MULTI-EFFECTOR NUCLEOBASE EDITORS AND METHODS OF USING SAME TO MODIFY A NUCLEIC ACID TARGET SEQUENCE
The invention features a multi-effector nucleobase editor capable of inducing changes at multiple different bases within a target nucleic acid and methods of using such editors.
1 . A multi-effector nucleobase editor polypeptide comprising a domain having nucleic acid sequence specific binding activity and two or more nucleobase editor domains selected from the group consisting of an adenosine deaminase, a cytidine deaminase, and an abasic editor.
2 . The polypeptide of claim 1 , further comprising one or more Nuclear Localization Signals (NLS) and/or one or more Uracil DNA glycosylase inhibitors (UGIs).
3 . The polypeptide of claim 2 , wherein the NLS is a bipartite NLS.
4 . The polypeptide of claim 3 , wherein the polypeptide comprises an N-terminal NLS and a C-terminal NLS.
5 - 6 . (canceled)
7 . The polypeptide of claim 1 , wherein the adenosine deaminase is a TadA deaminase.
8 . The polypeptide of claim 7 , wherein the TadA deaminase is a modified adenosine deaminase that does not occur in nature.
9 . The polypeptide of claim 1 , wherein the polypeptide comprises two adenosine deaminases that are the same or different.
10 . The polypeptide of claim 9 , wherein the two adenosine deaminases are capable of forming heterodimers or homodimers.
11 . The polypeptide of claim 10 , wherein the two adenosine deaminase domains are wild-type TadA and TadA7.10.
12 . The polypeptide of claim 1 , wherein the domain having nucleic acid sequence specific binding activity is a nucleic acid programmable DNA binding protein (napDNAbp).
13 . The polypeptide of claim 12 , wherein the napDNAbp domain comprises a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9.
14 . The polypeptide of claim 12 , wherein the napDNAbp is selected from the group consisting of Cas9, Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, and Cas12i, or active fragments thereof.
15 - 22 . (canceled)
23 . A multi-effector nucleobase editor polypeptide comprising one or more Nuclear Localization Signal (NLS), a napDNAbp, a Uracil DNA glycosylase inhibitor, an adenosine deaminase, and a cytidine deaminase.
24 - 27 . (canceled)
28 . A Multi-Effector Nucleobase Editor polypeptide comprising the following domains A-C, A-D, or A-E:
NH 2 -[A-B-C]-COOH,
NH 2 -[A-B-C-D]-COOH, or
NH 2 -[A-B-C-D-E]-COOH
wherein A and C or A, C, and E, each comprises one or more of the following:
an adenosine deaminase domain or an active fragment thereof,
a cytidine deaminase domain or an active fragment thereof,
a DNA glycosylase domain or an active fragment thereof; and
wherein B or B and D, each comprises one or more domains having nucleic acid sequence specific binding activity.
29 . The Multi-Effector Nucleobase Editor polypeptide of claim 28 , comprising:
NH 2 -[A n -B o -C n ]-COOH,
NH 2 -[A n -B o -C n -D o ]-COOH, or
NH 2 -[A n -B o -C p -D o -E q ]-COOH;
wherein A and C or A, C, and E, each comprises one or more of the following:
an adenosine deaminase domain or an active fragment thereof,
a cytidine deaminase domain or an active fragment thereof, and
a DNA glycosylase domain or an active fragment thereof; and
wherein n is an integer: 1, 2, 3, 4, or 5, wherein p is an integer: 0, 1, 2, 3, 4, or 5; wherein q is an integer 0, 1, 2, 3, 4, or 5; and
wherein B or B and D each comprises a domain having nucleic acid sequence specific binding activity; and wherein o is an integer: 1, 2, 3, 4, or 5.
30 - 48 . (canceled)
49 . A polynucleotide molecule encoding the multi-effector nucleobase editor polypeptide of claim 1 .
50 . (canceled)
51 . An expression vector comprising a polynucleotide molecule of claim 49 .
52 . The expression vector of claim 51 , wherein the expression vector is a mammalian expression vector; or wherein the vector is a viral vector selected from the group consisting of adeno-associated virus (AAV), retroviral vector, adenoviral vector, lentiviral vector, Sendai virus vector, and herpesvirus vector.
53 - 54 . (canceled)
55 . A cell comprising the polynucleotide of claim 49 .
56 . The cell of claim 55 , wherein the cell is a bacterial cell, plant cell, insect cell, or mammalian cell.
57 . A molecular complex comprising the multi-effector nucleobase editor polypeptide of claim 1 and one or more of a guide RNA, tracrRNA, or target DNA molecule.
58 . A kit comprising the multi-effector nucleobase editor polypeptide of claim 1 .
59 . A method of editing a nucleobase of a nucleic acid sequence, the method comprising contacting a nucleic acid sequence with a base editor comprising: the multi-effector nucleobase editor polypeptide of claim 1 and converting a first nucleobase of the nucleic acid sequence to a second nucleobase.
60 - 63 . (canceled)
64 . A method of editing a regulatory sequence present in the genome of a cell, the method comprising contacting a regulatory sequence with a base editor comprising: the multi-effector nucleobase editor polypeptide of claim 1 and converting a first and second nucleobase of the DNA sequence to a third and fourth nucleobase.
65 . A method of editing a genome of a cell, the method comprising contacting the genome with a base editor comprising: the multi-effector nucleobase editor polypeptide of claim 1 and converting a first and second nucleobase of the DNA sequence to a third and fourth nucleobase.
66 . (canceled)