes selected from R33A and K34A; or W90F and K34A, b) MKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEIIKQYLPVPWKKGVFRNQVDPETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTIYTARLYYFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREILQ, or said amino acid sequence with an amino acid change, wherein the amino acid change consists of the amino acid change F130L, c)MADSSEKMRGQYISRDTFEKNYKPIDGTKEAHLLCEIKWGKYGKPWLHWCQNQRMNIHAEDYFMNNIFKAKKHPVHCYVTWYLSWSPCADCASKIV KFLEERPYLKLTIYVAQLYYHTEEENRKGLRLLRSKKVIIRVMDISDYNYCWKVFVSNQNGNEDYWPLQFDPWVKENYSRLLDIFWESKCRSPNPW, and d) MDPQRLRQWPGPGPASRGGYGQRPRIRNPEEWFHELSPRTFSFHFRNLRFASGRNRSYICCQVEGKNCFFQGIFQNQVPPDPPCHAELCFLSWFQSWGLSPDEHYYVTWFISWSPCCECAAKVAQFLEENRNVSLSLSAARLYYFWKSESREGLRRLSDLGAQVGIMSFQDFQHCWNNFVHNLGMPFQPWKKLHKNYQRLVTELKQILREEPATYGSPQAQGKVRIGSTAAGLRHSHSHTRSEAHLRPNHSSRQHRILNPPREARARTCVLVDASWICYR, or said amino acid sequence with an amino acid change, wherein the amino acid change consists of the amino acid change R54Q; and (iii) uracil glycosylase inhibitor domain, wherein the cytidine base editor fusion protein has an increased ratio of cis-activity to trans-activity compared to a cytidine base editor fusion protein comprising rat APOBEC-1 cytidine deaminase and Cas9 nickase, wherein: A) the cis-activity refers to base editing of the target DNA, and B) The trans-activity refers to base editing in the secondary SaCas9-induced R-loop, where the base editor is not guided by its own single guide RNA.

2. The cytidine base editor fusion protein of claim 1, wherein the cytidine base editor fusion protein comprising rat APOBEC-1 cytidine deaminase and Cas9 nickase is BE3 or BE4.

3. The cytidine base editor fusion protein of claim 1, wherein the cytidine deaminase consists of the following amino acid sequence: MDPQRLRQWPGPGPASRGGYGQRPRIRNPEEWFHELSPRTFSFHFRNLRFASGRNRSYICCQVEGKNCFFQGIFQNQVPPDPPCHAELCFLSWFQSWGLSPDEHYYVTWFISWSPCCECAAKVAQFLEENRNVSLSLSAARLYYFWKSESREGLRRLSDLGAQVGIMSFQDFQHCWNNFVHNLGMPFQPWKKLHKNYQRLVTELKQILREEPATYGSPQAQGKVRIGSTAAGLRHSHSHTRSEAHLRPNHSSRQHRILNPPREARARTCVLVDASWICYR having an amino acid change, wherein the amino acid change consists of amino acid change R54Q.

4. The cytidine base editor fusion protein of claim 1, wherein the cytidine deaminase consists of the following amino acid sequence: MKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEIIKQYLPVPWKKGVFRNQVDPETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTIYTARLYYFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREILQ having an amino acid change consisting of amino acid change F130L.

5. The cytidine base editor fusion protein of claim 1, further comprising one or more nuclear localization signals.

6. The cytidine base editor fusion protein of claim 1, wherein the cytidine base editor fusion protein comprises an N-terminal nuclear localization signal and / or a C-terminal nuclear localization signal.

7. The cytidine base editor fusion protein of claim 5 or 6, wherein the nuclear localization signal is a bipartite nuclear localization signal.

8. The cytidine base editor fusion protein of claim 1, wherein the uracil glycosylase inhibitor domain is derived from Bacillus subtilis phage PBS1 and inhibits human uracil DNA glycosylase activity.

9. The cytidine base editor fusion protein of claim 8, wherein the cytidine base editor fusion protein comprises two uracil glycosylase inhibitors.

