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.

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.

MeSH terms

  • Animals
  • Bacterial Proteins / metabolism*
  • CRISPR-Associated Protein 9
  • Chromosome Mapping
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Endonucleases / metabolism*
  • Gene Targeting / methods*
  • Humans
  • Mice
  • Nucleosomes / metabolism*
  • RNA, Guide / metabolism*

Substances

  • Bacterial Proteins
  • Nucleosomes
  • RNA, Guide
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases