Construction and methods of use of a barcoded and gene edited DNA library
Provided herein, in some embodiments, are methods and compositions for gene editing using a trackable gene library, for example, a barcoded and gene edited library.
1. A bacterial cell composition comprising:
(a) a recipient plasmid comprising:
(i) a selection marker;
(ii) a barcode landing sequence flanked by a region that is complementary to a portion of a barcode sequence in donor plasmid of (b); and
(iii) a target nucleic acid sequence that encodes a gene of interest and is flanked by a region that is complementary to a portion of a modified target nucleic acid sequence; and
(b) a donor plasmid comprising:
(i) a selection marker and a counter selection marker, wherein the selection marker and the counter selection marker are not the same;
(ii) a first guide nucleic acid sequence comprising a region that is complementary to a portion of the barcode landing sequence of (a)(ii), wherein the first guide nucleic acid sequence is operably linked to a constitutively active promoter;
(iii) the barcode sequence; and
(iv) an editing cassette comprising the modified target nucleic acid sequence linked to a mutated protospacer adjacent motif (mutated PAM) and a second guide nucleic acid sequence comprising a region that is complementary to a portion of the editing cassette, wherein the second guide nucleic acid sequence is operably linked to an inducible promoter;
(c) a nucleic acid-guided DNA binding protein; and
(d) a recombination system.
2. The bacterial cell composition of claim 1 , wherein the modified target nucleic acid sequence comprises at least one inserted, deleted, or substituted nucleic acid as compared to the target nucleic acid sequence.
3. The bacterial cell composition of claim 1 , wherein the nucleic acid-guided DNA binding protein is a Cas9 protein.
4. The bacterial cell composition of claim 1 , wherein the recombination system is selected from the group consisting of a lambda red recombination system, a Cre/Lox recombination system, and an attB/attP recombination system.
5. The bacterial cell composition of claim 1 , wherein the nucleic acid -guided DNA binding protein; the recombination system; or the nucleic acid-guided DNA binding protein and the recombination system are integrated in a genome of a cell.
6. The bacterial cell composition of claim 1 , wherein the nucleic acid-guided DNA binding protein; the recombination system; or the nucleic acid-guided DNA binding protein and the recombination system are expressed from a plasmid in a cell.
7. The bacterial cell composition of claim 1 , wherein at least one of the selection markers in (a) and (b) comprises an antibiotic resistance gene.
8. The bacterial cell composition of claim 1 , wherein the counter selection marker comprises a nsfl gene.
9. A composition comprising:
(a) a recipient plasmid comprising:
(i) a selection marker;
(ii) a barcode landing sequence flanked by a region that is complementary to a portion of a barcode sequence; and
iii) a target nucleic acid sequence that encodes a gene of interest and is flanked by a region that is complementary to a portion of a modified target nucleic acid sequence; and
(b) a donor plasmid comprising:
(i) a selection marker and a counter selection marker wherein the selection marker and the counter selection marker are not the same;
(ii) a first guide nucleic acid sequence comprising a region that is complementary to a portion of the barcode landing sequence, wherein the first guide nucleic acid sequence is operably linked to a constitutively active promoter;
(iii) the barcode sequence; and
(iv) an editing cassette comprising the modified target nucleic acid sequence linked to a mutated protospacer adjacent motif (mutated PAM) and a second guide nucleic acid sequence comprising a region that is complementary to a portion of the editing cassette, wherein the second guide nucleic acid sequence is operably linked to an inducible promoter.
10. The composition of claim 9 , wherein the modified target nucleic acid sequence comprises at least one inserted, deleted, or substituted nucleic acid as compared to the target nucleic acid sequence.
11. The composition of claim 9 , wherein at least one of the selection markers in (a) and (b) comprises an antibiotic resistance gene.
