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Elife. 2016 Sep 23;5. pii: e19760. doi: 10.7554/eLife.19760.

Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation.

Horlbeck MA1,2,3,4, Gilbert LA1,2,3,4, Villalta JE1,2,3,4, Adamson B1,2,3,4, Pak RA1,5, Chen Y1,2,3,4, Fields AP1,2,3,4, Park CY1,5, Corn JE5,6, Kampmann M1,2,3,4,7, Weissman JS1,2,3,4.

Author information

1
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States.
2
Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.
3
California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States.
4
Center for RNA Systems Biology, University of California, San Francisco, San Francisco, United States.
5
Innovative Genomics Initiative, University of California, Berkeley, Berkeley, United States.
6
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.
7
Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, United states.

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.

KEYWORDS:

CRISPR; chromosomes; computational biology; genes; genetic screening; human; nucleosomes; systems biology

PMID:
27661255
PMCID:
PMC5094855
DOI:
10.7554/eLife.19760
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

MAH: patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449). JSW is a founder of, and MAH and LAG are consultants for, KSQ Therapeutics, a CRISPR functional genomics company. LAG: filed a patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449). JSW is a founder of, and MAH and LAG are consultants for, KSQ Therapeutics, a CRISPR functional genomics company. MK: patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449). JSW: filed a patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449). JSW is a founder of, and MAH and LAG are consultants for, KSQ Therapeutics, a CRISPR functional genomics company. The other authors declare that no competing interests exist.

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