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

  • Elife. 2016 Sep 23:5:e19760. doi: 10.7554/eLife.19760.
Max A Horlbeck  1  2  3  4 ,  Luke A Gilbert  1  2  3  4 ,  Jacqueline E Villalta  1  2  3  4 ,  Britt Adamson  1  2  3  4 ,  Ryan A Pak  1  5 ,  Yuwen Chen  1  2  3  4 ,  Alexander P Fields  1  2  3  4 ,  Chong Yon Park  1  5 ,  Jacob E Corn  5  6 ,  Martin Kampmann  1  2  3  4  7 ,  Jonathan S Weissman  1  2  3  4
Affiliations
  • 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.
PMID: 27661255 DOI: 10.7554/eLife.19760
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.