GENE EDITING SYSTEMS COMPRISING REVERSE TRANSCRIPTASES
The disclosure relates generally to gene editing systems comprising reverse transcriptases and fusion proteins of reverse transcriptases with nickases or nucleases, methods of making such reverse transcriptases and fusion proteins, and methods of using such reverse transcriptases and fusion proteins for site directed genome editing in cells.
1 . A fusion protein comprising a nickase linked to a reverse transcriptase using a linker, wherein the reverse transcriptase comprises at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.
2 . A fusion protein comprising a nuclease linked to a reverse transcriptase using a linker, wherein the reverse transcriptase comprises at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.
3 . A fusion protein comprising a catalytically dead nuclease linked to a reverse transcriptase using a linker, wherein the reverse transcriptase comprises at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.
4 . A gene editing system, comprising:
a) a nickase;
b) a guide nucleic acid configured to form a complex with the nickase and to hybridize to a target nucleic acid sequence; and
c) a reverse transcriptase having at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585 and configured to form a complex with the nickase.
5 . The gene editing system of claim 4 , wherein the nickase is a modified endonuclease.
6 . The gene editing system of claim 5 , wherein the modified endonuclease is a Type II CRISPR endonuclease.
7 . The gene editing system of claim 5 , wherein the modified endonuclease is a Type V CRISPR endonuclease.
8 . The gene editing system of any one of claims 6-7 , wherein the Type II CRISPR endonuclease or the Type V CRISPR endonuclease has nickase activity.
9 . The gene editing system of claim 5 , wherein the modified endonuclease is selected from the group consisting of: spCas9 (H840A), spCas9 (D10A), nMG3-6 (D13A), nMG3-6 (H586A), nMG3-6 (N609A), Cas12a, and MG29-1.
10 . The gene editing system of claim 5 , wherein the modified endonuclease comprises at least about 80% sequence identity to any one of SEQ ID NOs: 152-154.
11 . The gene editing system of any one of claims 4-10 , wherein the nickase and the reverse transcriptase are fused.
12 . The gene editing system of any one of claims 4-10 , wherein the nickase and the reverse transcriptase are linked by a linker.
13 . The gene editing system of claim 12 , wherein the linker comprises at least 10, 20, or 30 amino acids.
14 . The gene editing system of claim 12 , wherein the linker comprises about 30-35 amino acids.
15 . The gene editing system of claim 12 , wherein the linker comprises about 30 amino acids.
16 . The gene editing system of claim 12 , wherein the linker comprises at least 80% sequence identity to SEQ ID NO: 103.
17 . The gene editing system of claim 12 , wherein the linker comprises at least 80% sequence identity to any one of SEQ ID NOs: 155-160.
18 . The gene editing system of any one of claims 4-10 , wherein the nickase and the reverse transcriptase are not linked.
19 . The gene editing system of any one of claims 4-18 , wherein the guide nucleic acid comprises a spacer sequence and a crRNA.
20 . The gene editing system of any one of claims 4-19 , wherein the guide nucleic acid further comprises a reverse transcriptase template (RTT).
21 . The gene editing system of claim 20 , wherein a base in the RTT comprises a bulky modification selected from the group of complex sugars, or complex amino groups, and/or other modifications compatible with RNA.
22 . The gene editing system of any one of claims 4-21 , wherein the guide nucleic acid further comprises a primer binding site.
23 . The gene editing system of claim 22 , wherein the primer binding site is on a 3′ end of the guide nucleic acid.
24 . The gene editing system of any one of claims 22-23 , wherein the primer binding site comprises at least 2, 4, 6, 8, 10, 13, 16, 20, 24, 28, 32, 36, 40, 45, 50, 55, 60, or 65 nucleotides.
25 . The gene editing system of any one of claims 4-24 , wherein the nuclease is non-covalently linked to the guide nucleic acid.
26 . The gene editing system of any one of claims 4-24 , wherein the nuclease is covalently linked to the guide nucleic acid.
27 . The gene editing system of any one of claims 4-24 , wherein the nuclease is fused to the guide nucleic acid.
28 . The gene editing system of any one of claims 4-24 , further comprising a transposase, integrase, or homing endonuclease.
29 . The gene editing system of any one of claims 4-28 , further comprising a retrotransposon.
30 . The gene editing system of any one of claims 4-29 , wherein the reverse transcriptase comprises a processivity of at least about 2-fold more than Moloney Murine Leukemia Virus (MMLV) reverse transcriptase.
