Complementary information derived from CRISPR Cas9 mediated gene deletion and suppression

Nat Commun. 2017 May 23;8:15403. doi: 10.1038/ncomms15403.

Abstract

CRISPR-Cas9 provides the means to perform genome editing and facilitates loss-of-function screens. However, we and others demonstrated that expression of the Cas9 endonuclease induces a gene-independent response that correlates with the number of target sequences in the genome. An alternative approach to suppressing gene expression is to block transcription using a catalytically inactive Cas9 (dCas9). Here we directly compare genome editing by CRISPR-Cas9 (cutting, CRISPRc) and gene suppression using KRAB-dCas9 (CRISPRi) in loss-of-function screens to identify cell essential genes. CRISPRc identified 98% of previously defined cell essential genes. After optimizing library construction by analysing transcriptional start sites (TSS), CRISRPi identified 92% of core cell essential genes and did not show a bias to regions involved in copy number alterations. However, bidirectional promoters scored as false positives in CRISRPi. We conclude that CRISPRc and CRISPRi have different off-target effects and combining these approaches provides complementary information in loss-of-function genetic screens.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • A549 Cells
  • CRISPR-Cas Systems*
  • Catalysis
  • Cell Line
  • Cell Line, Tumor
  • Cell Proliferation
  • DNA / analysis
  • DNA Copy Number Variations
  • Endonucleases / metabolism
  • False Positive Reactions
  • Gene Deletion*
  • Gene Editing
  • Gene Expression
  • Gene Library
  • Genes, Essential*
  • Genome, Human*
  • HT29 Cells
  • Humans
  • Phenotype

Substances

  • DNA
  • Endonucleases