Methods of producing and using single-stranded deoxyribonucleic acids and compositions for use in practicing the same
Methods of producing single-stranded deoxyribonucleic acids (ssDNAs) are provided. Aspects of the methods include generating a double stranded deoxyribonucleic acid (dsDNA) and then selectively degrading one strand of the dsDNA to produce a ssDNA. ssDNAs produced using methods of the invention find use in a variety of applications, including genomic modification applications. Also provided are compositions, e.g., kits, that find use in practicing various embodiments of the invention.
1. A method of producing a single-stranded DNA (ssDNA), the method comprising:
(a) generating a double-stranded DNA (dsDNA) comprising a product strand and a complementary strand that differ structurally from one another; and
(b) selectively degrading the complementary strand based on the structural difference between the product strand and the complementary strand to produce the ssDNA, the selective degradation comprising:
(i) contacting the dsDNA with a first exonuclease that comprises exonuclease activity that is 5′-phosphate-dependent to generate a partially degraded complementary strand, and
(ii) contacting the partially degraded complementary strand with a second exonuclease.
2. The method according to claim 1 , wherein the dsDNA comprises a 5′-phosphate in the complementary strand, one or more protective base modifications in the product strand or both.
3. The method according to claim 1 , wherein the second enzyme is a 3′ to 5′ exonuclease.
4. The method according to claim 1 , wherein the first exonuclease is a lambda exonuclease.
5. The method of claim 4 , wherein the 3′ to 5′ exonuclease is an exonuclease III.
6. The method according to claim 1 , wherein the dsDNA is generated by a polymerase chain reaction (PCR).
7. The method according to claim 6 , wherein a primer comprising a 5′ phosphate is incorporated into the dsDNA during the PCR.
8. The method according to claim 6 , wherein one or more protective base modifications are incorporated into the dsDNA during the PCR.
9. The method according to claim 8 , wherein a primer comprising at least one of the one or more protective base modifications is incorporated into the dsDNA during the PCR.
10. The method according to claim 1 , wherein the dsDNA is generated by a ligation reaction comprising the ligation of two or more dsDNA segments.
11. The method according to claim 10 , wherein one of the two or more dsDNA segments comprises a 5′ phosphate that, following the ligation reaction, provides a 5′ phosphate to the generated complementary strand.
12. The method according to claim 10 , wherein at least one of the two or more dsDNA segments comprises a protective base modification.
13. The method according to claim 1 , wherein the complementary strand comprises a 5′-phosphate-dependent exonuclease pausing sequence.
14. The method according to claim 1 , wherein the ssDNA is greater than 200 bases in length.
15. The method according to claim 14 , wherein the ssDNA is greater than 500 bases in length.
16. The method according to claim 15 , wherein the ssDNA is greater than 1000 bases in length.
17. The method according to claim 15 , wherein the ssDNA ranges from 500 to 10,000 bases in length.
18. The method according to claim 1 , wherein the method further comprises testing for the presence of the ssDNA.
19. The method according to claim 1 , wherein the dsDNA is blunt-ended at both ends.
20. The method according to claim 1 , wherein the first exonuclease and the second exonuclease are present together in a reaction mixture.
21. The method according to claim 20 , wherein the second exonuclease is added to a reaction mixture comprising the first exonuclease.
22. A method of modifying genomic DNA, the method comprising:
(a) producing a ssDNA by a method according to claim 1 ; and
(b) contacting a genome with a nuclease and the produced ssDNA under conditions permissive for homology-directed repair in order to generate a modified genome comprising the ssDNA.
23. The method according to claim 22 , wherein the contacting step is performed in vitro.
24. The method according to claim 22 , wherein the contacting step is performed in vivo.
25. The method according to claim 22 , wherein the nuclease is selected from the group consisting of: a Cas9 nuclease, a zinc-finger nuclease and a transcription activator-like effector nuclease.
26. The method according to claim 22 , wherein the dsDNA comprises a coding sequence or portion thereof.
27. The method according to claim 26 , wherein the coding sequence comprises an exon.
28. The method according to claim 27 , wherein the coding sequence comprises a gene or a cDNA.
29. The method according to claim 22 , wherein the dsDNA comprises a non-coding sequence or portion thereof.
30. The method according to claim 29 , wherein the non-coding sequence comprises a promoter, an enhancer, an intron or a combination thereof.
31. The method according to claim 22 , wherein the genome comprises a deleterious mutation and the modified genome comprises a correction of the deleterious mutation.
32. The method according to claim 22 , further comprising testing for the presence of the ssDNA in the modified genome.