Functional genomics using CRISPR-Cas systems for saturating mutagenesis of non-coding elements, compositions, methods, libraries and applications thereof
Daniel E. Bauer, Cambridge, MA (US); Stuart H. Orkin, Brookline, MA (US); Neville Espi Sanjana, New York, NY (US); Ophir Shalem, Albany, CA (US); Jason Wright, Cambridge, MA (US); and Feng Zhang, Cambridge, MA (US)
Assigned to The Broad Institute, Inc., Cambridge, MA (US); Massachusetts Institute of Technology, Cambridge, MA (US); and Children's Medical Center Corporation, Boston, MA (US)
Filed by THE BROAD INSTITUTE, INC., Cambridge, MA (US); MASSACHUSETTS INSTITUTE OF TECHNOLOGY, Cambridge, MA (US); and CHILDREN'S MEDICAL CENTER CORPORATION, Boston, MA (US)
Filed on Nov. 8, 2017, as Appl. No. 15/807,007.
Application 15/807,007 is a continuation in part of application No. PCT/US2016/031164, filed on May 6, 2016.
Claims priority of provisional application 62/316,421, filed on Mar. 31, 2016.
Claims priority of provisional application 62/219,498, filed on Sep. 16, 2015.
Claims priority of provisional application 62/158,882, filed on May 8, 2015.
1. A deep scanning mutagenesis library to interrogate phenotypic changes in a population of cells comprising a plurality of Type II CRISPR-Cas system guide RNAs comprising guide sequences, each guide sequence capable of targeting a non-coding genomic sequence within at least one continuous genomic region, wherein the plurality of guide RNAs target at least 100 genomic non-coding sequences comprising non-overlapping cleavage sites upstream of a PAM sequence for every 1000 base pairs within the at least one continuous genomic region and wherein the plurality of guide RNAs are capable of targeting two or more continuous genomic regions that physically interact, the at least one continuous genomic region comprising noncoding sequences, and wherein the guide RNAs have a median adjacent genomic cleavage distance between 4 bp and 20 bp.