31 . The gene editing system of any one of claims 4-29 , wherein the reverse transcriptase comprises a processivity of at least about 2-fold less than Moloney Murine Leukemia Virus (MMLV) reverse transcriptase.
32 . The gene editing system of any one of claims 4-31 , wherein the reverse transcriptase comprises an error rate of less than about 2.5%, 2.0%, 1.5%, 1%, 0.5%, 0.25%, 0.10%, or 0.05%.
33 . The gene editing system of any one of claims 4-32 , wherein the reverse transcriptase comprises an error rate of less than about 2.5%, 2.0%, 1.5%, 1%, 0.5%, 0.25%, 0.10%, or 0.05% as compared to Moloney Murine Leukemia Virus (MMLV) reverse transcriptase.
34 . A gene editing system, comprising:
a) a nuclease;
b) a guide nucleic acid configured to form a complex with the nuclease and to hybridize to a target nucleic acid sequence; and
c) a reverse transcriptase having at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585 and configured to form a complex with the nuclease.
35 . The gene editing system of claim 34 , wherein the nuclease is a double strand nuclease.
36 . The gene editing system of any one of claims 34-35 , wherein the nuclease is a Type II CRISPR endonuclease.
37 . The gene editing system of claim 36 , wherein the CRISPR endonuclease is Cas9.
38 . The gene editing system of claim 37 , wherein the Cas9 is catalytically dead Cas9 (dCas9).
39 . The gene editing system of any one of claims 34-38 , wherein the nuclease and the reverse transcriptase are fused.
40 . The gene editing system of any one of claims 34-38 , wherein the nuclease and the reverse transcriptase are linked by a linker.
41 . The gene editing system of claim 40 , wherein the linker comprises at least 10, 20, or 30 amino acids.
42 . The gene editing system of claim 40 , wherein the linker comprises about 30-35 amino acids.
43 . The gene editing system of claim 40 , wherein the linker comprises about 30 amino acids.
44 . The gene editing system of claim 40 , wherein the linker comprises at least 80% sequence identity to SEQ ID NO: 103.
45 . The gene editing system of claim 40 , wherein the linker comprises at least 80% sequence identity to any one of SEQ ID NOs: 155-160.
46 . The gene editing system of any one of claims 34-38 , wherein the nuclease and the reverse transcriptase are not linked.
47 . The gene editing system of any one of claims 34-46 , wherein the guide nucleic acid further comprises a primer binding site.
48 . The gene editing system of claim 47 , wherein the primer binding site is on a 3′ end of the guide nucleic acid.
49 . The gene editing system of any one of claims 47-48 , wherein the primer binding site comprises at least 2, 4, 6, 8, 10, 13, 16, 20, 24, 28, 32, 36, 40, 45, 50, 55, 60, or 65 nucleotides.
50 . The gene editing system of any one of claims 34-49 , wherein the nuclease is non-covalently linked to the guide nucleic acid.
51 . The gene editing system of any one of claims 34-49 , wherein the nuclease is covalently linked to the guide nucleic acid.
52 . The gene editing system of any one of claims 34-49 , wherein the nuclease is fused to the guide nucleic acid.
53 . The gene editing system of any one of claims 34-52 , further comprising a transposase, integrase, or homing endonuclease.
54 . The gene editing system of any one of claims 34-53 , further comprising a retrotransposon.
55 . The gene editing system of any one of claims 34-54 , wherein the reverse transcriptase comprises a processivity of at least about 2-fold more than Moloney Murine Leukemia Virus (MMLV) reverse transcriptase.
56 . The gene editing system of any one of claims 34-54 , wherein the reverse transcriptase comprises a processivity of at least about 2-fold less than Moloney Murine Leukemia Virus (MMLV) reverse transcriptase.
57 . The gene editing system of any one of claims 34-56 , wherein the reverse transcriptase comprises an error rate of less than about 2.5%, 2.0%, 1.5%, 1%, 0.5%, 0.25%, 0.10%, or 0.05%.
58 . The gene editing system of any one of claims 34-56 , wherein the reverse transcriptase comprises an error rate of less than about 2.5%, 2.0%, 1.5%, 1%, 0.5%, 0.25%, 0.10%, or 0.05% as compared to Moloney Murine Leukemia Virus (MMLV) reverse transcriptase.
59 . A gene editing system, comprising:
a) a nickase;
b) a guide nucleic acid configured to form a complex with the nickase and to hybridize to a target nucleic acid sequence; and
c) a reverse transcriptase configured to form a complex with the nickase, the reverse transcriptase having a X 1 X 2 DD motif, wherein X 1 is F or Y, and wherein when X 1 is Y, X 2 is A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, V, W, or Y.
