Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation

Nat Biotechnol. 2014 Dec;32(12):1262-7. doi: 10.1038/nbt.3026. Epub 2014 Sep 3.


Components of the prokaryotic clustered, regularly interspaced, short palindromic repeats (CRISPR) loci have recently been repurposed for use in mammalian cells. The CRISPR-associated (Cas)9 can be programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there are few known rules governing on-target efficacy of this system. We created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. We discovered sequence features that improved activity, including a further optimization of the protospacer-adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens. We provide an online tool for the design of highly active sgRNAs for any gene of interest.

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • Flow Cytometry
  • Gene Silencing*
  • Gene Targeting*
  • Humans
  • Mice
  • RNA / genetics*
  • Streptococcus pyogenes / genetics


  • RNA

Associated data

  • SRA/SRP045596