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

11. The cell of claim 10, wherein the cell is a bacterial cell, a plant cell, an insect cell, or a mammalian cell.

12. A molecular complex comprising a cytidine base editor fusion protein as described in any one of claims 1 to 9 and one or more guide RNA sequences, tracrRNA sequences, or target DNA sequences.

13. A method for editing a nucleobase of a nucleic acid sequence in vitro, the method comprising: a) contacting the nucleic acid sequence with a cytidine base editor fusion protein as described in any one of claims 1 to 9; and b) converting cytosine of the nucleic acid sequence to thymidine.

14. The method of claim 13, further comprising contacting the nucleic acid sequence with a guide polynucleotide.

15. The method of claim 13, wherein the cytidine base editor fusion protein deaminates the target nucleobase with higher efficiency compared to the cytidine base editor fusion protein comprising rat APOBEC-1 cytidine deaminase and Cas9 nickase. The method of claim 13 , wherein the contacting is performed in a cell.

17. The method of claim 16, wherein the cell is a mammalian cell.

18. A fusion protein comprising: a) Staphylococcus aureus Cas9 nickase polynucleotide programmable DNA binding domain; b) cytidine deaminase, wherein the cytidine deaminase consists of the following amino acid sequence: MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVARL FWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR; and c) a uracil glycosylase inhibitor domain, wherein the fusion protein has an increased ratio of cis-activity to trans-activity compared to a cytidine base editor fusion protein comprising rat APOBEC-1 cytidine deaminase and Cas9 nickase, wherein: A) the cis-activity refers to base editing of the target DNA, and B) The trans-activity refers to base editing in the secondary SaCas9-induced R-loop, where the base editor is not guided by its own single guide RNA.

19. A fusion protein comprising: a) Staphylococcus aureus Cas9 nickase polynucleotide programmable DNA binding domain; b) cytidine deaminase, wherein the cytidine deaminase consists of the following amino acid sequence: MKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEIIKQYLPVPWKKGVFRNQVDPETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTIYTARLYYFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREILQ; and c) a uracil glycosylase inhibitor domain, wherein the fusion protein has an increased ratio of cis-activity to trans-activity compared to a cytidine base editor fusion protein comprising rat APOBEC-1 cytidine deaminase and Cas9 nickase, wherein: A) the cis-activity refers to base editing of the target DNA, and B) The trans-activity refers to base editing in the secondary SaCas9-induced R-loop, where the base editor is not guided by its own single guide RNA.

20. A fusion protein comprising: a) Staphylococcus aureus Cas9 nickase polynucleotide programmable DNA binding domain; b) cytidine deaminase, wherein the cytidine deaminase consists of the following amino acid sequence: MADSSEKMRGQYISRDTFEKNYKPIDGTKEAHLLCEIKWGKYGKPWLHWCQNQRMNIHAEDYFMNNIFKAKKHPVHCYVTWYLSWSPCADCASKIVKFLEERPYLKLTIYVAQLYYHTEEENRKGLRLLRSKKVIIRVMDISDYNYCWKVFVSNQNGNEDYWPLQFDPWVKENYSRLLDIFWESKCRSPNPW; and c) a uracil glycosylase inhibitor domain, wherein the fusion protein has an increased ratio of cis-activity to trans-activity compared to a cytidine base editor fusion protein comprising rat APOBEC-1 cytidine deaminase and Cas9 nickase, wherein: A) the cis-activity refers to base editing of the target DNA, and B) The trans-activity refers to base editing in the secondary SaCas9-induced R-loop, where the base editor is not guided by its own single guide RNA.

21. A fusion protein comprising: a) Staphylococcus aureus Cas9 nickase polynucleotide programmable DNA binding domain; b) cytidine deaminase, wherein the cytidine deaminase consists of the following amino acid sequence: MDPQRLRQWPGPGPASRGGYGQRPRIRNPEEWFHELSPRTFSFHFRNLRFASGRNRSYICCQVEGKNCFFQGIFQNQVPPDPPCHAELCFLSWFQSWGLSPDEHYYVTWFISWSPCCECAAKVAQFLEENRNVSLSLSAARLYYFWKSESREGLRRLSDLGAQVGIMSFQDFQHCWNNFVHNLGMPFQPWKKLHKNYQRLVTELKQILREEPATYGSPQAQGKVRIGSTAAGLRHSHSHTRSEAHLRPNHSSRQHRILNPPREARARTCVLVDASWICYR; and c) a uracil glycosylase inhibitor domain, wherein the fusion protein has an increased ratio of cis-activity to trans-activity compared to a cytidine base editor fusion protein comprising rat APOBEC-1 cytidine deaminase and Cas9 nickase, wherein: A) the cis-activity refers to base editing of the target DNA, and B) The trans-activity refers to base editing in the secondary SaCas9-induced R-loop, where the base editor is not guided by its own single guide RNA.

