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

Max A. Horlbeck, Luke A. Gilbert, Jacqueline E. Villalta, Britt Adamson, Ryan A. Pak, Yuwen Chen, Alexander P. Fields, Chong Yon Park, Jacob E. Corn, Martin Kampmann, Jonathan S. Weissman

Research output: Contribution to journalArticle

161 Scopus citations

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.

Original languageEnglish (US)
Article numbere19760
JournaleLife
Volume5
Issue numberSeptember2016
DOIs
StatePublished - Sep 23 2016

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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    Horlbeck, M. A., Gilbert, L. A., Villalta, J. E., Adamson, B., Pak, R. A., Chen, Y., Fields, A. P., Park, C. Y., Corn, J. E., Kampmann, M., & Weissman, J. S. (2016). Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife, 5(September2016), [e19760]. https://doi.org/10.7554/eLife.19760