US 12,441,995 B2
Functional genomics using CRISPR-Cas systems, compositions, methods, screens and applications thereof
Feng Zhang, Cambridge, MA (US); Ophir Shalem, Cambridge, MA (US); Neville Espi Sanjana, Cambridge, MA (US); John Doench, Cambridge, MA (US); David Root, Cambridge, MA (US); and Ella Nicole Biewener Hartenian, Cambridge, MA (US)
Assigned to THE BROAD INSTITUTE, INC., Cambridge, MA (US); and MASSACHUSETTS INSTITUTE OF TECHNOLOGY, Cambridge, MA (US)
Filed by The Broad Institute, Inc., Cambridge, MA (US); and Massachusetts Institute of Technology, Cambridge, MA (US)
Filed on Sep. 21, 2020, as Appl. No. 17/027,151.
Application 17/027,151 is a continuation of application No. 14/973,062, filed on Dec. 17, 2015, granted, now 10,781,444.
Application 14/973,062 is a continuation in part of application No. PCT/US2014/041806, filed on Jun. 10, 2014.
Claims priority of provisional application 61/836,123, filed on Jun. 17, 2013.
Claims priority of provisional application 61/960,777, filed on Sep. 25, 2013.
Claims priority of provisional application 61/995,636, filed on Apr. 15, 2014.
Prior Publication US 2021/0108196 A1, Apr. 15, 2021
Int. Cl. C12N 15/10 (2006.01); C12N 9/22 (2006.01); C12N 15/63 (2006.01); C12N 15/90 (2006.01)
CPC C12N 15/1093 (2013.01) [C12N 9/22 (2013.01); C12N 15/102 (2013.01); C12N 15/1082 (2013.01); C12N 15/63 (2013.01); C12N 15/907 (2013.01); C12N 2310/20 (2017.05)] 17 Claims
 
1. A method for identifying one or more genomic loci associated with drug resistance, comprising:
contacting a population of cells with a composition comprising one or more vectors that encode (a) a Cas9 protein and (b) a plurality of CRISPR-Cas system guide RNAs targeting a plurality of different genomic loci, wherein the plurality of CRISPR-Cas system guide RNAs comprise guide RNAs encoded by plasmid library of ATCC Deposit No. PTA121339, guide RNAs encoded by plasmid library of ATCC Deposit No. 121340, guide RNAs encoded by plasmid library of ATCC Deposit No. 121341, guide RNAs encoded by plasmid library of ATCC Deposit No. 121342, guide RNAs encoded by plasmid library of ATCC Deposit No. 121343, or a combination thereof,
selecting cells successfully transduced with the vectors, wherein the selected cells each comprises a CRISPR-Cas system comprising the Cas9 protein and one of the guide RNAs;
treating the selected cells with a drug to obtain cells having resistance to the drug; and
profiling the depletion or enrichment of the guide RNAs in the cells having resistance to the drug by high-throughput sequencing to identify one or more genomic loci associated with the resistance to the drug.