Novel base editors and uses thereof

The fusion protein of any preceding claim, wherein the conversion of the first deoxyribonucleotide to the fourth deoxyribonucleotide is dG-to-dA, dG-to-dT, dG-to-dC, dT-to-dA, dT-to-dC, dT-to-dG, dC-to-dA, dC-to-dT, or dC-to-dG.

The fusion protein of any preceding claim, wherein the base excising domain comprises a glycosylase.

The fusion protein of any preceding claim, wherein the glycosylase is selected from the group consisting of N-methylpurine DNA glycosylase (MPG), 8-oxoguanine DNA glycosylase (OGG1), methyl-CpG binding domain 4, DNA glycosylase (MBD4), thymine DNA glycosylase (TDG), uracil DNA glycosylase (UNG), single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), mutY DNA glycosylase (MUTYH), nth like DNA glycosylase 1 (NTHL1), nei like DNA glycosylase 1 (NEIL1), nei like DNA glycosylase 2 (NEIL2), nei like DNA glycosylase 3 (NEIL3), and mutants thereof capable of recognizing and excising a base from a nucleotide of a nucleic acid.

The fusion protein of any preceding claim, wherein the base excising domain comprises an N-methylpurine DNA glycosylase (MPG).

The fusion protein of any preceding claim, wherein the MPG comprises an amino acid substitution relative to a reference MPG of SEQ ID NO:

• 7 at a position that is corresponding to a position or that is a position selected from the group consisting of N169, D175, C178, and/or Q294 of the reference MPG, wherein the position is numbered according to SEQ ID NO:
• 1.

The fusion protein of any preceding claim, wherein the amino acid substitution is a substitution with R, A, N, or G.

The fusion protein of any preceding claim, wherein the MPG comprises an amino acid substitution relative to a reference MPG of SEQ ID NO:

• 7 that is corresponding to a substitution or that is a substitution selected from the group consisting of N169G, D175R, C178N, Q294R, and a combination of any two or more substitutions thereof, wherein the position is numbered according to SEQ ID NO:
• 1.

The fusion protein of any preceding claim, wherein the MPG comprises a combination substitution relative to a reference MPG of SEQ ID NO:

• 7 that is corresponding to a combination substitution of N169G, D175R, C178N, and Q294R, wherein the position is numbered according to SEQ ID NO:
• 1.

The fusion protein of any preceding claim, wherein the MPG comprises, consists essentially of, or consists of an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to SEQ ID NO:

• 8, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38.

The fusion protein of any preceding claim, wherein the MPG is (substantially) capable of excising guanine of dG.

The fusion protein of any preceding claim, wherein the base excising domain comprises an uracil-DNA glycosylase (UNG).

The fusion protein of any preceding claim, wherein the UNG comprises an amino acid substitution relative to a reference UNG of SEQ ID NO:

• 135 or 137 at a position that is corresponding to a position or that is a position selected from the group consisting of K184, A214, Q259, and/or Y284 of the reference UNG, wherein the position is numbered according to SEQ ID NO:
• 133.

The fusion protein of any preceding claim, wherein the amino acid substitution is a substitution with A, D, V, or T.

The fusion protein of any preceding claim, wherein the UNG comprises an amino acid substitution relative to a reference UNG of SEQ ID NO:

• 135 or 137 that is corresponding to a substitution or that is a substitution selected from the group consisting of K184A, A214V, A214T, Q259A, Y284D, and a combination of any two or more substitutions thereof, wherein the position is numbered according to SEQ ID NO:
• 133.

The fusion protein of any preceding claim, wherein the UNG comprises a deletion of amino acids at positions that are corresponding to positions or that are positions 1-65, 1-66, 1-67, 1-68, 1-69, 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-76, 1-77, 1-78, 1-79, 1-80, 1-81, 1-82, 1-83, 1-84, 1-85, 1-86, 1-87, 1-88, 1-89, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, 1-96, 1-97, 1-98, 1-99, or 1-100 of the reference UNG of SEQ ID NO:

• 135 or 137, wherein the position is numbered according to SEQ ID NO:
• 133.

The fusion protein of any preceding claim, wherein the UNG comprises an amino acid substitution relative to a reference UNG of SEQ ID NO:

• 135 that is corresponding to a substitution or that is a substitution selected from the group consisting of A214T, Q259A, Y284D, and a combination of any two or more substitutions thereof, and comprises a deletion of amino acids at positions that are corresponding to positions or that are positions 1-88 of the reference UNG, wherein the position is numbered according to SEQ ID NO:
• 133.

The fusion protein of any preceding claim, wherein the UNG comprises an amino acid substitution relative to a reference UNG of SEQ ID NO:

• 137 that is corresponding to a substitution or that is a substitution selected from the group consisting of K184A, A214V, and a combination of the two substitutions, and comprises a deletion of amino acids at positions that are corresponding to positions or that are positions 1-88 of the reference UNG, wherein the position is numbered according to SEQ ID NO:
• 133.

