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 vector comprising:
(i) an editing cassette comprising:
(a) a region homologous to a target region of a nucleic acid in a cell comprising a mutation of at least one nucleotide relative to said target region in the cell, and
(b) a first protospacer adjacent motif (PAM) mutation; and
(ii) at least one nucleic acid encoding at least a target region specific targeting portion of a guide RNA (gRNA) targeting the target region, wherein the editing cassette is covalently linked to the at least one nucleic acid.
2. A vector library comprising a plurality of vectors of claim 1 .
3. The vector library of claim 2 , wherein said at least one nucleic acid encoding at least a target region-specific targeting portion of a gRNA further comprises a region that interacts with, or recruits, an endonuclease.
4. The vector library of claim 2 , wherein said vector further comprises a nucleic acid encoding an endonuclease.
5. A population of cells comprising:
(a) nucleic acids comprising target regions,
(b) the vector library of claim 2 , and
(c) an endonuclease compatible with said gRNA.
6. The vector library of claim 2 , wherein said plurality of vectors encode a plurality of gRNAs that target at least two different target regions.
7. The vector library of claim 2 , wherein said plurality of vectors comprise different mutations relative to said target region.
8. The vector library of claim 2 , wherein said plurality of vectors comprise at least 8,000 different mutations relative to said targeting region.
9. The vector library of claim 2 , comprising at least 27,000 different vectors.
10. The vector library of claim 2 , comprising about 10 4 -10 6 different vectors.
11. The vector library of claim 2 , comprising about 10 4 different vectors.
12. The vector library of claim 2 , comprising about 10 5 different vectors.
13. The vector of claim 1 , wherein said at least one nucleic acid encoding a gRNA further comprises a region that interacts with, or recruits, an endonuclease.
14. The vector of claim 13 , wherein said endonuclease is a Cas9 nuclease.
15. The vector of claim 1 , further comprising a barcode.
16. The vector of claim 1 , wherein said region homologous to said target region of (i) is within a non-coding region of said target region.
17. The vector of claim 1 , wherein said region homologous to said target region of (i) is within a gene of interest of said target region.
18. The vector of claim 17 , wherein said mutation of said at least one nucleotide is within at least one codon of said gene of interest.
19. The vector of claim 17 , wherein said first protospacer adjacent motif (PAM) mutation is outside of a reading frame of said gene of interest.
20. The vector of claim 17 , wherein said gene of interest is a prokaryotic gene.
21. The vector of claim 17 , wherein said gene of interest is a eukaryotic gene.
22. The vector of claim 1 , wherein said mutation of at least one nucleotide relative to said target region is within 100 nucleotides of said first protospacer adjacent motif (PAM) mutation.
23. The vector of claim 1 , wherein said gRNA comprises a single chimeric gRNA.
24. The vector of claim 1 , wherein said gRNA comprises a crRNA and a trRNA.
25. The vector of claim 1 , further comprising at least two priming sites.
26. The vector of claim 1 , further comprising a promoter.