DNA-DEPENDENT DNA POLYMERASE COMPOSITIONS, METHODS, AND USES THEREOF

1 . A composition comprising:

(a) a phi29 DNA-dependent DNA polymerase or a variant thereof and a nickase; or

(b) a polynucleotide encoding the phi29 DNA-dependent DNA polymerase or a variant thereof and a polynucleotide encoding the nickase; and, in addition to (a) or (b),

(c) a guide polynucleotide comprising:

(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 nickase; and

(iii) a DNA-dependent DNA polymerase synthesis template (DST), wherein the DNA-dependent DNA polymerase synthesis template (DST) region comprises: a sequence that has complementarity to the target nucleic acid and at least one alteration relative to the target nucleic acid or at least one nucleic acid strand thereof, and

(iv) a hybridization region, wherein the hybridization region comprises:

deoxyribonucleotides and ribonucleotides.

2 . The composition of claim 1 , wherein the nickase comprises a Cas9, a Cas1, a Cas2, or a Csn2 protein.

3 . The composition of claim 2 , wherein the Cas9 protein comprises a Streptococcus pyogenes Cas9 protein or a variant thereof, or

a Streptococcus thermophilus Cas9 protein or a variant thereof.

4 . The composition of claim 1 , wherein the phi29 DNA-dependent DNA polymerase comprises a sequence that is at least 90% identical to SEQ ID NO: 63.

5 . The composition of claim 1 , wherein the phi29 DNA-dependent DNA polymerase variant comprises a mutation, wherein the mutation comprises an amino acid substitution of an amino acid residue of position: 8, 12, 51, 66, 97, 137, 197, 221, 369, 372, 375, 377, 378, 497, 512, or 526 as compared to SEQ ID NO: 63.

6 . The composition of claim 5 , wherein the amino acid substitution at any one of positions 8, 12, 51, 66, 97, 137, 197, 221, 369, 372, 375, 377, 378, 497, 512, or 526 comprises a substitution of an amino acid residue to a different amino acid residue, wherein the different amino acid residue is a hydrophobic amino acid residue, a hydrophilic amino acid residue, a charged amino acid residue that is a basic amino acid residue or an acidic amino acid residue, or an aliphatic amino acid residue as compared to SEQ ID NO: 63.

7 . The composition of claim 5 , wherein the amino acid substitution at any one of positions 8, 12, 51, 66, 97, 137, 197, 221, 369, 372, 375, 377, 378, 497, 512, or 526 comprises a substitution of an amino acid residue to a different amino acid residue, wherein the different amino acid residue is an arginine (Arg, R), an alanine (Ala, A), a glutamic acid (Glu, E), a proline (Pro, P), a threonine (Thr, T), an aspartic acid (Asp, D), a cysteine (Cys, C), a leucine (Leu, L) or a lysine (Lys, K) as compared to SEQ ID NO: 63.

8 . The composition of claim 6 , wherein the amino acid substitution comprises: M8R, D12A, V51A, D66A, M97T, F137C, G197D, E221K, Y369E, T372N, E375D, A377C, I378R, Q497P, K512E, F526L, or any combination thereof as compared to SEQ ID NO: 63.

9 . The composition of claim 1 , wherein the phi29 DNA-dependent DNA polymerase variant comprises any one of SEQ ID NO: 99-SEQ ID NO: 106.

10 . The composition of claim 1 , wherein the phi29 DNA-dependent DNA polymerase variant comprises SEQ ID NO: 102.

11 . The composition of claim 1 , wherein the phi29 DNA-dependent DNA polymerase variant comprises SEQ ID NO: 103.

12 . The composition of claim 1 , wherein the phi29 DNA-dependent DNA polymerase variant comprises SEQ ID NO: 104.

13 . The composition of claim 1 , wherein the polynucleotide encoding the phi29 DNA-dependent DNA polymerase or a variant thereof is linked to the polynucleotide encoding the nickase.

14 . The composition of claim 1 , wherein the phi29 DNA-dependent DNA polymerase or a variant thereof is linked to the nickase.

15 . The composition of claim 1 , wherein the hybridization region comprises at least about 5 nucleotides up to 10 nucleotides.

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

17 . The composition of claim 1 , wherein the DST region comprises at least about 5 nucleotides up to 10,000 nucleotides.

18 . The composition of claim 1 , further comprising a nicking gRNA.

19 . A composition comprising:

(a) a DNA-dependent DNA polymerase or a variant thereof and a Streptococcus pyogenes Cas9 nickase or a variant thereof, wherein the variant comprises an amino acid substitution at position: 61, 221, 394, 840, 1111, 1135, 1136, 1137, 1218, 1219, 1317, 1322, 1333, 1335, or 1337 as compared to SEQ ID NO: 92; or

(b) a polynucleotide encoding the DNA-dependent DNA polymerase or a variant thereof and a polynucleotide encoding the Streptococcus pyogenes Cas9 nickase or the variant thereof, and, in addition to (a) or (b),

(c) a guide polynucleotide comprising:

(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 protein; and

(iii) a DNA-dependent DNA polymerase synthesis template (DST), wherein the DNA-dependent DNA polymerase synthesis template (DST) region comprises: a sequence that has complementarity to the target nucleic acid and at least one alteration relative to the target nucleic acid or at least one nucleic acid strand thereof, and

(iv) a hybridization region, wherein the hybridization region comprises:

deoxyribonucleotides and ribonucleotides.

20 . A system for synthesizing a nucleic acid sequence, the system comprising:

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

(i) a nickase region or a variant thereof, and

(ii) a DNA-dependent DNA polymerase (DdDP) or a variant thereof, and

(b) 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; and

(iii) a DNA-dependent DNA polymerase synthesis template (DST) region, wherein the DNA-dependent DNA polymerase synthesis template (DST) region comprises a sequence that has complementarity to the target nucleic acid and at least one alteration relative to the target nucleic acid; and

(iv) a hybridization region, wherein the hybridization region comprises: a deoxyribonucleotide and a ribonucleotide, wherein the hybridization 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 synthesizes a nucleic acid sequence that is incorporated into the target nucleic acid.

21 . A method of synthesizing a nucleic acid, the method comprising:

contacting a cell or a cell-free system with:

(a) 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;

(iii) a DNA-dependent DNA polymerase synthesis template (DST) region, wherein the DST region comprises a sequence that has complementarity to a target nucleic acid and at least one alteration relative to the target nucleic acid; and

(iv) a hybridization region, wherein the hybridization region comprises a deoxyribonucleotide and a ribonucleotide; and

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

(i) a nuclease region; and

(ii) a DdDP 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 sequence forms a complex with a complementary strand of the cleaved target nucleic acid, the hybridization region forms a complex with the leading strand,

and wherein the guide polynucleotide associates with the DdDP region of the engineered protein, thereby synthesizing a new nucleic acid.