CRISPR enabled multiplexed genome engineering
View Patent ↗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 library of cells with genome edits, comprising:
(a) contacting cells with a library of synthesized oligonucleotides, wherein the oligonucleotides comprise:
(i) a region homologous to a target region of a nucleic add and a sequence mutation relative to the target region;
(ii) a protospacer adjacent motif (PAM) mutation;
(iii) a barcode; and
(iv) a nucleic acid encoding at least a target region-specific targeting portion of a guide RNA (gRNA) targeting the target region,
(b) enriching for the cells comprising the change in the sequence relative to the target region, the PAM mutation, and the barcode; and
(c) sequencing the barcodes of one or more cells of the library of cells, thereby identifying the changes in the sequence within those cells of the library.
2. The method of claim 1 , wherein the library of cells comprises cells with a genome edit in at least one gene of interest.
3. The method of claim 1 , wherein the library of cells comprises cells with a genome edit in at least one non-coding region of interest.
4. The method of claim 1 , wherein the enriching comprises using a nuclease compatible with said guide RNA.