DNA LIGASE COMPOSITIONS, METHODS, AND USES THEREOF

1 . A composition comprising:

(a) a donor nucleic acid;

(b) a polynucleotide encoding an engineered protein construct comprising a nickase or a variant thereof and a DNA ligase or a functional fragment thereof; and

(c) a guide polynucleotide, wherein the guide polynucleotide comprises:

(i) a targeting region that has complementarity to a target nucleic acid;

(ii) a protein binding region, wherein the protein binding region comprises a secondary structure that binds to the engineered protein construct comprising a nickase region;

(iii) a ligation splint 2 region, wherein the ligation splint region has complementarity to the donor nucleic acid and has complementarity to the target nucleic acid, and wherein the ligation splint region comprises at least one alteration relative to the target nucleic acid; and

(iv) a ligation splint 1 region, wherein the ligation splint 1 region comprises: a deoxyribonucleotide and a ribonucleotide, wherein the ligation splint 1 region has complementarity to the target nucleic acid.

2 . The composition of claim 1 , wherein the ligation splint 1 region forms a DNA-RNA (DR)-loop upon association with the target nucleic acid and a DNA ligase.

3 . The composition of claim 2 , wherein the ligation splint 1 region comprises a ratio of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs) of: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 2:1, 2:3, 2:5, 2:7, 2:9, 2:11, 2:13, 2:15, 2:17, 2:19, 3:1, 3:2, 3:4, 3:5, 3:7, 3:8, 3:10, 3:11, 3:13, 3:14, 3:15, 3:16, 3:17, 3:19, 4:1, 4:3, 4:5, 4:7, 4:9, 4:11, 4:13, 4:15, 4:17, 4:19, 5:1, 5:2, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 5:11, 5:12, 5:13, 5:14, 5:16, 6:1, 6:5, 6:7, 6:9, 6:11, 6:13, 6:15, 7:1, 7:2, 7:3, 7:4, 7:5, 7:6, 7:8, 7:9, 7:10, 7:11, 7:12, 7:13, 7:15, 8:1, 8:3, 8:5, 8:7, 8:9, 8:11, 8:13, 8:15 9:1, 9:2, 9:4, 9:5, 9:7, 9:8, 9:10, 9:11, 9:13, 9:15, 9:17, 9:19, 9:20, 10:1, 10:3, 10:7, 10:9, 10:11, 10:13, 10:15, 10:17, 10:19, 11:1, 11:2, 11:3, 11:4, 11:5, 11:6, 11:7, 11:8, 11:9, 11:10, 11:12, 11:13, 11:15, 12:1, 12:5, 12:7, 12:9, 12:11, 12:13, 13:1, 13:2, 13:3, 13:4, 13:5, 13:6, 13:7, 13:8, 13:9, 13:10, 13:11, 13:12, 13:14, 14:1, 14:3, 14:5, 14:9, 14:11, 14:13, 15:1, 15:2, 15:4, 15:6, 15:8, 15:11, 15:13, 16:1, 16:3, 16:5, 16:7, 16:9, 16:11, 16:13, 16:15, 17:1, 17:2, 17:3, 17:4, 17:5, 17:6, 17:7, 17:8, 17:9, 17:10, 17:11, 17:12, 17:13, 17:14, 17:15, 17:16, 18:1, 18:5, 18:7, 18:11, 18:13, 18:17, 19:1, 19:2, 19:3, 19:4, 19:5, 19:6, 19:7, 19:8, 19:9, 19:10, 19:11, 19:12, 19:13, 19:14, 19:15, 19:16, 19:17, 19:18 or 20:1.

4 . The composition of claim 1 , wherein the ligation splint 2 region comprises at least about 5 nucleotides up to 10,000 nucleotides.

5 . The composition of claim 1 , wherein the ligation splint 1 region comprises at least about 5 nucleotides up to 20 nucleotides.

6 . The composition of claim 1 , wherein the ligation splint 2 region comprises a reverse complement sequence of

(1) a non-coding polynucleotide sequence or a variant thereof;

(2) a sequence encoding a coding region of a polynucleotide sequence or a variant thereof; or

(3) a sequence encoding for an exon or an intron.

7 . The composition of claim 1 , wherein the ligation splint 2 region comprises a complement sequence of a sequence encoding

(1) a non-coding polynucleotide sequence or a variant thereof;

(2) a coding region of a polynucleotide sequence or a variant thereof; or

(3) a sequence encoding for an exon or an intron.

