@article{95a99b9217ad4a1b99bddb066c7fbfa3,
title = "Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation",
abstract = "We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites.",
author = "Horlbeck, {Max A.} and Gilbert, {Luke A.} and Villalta, {Jacqueline E.} and Britt Adamson and Pak, {Ryan A.} and Yuwen Chen and Fields, {Alexander P.} and Park, {Chong Yon} and Corn, {Jacob E.} and Martin Kampmann and Weissman, {Jonathan S.}",
note = "Funding Information: We would like to thank Dr. Manuel Leonetti, Ben Barsi-Rhyne, Dr. Jonathan Friedman, and Dr. Jodi Nunnari for generously sharing unpublished screening data for determination of sgRNA activity. We would also like to thank Dr. Xuebing Wu and members of the Weissman lab, particularly Dr. Joshua Dunn and Manny DeVera, for helpful discussions and assistance. We thank Dr. Laurakay Bruhn, Dr. Daniel Ryan, Dr. Luke Fairbairn, and Dr. Peter Tsang of Agilent Technologies for their assistance on the design and synthesis of oligonucleotide pools. MAH, LAG, JEV, BA, YC, APF, and JSW were sup- ported by the Howard Hughes Medical Institutes and the National Institutes of Heath (P50 GM102706, U01 CA168370, R01 DA036858). LAG was supported by the Leukemia and Lymphoma Society. RAP, CYP, and JEC were supported by the Li Ka Shing Foundation. MK was supported by NIH/NIGMS DP2Howard Hughes Medical Institute Max A Horlbeck Luke A Gilbert Jacqueline E Villalta Britt Adamson Yuwen Chen Alexander P Fields GM119139. Leukemia and Lymphoma Society Luke A Gilbert National Cancer Institute Pathway to Independence Award, K99 CA204602 Luke A Gilbert Li Ka Shing Foundation Ryan A Pak Chong Yon Park Jacob E Corn National Institute of General Medical Sciences DP2 GM119139 Martin Kampmann Publisher Copyright: {\textcopyright} Horlbeck et al.",
year = "2016",
month = sep,
day = "23",
doi = "10.7554/eLife.19760",
language = "English (US)",
volume = "5",
journal = "eLife",
issn = "2050-084X",
publisher = "eLife Sciences Publications",
number = "September2016",
}