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

Analysis of the genome editing activity of more than 1800 sgRNAs in mouse and human cells yields rules to facilitate design of highly active RNA guides for Cas-9 genome editing. Components of the prokaryotic clustered, regularly interspaced, short palindromic repeats (CRISPR) loci have recently been repurposed for use in mammalian cells1,2,3,4,5,6. 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 system7,8. 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.