12. The bacterial cell composition of claim 1 , wherein the counter selection marker comprises a nsfl gene.
13. A method of gene editing, the method comprising:
combining a population of cells comprising a nucleic acid-guided DNA binding protein and a recombination system with:
(a) a recipient plasmid comprising:
(i) a selection marker;
(ii) a barcode landing sequence flanked by a region that is complementary to a portion of a barcode sequence; and
(iii) a target nucleic acid sequence that encodes a gene of interest and is flanked by a region that is complementary to a portion of a modified target nucleic acid sequence; and
(b) a donor plasmid comprising:
(i) a selection marker and a counter selection marker, wherein the selection marker and the counter selection marker are not the same;
(ii) a first guide nucleic acid sequence comprising a region that is complementary to a portion of the barcode landing sequence, wherein the first guide nucleic acid sequence is operably linked to a constitutively active promoter;
(iii) the barcode sequence; and
(iv) an editing cassette comprising the modified target nucleic acid sequence linked to a mutated protospacer adjacent motif (mutated PAM) and a second guide nucleic acid sequence comprising a region that is complementary to a portion of the editing cassette, wherein the second guide nucleic acid sequence is operably linked to an inducible promoter, thereby producing a combination;
maintaining the combination under conditions under which the first guide nucleic acid sequence and the nucleic acid-guided DNA binding protein create a barcode edit in the barcode landing sequence;
selecting for a population of cells comprising the recipient plasmid and the donor plasmid, thereby producing a selected population of cells;
maintaining the selected population of cells under conditions under which expression of the second nucleic acid sequence is induced and the expressed second guide nucleic acid sequence and the nucleic acid-guided DNA binding protein create a gene edit in the target nucleic acid sequence; and
counter selecting for a population of cells comprising the barcode edit in the barcode landing sequence and the gene edit in the target nucleic acid sequence.
14. The method of claim 13 , wherein the modified target nucleic acid sequence comprises at least one inserted, deleted, or substituted nucleic acid as compared to the target nucleic acid sequence.
15. The method of claim 13 , wherein the nucleic acid-guided DNA binding protein is a Cas 9 protein.
16. The method of claim 13 , wherein the recombination system is selected from the group consisting of a lambda red recombination system, a Cre/Lox recombination system, and an attB/attP recombination system.
17. The method of claim 13 , wherein the nucleic acid-guided DNA binding protein;
the recombination system; or the nucleic acid-guided DNA binding protein and the recombination system are integrated in a genome of a cell.
18. The method of claim 13 , wherein the nucleic acid-guided DNA binding protein;
the recombination system; or the nucleic acid-guided DNA binding protein and the recombination system are expressed from a plasmid in a cell.
19. The composition of claim 13 , wherein at least one of the selection markers in (a) and (b) comprises an antibiotic resistance gene.
20. The bacterial cell composition of claim 1 , wherein the counter selection marker comprises a nsfl gene.
21. A bacterial cell composition comprising:
(a) a recipient plasmid comprising:
(i) a selection marker;
(ii) a barcode landing sequence flanked by a region that is complementary to a portion of a barcode sequence in donor plasmid of (b); and
(iii) a target nucleic acid sequence that encodes a gene of interest and is flanked by a region that is complementary to a portion of a modified target nucleic acid sequence; and
(b) a donor plasmid comprising:
i) a selection marker and a counter selection marker, wherein the selection marker and the counter selection marker are not the same;
(ii) a first guide nucleic acid sequence comprising a region that is complementary to a portion of the barcode landing sequence of (aii), wherein the first guide nucleic acid sequence is operably linked to a constitutively active promoter;
(iii) the barcode sequence; and
(iv) an editing cassette comprising the modified target nucleic acid sequence linked to a mutated protospacer adjacent motif (mutated PAM) and a second guide nucleic acid sequence comprising a region that is complementary to a portion of the editing cassette, wherein the second guide nucleic acid sequence is operably linked to an inducible promoter.
22. The bacterial cell composition of claim 21 , further comprising a nucleic acid-guided DNA binding protein and a recombination system.