8 . The composition of claim 1 , wherein the ligation splint 2 region comprises a sequence comprising at least one nucleobase that is complementary to or mismatched with a sequence encoding a splice acceptor site.

9 . The composition of claim 1 , wherein the nickase is an engineered Cas protein or a functional variant thereof comprising at least one amino acid substitution at position 840 corresponding to SEQ ID NO: 69.

10 . The composition of claim 9 , wherein the engineered Cas protein or a functional variant thereof further comprises at least one amino acid substitution at position 221, 394, or a combination thereof, corresponding to SEQ ID NO: 69.

11 . The composition of claim 9 , wherein the engineered Cas protein or a functional variant thereof comprises amino acid substitutions of R221K, N394K, H840A, or any combination thereof.

12 . The composition of claim 9 , wherein the engineered Cas protein or a functional variant thereof comprises a sequence 97%, 98%, 99%, or 100% identical to SEQ ID NO: 70 or SEQ ID NO: 71.

13 . The composition of claim 1 , wherein the DNA ligase or a functional fragment comprises an E. coli DNA ligase, a Taq DNA ligase, a T3 DNA ligase, a T4 DNA ligase, a T7 DNA ligase, a Chlorella virus DNA ligase, a human DNA ligase I, a human DNA ligase II, a human DNA ligase III, a human DNA ligase IV, a human ligase V, a variant, or a combination thereof.

14 . The composition of claim 1 , wherein the DNA ligase or a functional fragment thereof comprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NOS: 58-66.

15 . The composition of claim 1 , wherein the DNA ligase or a functional fragment thereof is linked to the nickase or a variant thereof.

16 . The composition of claim 1 , wherein the engineered protein construct comprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NOS: 89-101.

17 . An engineered fusion protein comprising:

(a) a DNA ligase or a functional fragment thereof; and

(b) an engineered nickase that comprises three amino acid substitutions at positions corresponding to amino acid positions 221, 394, and 840 of a nuclease comprising a sequence of SEQ ID NO: 69.

18 . A system for modifying a target nucleic acid, the system comprising:

(a) a donor nucleic acid;

(b) an engineered protein construct comprising a nickase region or a variant thereof and a DNA ligase or a functional fragment thereof, or a polynucleotide encoding the engineered protein construct; and

(c) a guide polynucleotide, wherein the guide polynucleotide comprises:

(i) a targeting region that has complementarity to a target nucleic acid;

(ii) a protein binding region, wherein the protein binding region comprises a secondary structure that binds to the engineered protein construct comprising a nickase region;

(iii) a ligation splint 2 region, wherein the ligation splint region has complementarity to the donor nucleic acid and has complementarity to the target nucleic acid, and wherein the ligation splint region comprises at least one alteration relative to the target nucleic acid; and

(iv) a ligation splint 1 region, wherein the ligation splint 1 region comprises: a deoxyribonucleotide and a ribonucleotide, wherein the ligation splint 1 region has complementarity to the target nucleic acid,

wherein upon introduction of the system to a cell, a nucleus, or a cell-free system, the system incorporates the donor nucleic acid into the target nucleic acid, thereby modifying the target nucleic acid.

19 . A method of ligating a donor nucleic acid with a target nucleic acid, the method comprising:

contacting a cell or a cell-free system with:

(a) a donor nucleic acid

(b) a guide polynucleotide or a polynucleotide encoding the guide polynucleotide, wherein the guide polynucleotide comprises:

(i) a targeting region that has complementarity to a target nucleic acid;

(ii) a protein binding region, wherein the protein binding region comprises a secondary structure that binds to a nuclease; and

(iii) a ligation splint 2 region, wherein the ligation splint 2 region is complementary to the donor nucleic acid and has complementarity to the target nucleic acid and at least one alteration relative to the target nucleic acid;

(iv) a ligation splint 1 region, wherein the ligation splint 1 region comprises: a deoxyribonucleotide and a ribonucleotide;

(c) an engineered protein or a polynucleotide encoding the engineered protein, wherein the engineered protein comprises:

(i) a nuclease region; and

(ii) a DNA ligase region;

wherein:

the guide polynucleotide forms a complex with the engineered protein via the protein binding region,

the nuclease region of the engineered protein generates a break in the target nucleic acid to generate a leading strand and a complementary strand,

the targeting region forms a complex with the complementary strand,

the ligation splint 1 region forms a complex with the leading strand, and

wherein the DNA ligase region attaches the donor nucleic acid to the leading strand, thereby ligating the donor nucleic acid to the target nucleic acid.

20 . The method of claim 19 , wherein the targeting strand dissociates from the complementary strand.