The fusion protein of any preceding claim, wherein the UNG comprising said amino acid mutation comprises, consists essentially of, or consists of an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of any one of SEQ ID NOs:

• 56, 58, 60, 62, 135, 137, 139, 141, 144, 146, 148, 150, 152, 155, 157, and 159 or an N-terminal truncation thereof lacking the most N-terminal Methionine (M) (coded by start codon ATG).

The fusion protein of any preceding claim, wherein the UNG is (substantially) capable of excising thymine of dT.

The fusion protein of any preceding claim, wherein the UNG is (substantially) capable of excising cytosine of dC.

The fusion protein of any preceding claim, wherein the napDNAbd is RNA programmable DNA binding protein.

The fusion protein of any preceding claim, wherein the napDNAbd is selected from the group consisting of CRISPR-associated (Cas) protein, IscB, IsrB, Argonaute, and TnpB.

The fusion protein of any preceding claim, wherein the napDNAbd is a nickase, e.g., a Cas9 nickase, an IscB nickase.

The fusion protein of any preceding claim, wherein the napDNAbd is nuclease-inactive, e.g., dead Cas9, dead Cas12i.

The fusion protein of any preceding claim, wherein the napDNAbd comprise an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to SEQ ID NO:

• 2, 48, 50, 52, or 163.

The fusion protein of any preceding claim, wherein the fusion protein comprises, from N-terminal to C-terminal, (1) the napDNAbp and the base excising domain;

• or (2) the base excising domain and the napDNAbp.

The fusion protein of any preceding claim, wherein the napDNAbd (e.g., Cas9) is a two-part napDNAbd, for example, a two-part split Cas9, comprising a N-terminal portion and a C-terminal portion, and wherein the fusion protein comprises, from N-terminal to C-terminal, (1) the N-terminal portion of the napDNAbd, the base excising domain, and the C-terminal portion of the napDNAbd;

• (2) the C-terminal portion of the napDNAbd, the base excising domain, and the N-terminal portion of the napDNAbd;
• or (3) the base excising domain, the C-terminal portion of the napDNAbd (e.g., amino acids at positions 1249-1368), and the N-terminal portion (e.g., amino acids at positions 1-1248) of the napDNAbd.

The fusion protein of any preceding claim, wherein the napDNAbd is SpCas9 (e.g., a SpCas9 nickase) or a mutant thereof (e.g., a SpG Cas9 nickase).

The fusion protein of any preceding claim, wherein the N-terminal portion of the napDNAbd is the amino acids of the napDNAbp at positions 1 or 2 to 1012, 1028, 1041, 1046, 1047, 1248, 1249, or 1300.

The fusion protein of any preceding claim, wherein the C-terminal portion of the napDNAbd is the amino acids of the napDNAbp at positions 1013, 1029, 1042, 1047, 1048, 1249, 1063, 1064, 1230, 1249, or 1301 to 1368.

The fusion protein of any preceding claim, wherein the fusion protein comprises an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to any one of SEQ ID NOs:

• 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 55, 57, 59, 61, 63, 136, 138, 140, 142, 143, 145, 147, 149, 151, 153, 154, 156, 158, 160, 161, 162, and 164.

The fusion protein of any preceding claim, wherein the guide nucleic acid is a guide RNA (gRNA).

The fusion protein or system of any preceding claim, wherein the fusion protein or system further comprises a translesion synthesis (TLS) polymerase or a recruiting domain or component capable of recruiting a TLS polymerase.

The fusion protein or system of any preceding claim, wherein TLS polymerase is selected from the group consisting of Polα (alpha), Polβ (beta), Polδ (delta) (PCNA), Polγ (gamma), Polη (eta), Polι (iota), Polκ (kappa), Polλ (lamda), Polμ (mu), Polν (nu), Polθ (theta), and REV1.

A method of modifying a target dsDNA, comprising contacting the target dsDNA with the system of any preceding claim, the target dsDNA comprising:

• (a) a first deoxyribonucleotide (e.g., dG (deoxyguanosine), dT (thymidine), dC (deoxycytidine)) in a protospacer sequence on a nontarget strand (edited strand) of the target dsDNA, and
• (b) a second deoxyribonucleotide (e.g., dC (deoxycytidine), dA (deoxyadenosine), dG (deoxyguanosine)) base pairing with the first deoxyribonucleotide (e.g., dG, dT, dC) and in a target sequence on the target strand (non-edited strand) of the target dsDNA, wherein the protospacer sequence is fully reverse complementary to the target sequence;
• wherein the method does not include deamination of the base of the first deoxyribonucleotide before the excision of the base of the first deoxyribonucleotide.

The method of any preceding claim, wherein the method does not include deamination of the base of the first deoxyribonucleotide.

An MPG as defined in any preceding claim.

An UNG as defined in any preceding claim.