US 12,454,687 B2
Functional genomics using CRISPR-Cas systems, compositions, methods, knock out libraries and applications thereof
Feng Zhang, Cambridge, MA (US); Neville Espi Sanjana, Cambridge, MA (US); and Ophir Shalem, Albany, CA (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 Jul. 29, 2019, as Appl. No. 16/525,531.
Application 16/525,531 is a continuation of application No. 14/463,253, filed on Aug. 19, 2014, abandoned.
Application 14/463,253 is a continuation of application No. PCT/US2013/074800, filed on Dec. 12, 2013.
Claims priority of provisional application 61/802,174, filed on Mar. 15, 2013.
Claims priority of provisional application 61/736,527, filed on Dec. 12, 2012.
Prior Publication US 2020/0165601 A1, May 28, 2020
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/10 (2006.01); C12N 15/63 (2006.01); C12N 15/90 (2006.01)
CPC C12N 15/1034 (2013.01) [C12N 15/102 (2013.01); C12N 15/1093 (2013.01); C12N 15/63 (2013.01); C12N 15/907 (2013.01); C12N 15/1082 (2013.01); C12N 15/635 (2013.01); C12N 2750/14143 (2013.01)] 13 Claims
 
1. A method for generating a library of CRISPR-containing eukaryotic cells for functional genomic screening, comprising:
providing a population of eukaryotic cells each comprising polynucleotides encoding a Cas9 protein;
introducing a library of CRISPR-Cas9 system guide RNAs or polynucleotides encoding the CRISPR-Cas9 system guide RNAs into the population of eukaryotic cells in a single mixture to obtain the library of CRISPR-containing eukaryotic cells for functional genomic screening, wherein the population of eukaryotic cells are human cells, wherein the library of CRISPR-Cas9 system guide RNAs target each gene in human genome; wherein each of the CRISPR-Cas9 system guide RNAs comprises an engineered guide sequence that targets a genomic locus in a eukaryotic cell, forms a CRISPR-Cas9 complex with the Cas9 protein in the eukaryotic cell, and directs sequence-specific binding of the CRISPR-Cas9 complex to a target sequence in the genomic locus; and
performing whole exome sequencing to identify a mutation in each targeted gene in the library of CRISPR-containing eukaryotic cells.