Methods for generating single- or multi-site mutagenesis
Deep mutational scanning is a foundational tool for addressing functional consequences of large numbers of mutants, yet a more efficient and accessible method for construction of user-defined mutagenesis libraries is needed. Provided herein are nicking saturation mutagenesis, a single-day, single-pot saturation mutagenesis method using routinely prepped plasmid dsDNA as input substrate. Reproducibility and convenience of the method are demonstrated through validation by an external research laboratory.
1. A method comprising the steps of:
(a) providing a double stranded nucleic acid molecule, wherein the double stranded nucleic acid molecule comprises a BbvCI nickase recognition site;
(b) providing a first nickase, wherein said first nickase nicks one strand of the nucleic acid molecule to create a first nicked strand and a remaining wild-type strand;
(c) providing a first exonuclease, wherein said first exonuclease digests the first nicked strand;
(d) providing at least one first mutagenic oligonucleotide, wherein said at least one first mutagenic oligonucleotide anneals to the remaining wild-type strand;
(e) providing a first polymerase, wherein said first polymerase extends said at least one first mutagenic oligonucleotide around the remaining wild-type strand;
(f) providing a first ligase, wherein said first ligase ligates the extended strand to form a double stranded nucleic acid comprising a mutant strand and a wild-type strand;
(g) purifying the double stranded nucleic acid from step (f);
(h) providing a second nickase, wherein said second nickase nicks the wild-type strand to create a second nicked strand and a remaining mutant strand;
(i) providing a second exonuclease, wherein said second exonuclease digests the second nicked strand;
(j) providing at least one second mutagenic oligonucleotide, wherein said at least one second mutagenic oligonucleotide anneals to the remaining mutant strand;
(k) providing a second polymerase, wherein said second polymerase extends said at least one second mutagenic oligonucleotide around the remaining mutant strand;
(l) providing a second ligase, wherein said second ligase ligates the extended strand to form a double stranded nucleic acid comprising a double stranded mutant nucleic acid molecule; and
(m) purifying the double stranded mutant nucleic acid molecule from step (l).
2. The method of claim 1 , wherein the nucleic acid molecule is DNA, cDNA, or genomic DNA.
3. The method of claim 1 , wherein the BbvCI restriction site is at least 7 base pairs.
4. The method of any one of claim 1 or 2 - 3 , wherein the first or second nickase is Nt.BbvCI or Nb.BbvCI.
5. The method of any one of claim 1 or 2 - 3 , wherein the first or second exonuclease is Exonuclease I, Exonuclease III, or both.
6. The method of any one of claims 1 - 2 or 3 - 5 , wherein the first or second polymerase is Q5 DNA Polymerase.
7. The method of any one of claims 1 - 2 or 3 - 6 , wherein the first or second ligase is Taq DNA ligase.
8. The method of any one of claims 1 - 2 or 3 - 6 , wherein the at least one first or second mutagenic oligonucleotide is provided at a primer:template ratio of between 1:5 to 1:50, wherein the at least one first or second mutagenic oligonucleotide is the template.
9. The method of claim 8 , wherein the primer to template ratio is 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.
10. The method of any one of claims 1 - 2 or 3 - 9 , further comprising the step of providing an enzyme to remove methylated nucleic acid molecules, hemimethylated nucleic acid molecules, or both.
11. The method of any one of claims 1 - 2 or 3 - 10 , wherein the mutation efficiency is enhanced to about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, or about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, wherein the enhanced mutation efficiency is characterized with increased transformants or transformation output.
12. A method of generating a double stranded mutant nucleic acid molecule comprising the steps of:
(a) providing a double stranded wild-type nucleic acid molecule, wherein the double stranded wild-type nucleic acid molecule comprises a BbvCI nickase recognition site;
(b) nicking one strand of the nucleic acid molecule with a first nickase to create a first nicked strand and a remaining wild-type strand;
(c) digesting said first nicked strand with a first exonuclease;
(d) annealing at least one first mutagenic oligonucleotide to the remaining wild-type strand;
(e) extending said at least one first mutagenic oligonucleotide around the remaining wild-type strand with a first polymerase;
(f) ligating the extended strand with a first ligase to form a double stranded nucleic acid comprising a mutant strand and a wild-type;
(g) purifying the double stranded nucleic acid from step (f);
(h) nicking the wild-type strand with a second nickase to create a second nicked strand and a remaining mutant strand;
(i) digesting said second nicked strand with a second exonuclease;
(j) annealing at least one second mutagenic oligonucleotide to the remaining mutant strand;
(k) extending said at least one second mutagenic oligonucleotide around the remaining mutant strand with a second polymerase;
(l) ligating the extended strand with a second ligase to form a double stranded mutant nucleic acid molecule; and
(m) purifying the double stranded mutant nucleic acid molecule from step (l).
13. The method of claim 12 , wherein the double stranded wild-type nucleic acid molecule is DNA, cDNA, or genomic DNA.
14. The method of claim 12 , wherein the BbvCI restriction site is at least 7 base pairs.
15. The method of any one of claims 12 - 13 or 14 , wherein the first or second nickase is Nt.BbvCI or Nb.BbvCI.
16. The method of any one of claims 12 - 13 or 14 - 15 , wherein the first or second exonuclease is Exonuclease I, Exonuclease III, or both.
17. The method of any one of claims 12 - 13 or 14 - 16 , wherein the first or second polymerase is Q5 DNA Polymerase.
18. The method of any one of claims 12 - 13 or 14 - 17 , wherein the first or second ligase is Taq DNA ligase.
19. The method of any one of claims 12 - 13 or 14 - 18 , wherein the at least one first or second mutagenic oligonucleotide is provided at a primer:template ratio of between 1:5 to 1:50, wherein the at least one first or second mutagenic oligonucleotide is the template.
20. The method of claim 19 , wherein the primer to template ratio is 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.
21. The method of any one of claims 12 - 13 or 14 - 20 , further comprising the step of providing an enzyme to remove methylated nucleic acid molecules, hemimethylated nucleic acid molecules, or both.
22. The method of any one of claims 12 - 13 or 14 - 21 , wherein the mutation efficiency is enhanced to about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, or about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, wherein the enhanced mutation efficiency is characterized with increased transformants or transformation output.
23. The method of any one of claims 12 - 22 for generating single-site saturation mutagenesis.
24. The method of any one of claims 12 - 22 for generating multi-site saturation mutagenesis.
25. The method of any one of claims 23 - 24 , wherein the mutagenesis is a three single or one triple-mutation.