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 single vector comprising at least two synthetic oligonucleotides, wherein each synthetic oligonucleotide comprises the following covalently-linked components: (i) a nucleic acid encoding a guide RNA (gRNA) sequence targeting a target region in a cell; (ii) a region homologous to the target region comprising a change in sequence relative to the target region; and (iii) a site conferring immunity to nuclease-mediated editing; and wherein the at least two synthetic oligonucleotides are homologous to different regions in the cell.
2. The single vector of claim 1 , comprising at least three synthetic oligonucleotides.
3. The single vector of claim 2 , comprising at least four synthetic oligonucleotides.
4. The single vector of claim 3 , comprising at least five synthetic oligonucleotides.
5. The single vector of claim 4 , comprising at least six synthetic oligonucleotides.
6. The single vector of claim 5 , comprising at least seven synthetic oligonucleotides.
7. The single vector of claim 1 , further comprising at least one promoter.
8. The single vector of claim 1 , wherein at least one of the gRNAs is a single chimeric gRNA.
9. The single vector of claim 1 , wherein the site conferring immunity to nuclease-mediated editing is a protospacer adjacent motif (PAM) mutation.
10. A single vector comprising at least two synthetic oligonucleotides, wherein each synthetic oligonucleotide comprises the following covalently-linked components:
(i) a nucleic acid encoding a guide RNA (gRNA) sequence targeting a target region in a cell; and
(ii) a region homologous to the target region comprising a change in sequence relative to the target region; wherein the at least two synthetic oligonucleotides are homologous to different target regions in the cell.
11. The single vector of claim 10 , comprising at least three synthetic oligonucleotides.
12. The single vector of claim 11 , comprising at least four synthetic oligonucleotides.
13. The single vector of claim 12 , comprising at least five synthetic oligonucleotides.
14. The single vector of claim 13 , comprising at least six synthetic oligonucleotides.
15. The single vector of claim 14 , comprising at least seven synthetic oligonucleotides.
16. The single vector of claim 10 , further comprising at least one promoter.
17. The single vector of claim 10 , further comprising a site conferring immunity to nuclease-mediated editing.
18. The single vector of claim 10 , wherein at least one of the target regions is within a non-coding region.
19. The single vector of claim 10 , wherein at least one of the target regions is within a coding region.
20. The single vector of claim 10 , wherein both of the target regions are within a coding region.