60 . The gene editing system of claim 59 , wherein the X 2 is A or I.
61 . The gene editing system of claim 59 , wherein the X 1 X 2 DD motif is YADD (SEQ ID NO: 2572) or YIDD (SEQ ID NO: 2573).
62 . The gene editing system of claim 59 , wherein the X 1 X 2 DD motif is FADD (SEQ ID NO: 2574), FVDD (SEQ ID NO: 2575), FIDD (SEQ ID NO: 2576), or FLDD (SEQ ID NO: 2577).
63 . The gene editing system of any one of claims 59-62 , wherein the reverse transcriptase has at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.
64 . A gene editing system, comprising:
a) a nuclease;
b) a guide nucleic acid configured to form a complex with the nuclease and to hybridize to a target nucleic acid sequence; and
c) a reverse transcriptase configured to form a complex with the nuclease, the reverse transcriptase having a X 1 X 2 DD motif, wherein X 1 is F or Y, and wherein when X 1 is Y, X 2 is A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, V, W, or Y.
65 . The gene editing system of claim 64 , wherein the X 2 is A or I.
66 . The gene editing system of claim 64 , wherein the X 1 X 2 DD motif is YADD (SEQ ID NO: 2572) or YIDD (SEQ ID NO: 2573).
67 . The gene editing system of claim 64 , wherein the X 1 X 2 DD motif is FADD (SEQ ID NO: 2574), FVDD (SEQ ID NO: 2575), FIDD (SEQ ID NO: 2576), or FLDD (SEQ ID NO: 2577).
68 . The gene editing system of any one of claims 64-67 , wherein the reverse transcriptase has at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.
69 . An isolated reverse transcriptase having at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.
70 . A nucleic acid encoding for the fusion protein of any one of claims 1-3 or the gene editing system of any one of claims 4-68 .
71 . The nucleic acid of claim 70 , wherein the nucleic acid is a DNA or an RNA.
72 . The nucleic acid of claim 71 , wherein the RNA is an mRNA.
73 . A vector comprising the nucleic acid of any one of claims 70-72 .
74 . An adeno-associated virus or a lipid nanoparticle comprising the nucleic acid of any one of claims 70-72 or the vector of claim 73 .
75 . A cell comprising the nucleic acid of any one of claims 70-72 or the vector of claim 73 .
76 . The cell of claim 75 , wherein the cell is a human cell.
77 . The cell of claim 75 , wherein the cell is a eukaryotic cell.
78 . The cell of claim 75 , wherein the cell is a mammalian cell.
79 . The cell of claim 75 , wherein the cell is an immortalized cell.
80 . The cell of claim 75 , wherein the cell is an insect cell.
81 . The cell of claim 75 , wherein the cell is a yeast cell.
82 . The cell of claim 75 , wherein the cell is a plant cell.
83 . The cell of claim 75 , wherein the cell is a fungal cell.
84 . The cell of claim 75 , wherein the cell is a prokaryotic cell.
85 . The cell of claim 75 , wherein the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary cell, or a derivative thereof.
86 . The cell of claim 75 , wherein the cell is an engineered cell.
87 . The cell of claim 75 , wherein the cell is a stable cell.
88 . A method for modifying a double- and/or single-stranded nucleic acid, comprising contacting a cell using the fusion protein of any one of claims 1-3 or the gene editing system of any one of claims 4-68 .
89 . A method for modifying a double- and/or single-stranded nucleic acid, comprising:
a) providing a cell with a guide nucleic acid to bind to a target strand of the nucleic acid;
b) providing the cell with a nuclease or nickase to cleave the nucleic acid at a location of binding of the guide nucleic acid;
c) providing the cell with a reverse transcriptase to synthesize a modification in the target strand of the nucleic acid at a location of cleavage by the nickase and/or nuclease.
90 . The method of claim 89 , wherein the reverse transcriptase has at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.
91 . The method of claim 89 , wherein the modification is an insertion, deletion, or mutation.
92 . The method of claim 89 , further comprising providing an RNA or DNA template.
93 . The method of claim 89 , wherein the nucleic acid is a genome or a vector.
94 . The method of claim 89 , further comprising providing the cell with a transposase, integrase, or homing endonuclease.
95 . The method of claim 89 , further comprising providing the cell with a retrotransposon.