22. A fusion protein, comprising: a) Staphylococcus aureus Cas9 nickase polynucleotide programmable DNA binding domain; b) a cytidine deaminase, wherein the cytidine deaminase consists of the amino acid sequence MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVARLFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR with an amino acid change, wherein the amino acid change consists of the amino acid change H122A; and c) a uracil glycosylase inhibitor domain, wherein the fusion protein has an increased ratio of cis-activity to trans-activity compared to a cytidine base editor fusion protein comprising rat APOBEC-1 cytidine deaminase and Cas9 nickase, wherein: A) The cis-activity refers to DNA base editing at the target, B) The trans-activity refers to base editing in the secondary SaCas9-induced R-loop, where the base editor is not guided by its own single guide RNA.

23. The fusion protein of any one of claims 18 to 22, further comprising one or more nuclear localization signals.

24. The fusion protein of any one of claims 18 to 22, wherein the fusion protein comprises an N-terminal nuclear localization signal and / or a C-terminal nuclear localization signal. The fusion protein of claim 23 , wherein the nuclear localization signal is a bipartite nuclear localization signal.

26. The fusion protein of any one of claims 18 to 22, wherein the uracil glycosylase inhibitor domain is derived from Bacillus subtilis phage PBS1 and inhibits human uracil DNA glycosylase activity.

27. The fusion protein of any one of claims 18 to 22, wherein the fusion protein comprises two uracil glycosylase inhibitors.

28. The fusion protein of any one of claims 18 to 22, wherein the cytidine base editor fusion protein comprising rat APOBEC-1 cytidine deaminase and Cas9 nickase is BE3 or BE4.

29. A polynucleotide molecule encoding the cytidine base editor fusion protein of any one of claims 1 to 9.

30. The polynucleotide molecule of claim 29, wherein the polynucleotide is codon optimized.

31. An expression vector comprising the polynucleotide molecule of claim 29 or 30.

32. The expression vector of claim 31, wherein the expression vector is a mammalian expression vector.

33. The expression vector of claim 32, wherein the vector is a viral vector selected from the group consisting of an adeno-associated viral vector, a retroviral vector, an adenoviral vector, a lentiviral vector, a Sendai viral vector, and a herpes viral vector.

34. The expression vector of any one of claims 31 to 33, wherein the vector comprises a promoter.

35. A cell comprising a polynucleotide molecule according to claim 29 or 30 or a vector according to any one of claims 31 to 34.

36. The cell of claim 35, wherein the cell is a bacterial cell, a plant cell, an insect cell, or a mammalian cell.

37. A molecular complex comprising the fusion protein of any one of claims 18 to 28 and one or more guide RNA sequences, tracrRNA sequences, or target DNA sequences.

38. A kit comprising: The fusion protein of any one of claims 18 to 28, the polynucleotide molecule of claim 29 or 30, the expression vector of any one of claims 31 to 34, or the molecular complex of claim 37.

39. A method for editing the nucleobase of a nucleic acid sequence in vitro, the method comprising contacting the nucleic acid sequence with a base editor comprising a fusion protein as described in any one of claims 18 to 28, and converting the cytosine of the nucleic acid sequence to thymidine.

40. The method of claim 39, further comprising contacting the nucleic acid sequence with a guide polynucleotide.

41. The method of claim 39, wherein the base editor deaminates the target nucleobase with greater efficiency than the cytidine base editor fusion protein comprising rat APOBEC-1 cytidine deaminase and Cas9 nickase.

42. The method of claim 39, wherein the contacting is performed in a cell.

43. The method of claim 42, wherein the cell is a mammalian cell.

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