CRISPR enabled multiplexed genome engineering
Described herein are methods and vectors for rational, multiplexed manipulation of chromosomes within open reading frames (e.g., in protein libraries) or any segment of a chromosome in a cell or population of cells, in which various CRISPR systems are used.
1. A library of at least two vectors, each vector comprising:
(i) a nucleic acid encoding a guide RNA (gRNA) comprising a region complementary to a target region of a nucleic acid in one or more cells covalently linked to
(ii) an editing cassette comprising
(a) a region homologous to the target region in the one or more cells with a mutation of at least one nucleotide relative to the target region in the one or more cells, and
(b) a protospacer adjacent motif (PAM) mutation;
wherein the at least two vectors have different mutations relative to the target region in the one or more cells.
2. The library of claim 1 , wherein at least one of the vectors encoding a gRNA further comprises a region that interacts with, or recruits, a Cas9 endonuclease.
3. The library of claim 1 , wherein the target region in the one or more cells is within a non-coding region.
4. The library of claim 1 , wherein the target region in the one or more cells is within a gene of interest within the one or more cells.
5. The library of claim 4 , wherein the mutation of at least one nucleotide is within at least one codon of the gene of interest.
6. The library of claim 5 , wherein the mutation of at least one codon is within 100 nucleotides of the PAM mutation.
7. The library of claim 4 , wherein the PAM mutation is outside of a reading frame of the gene of interest.
8. The library of claim 4 , wherein the gene of interest is a prokaryotic gene.
9. The library of claim 4 , wherein the gene of interest is a eukaryotic gene.
10. The library of claim 1 , wherein in at least one of the vectors the gRNA comprises a single chimeric gRNA.
11. The library of claim 1 , wherein the library of vectors comprises at least 27,000 different editing cassettes.
12. The library of claim 1 , wherein the library of vectors comprises about 10 4 -10 6 different editing cassettes.
13. The library of claim 1 , wherein the library of vectors comprises at least 10 4 different editing cassettes.
14. The library of claim 1 , wherein the library of vectors comprises at least 10 5 different editing cassettes.
15. The library of claim 1 , wherein the library of vectors comprises at least 10 6 different editing cassettes.
16. The library of claim 1 , wherein the library of vectors comprises linear vectors.
17. The library of claim 1 , wherein the library of vectors comprises circular vectors.
18. A library comprising a plurality vectors, wherein each vector comprises:
(i) an editing cassette comprising:
(a) a region homologous to a target region in one or more cells with a mutation of at least one nucleotide relative to the target region in the one or more cells, and
(b) a first protospacer adjacent motif (PAM) mutation; and
(ii) a nucleic acid encoding a guide RNA (gRNA) complementary to the target region of the one or more cells, wherein the editing cassette is covalently linked to the nucleic acid encoding the guide RNA, and wherein the plurality of vectors have different mutations relative to the target region in the one or more cells.
19. The library of claim 18 , wherein said nucleic acid encoding a gRNA further comprises a region that interacts with, or recruits, a Cas9 endonuclease.
20. The library of claim 18 , wherein the target region of at least one of the plurality of vectors is within a non-coding region.
21. The library of claim 18 , wherein the target region of at least one of the plurality of vectors is within a gene of interest within the one or more cells.
22. The library of claim 21 , wherein at least one of the plurality of vectors comprises a mutation of at least one nucleotide within at least one codon of the gene of interest.
23. The library of claim 22 , wherein the mutation of at least one codon is within 100 nucleotides of the PAM mutation.
24. The library of claim 21 , wherein the PAM mutation is outside of a reading frame of the gene of interest.
25. The library of claim 21 , wherein the gene of interest is a prokaryotic gene.
26. The library of claim 21 , wherein the gene of interest is a eukaryotic gene.
27. The library of claim 18 , wherein at least one of the gRNAs comprises a single chimeric gRNA.
28. The library of claim 18 , wherein the library of vectors comprises at least 27,000 different editing cassettes.
29. The library of claim 18 , wherein the library of vectors comprises about 10 4 -10 6 different editing cassettes.
30. The library of claim 18 , wherein the library of vectors comprises at least 10 4 different editing cassettes.
31. The library of claim 18 , wherein the library of vectors comprises at least 10 5 different editing cassettes.
32. The library of claim 18 , wherein the library of vectors comprises at least 10 6 different editing cassettes.
33. The library of claim 18 , wherein the library of vectors comprises linear vectors.
34. The library of claim 18 , wherein the library of vectors comprises circular vectors.