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US 9,982,278 B2Granted
CRISPR enabled multiplexed genome engineering
AssigneeThe Regents of the University of Colorado, a body corporate
InventorsRyan T. Gill, Andrew Garst
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Abstract
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.
Claims4
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.
6 more elements
Dependencies are inferred from the claim text.