eLife (Sep 2016)

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

DOI
https://doi.org/10.7554/eLife.19760
Journal volume & issue
Vol. 5

Abstract

Read online

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.

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