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 method of generating a cell having a genome edit in one or more target regions, said method comprising:
(a) contacting said cell with an editing cassette, wherein said editing cassette comprises a sequence having:
(i) a nucleic acid encoding a gRNA sequence targeting a target region,
(ii) a region homologous to said target region comprising a change in sequence relative to said target region,
(iii) a protospacer adjacent motif (PAM) mutation; and
(iv) a barcode, wherein said barcode identifies said change in sequence relative to said target region; and
(b) creating the genome edits using a nuclease compatible with said gRNA sequence; thereby generating a cell having a genome edit.
2. The method of claim 1 , wherein said gRNA sequence is a chimeric guide RNA.
3. The method of claim 1 , wherein said gRNA sequence comprises a crRNA and a tracrRNA.
4. The method of claim 1 , wherein said cell is a prokaryotic cell.
5. The method of claim 1 , wherein said cell is a eukaryotic cell.
6. The method of claim 1 , wherein the change in sequence relative to the target region is within a coding region of the target region.
7. The method of claim 1 , where in the change in sequence relative to the target region is within a non-coding region of the target region.
8. The method of claim 1 , wherein said cell comprises a change in sequence in at least two different target regions.
9. The method of claim 1 , wherein said cell comprises different changes within the same target region.
10. The method of claim 1 , wherein said region homologous to said target region is 100 to 120 nucleotides in length.
11. The method of claim 1 , wherein said PAM mutation is 17 to 59 base pairs from said change in sequence relative to said target region.
12. The method of claim 1 , wherein said PAM mutation is within a coding region of the target region, and the PAM mutation is a synonymous mutation.
13. The method of claim 1 , wherein said gRNA targets a region within a coding region of the target region.
14. The method of claim 1 , wherein said gRNA targets a region within a non-coding region of the target region.
15. A method of generating a library of cells with genome edits, said method comprising:
(a) obtaining a library of editing cassettes, wherein said library of editing cassettes comprises one or more editing cassettes comprising:
(i) a nucleic acid encoding a gRNA sequence targeting a target region,
(ii) a change in a sequence relative to said target region;
(iii) a protospacer adjacent motif (PAM) mutation; and
(iv) a barcode, wherein said barcode identifies said change in said sequence relative to said target region;
(b) introducing said one or more editing cassettes from said library of editing cassettes into cells,
(c) enriching for one or more transformed cells using a nuclease compatible with said nucleic acid, thereby providing a population of enriched cells, and
(d) growing said population of enriched cells, thereby generating said library of cells with genome edits.
16. The method of claim 15 , wherein the target region is in a non-coding region.
17. The method of claim 15 , wherein the target region is in a coding region.
18. The method of claim 15 , wherein the sequence change is in at least one codon of the target region.
19. The method of claim 15 , wherein the cells are prokaryotic cells.
20. The method of claim 15 , wherein the cells are eukaryotic cells.
21. The method of claim 15 , wherein the library comprises cells with a change in sequence in at least two different target regions.
22. The method of claim 15 , wherein the library comprises cells with different changes within the same target region.