PRJNA258441
Rational sgRNA Design for CRISPR/Cas9-mediated Gene Inactivation
Components of clustered regularly interspersed palindromic repeat (CRISPR) loci, which function as an adaptable immune system in many prokaryotes, have recently been repurposed for use in mammalian cells. The Cas9 protein can be easily programed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but as yet 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 endogenous genes. By quantitatively assessing the ability of these sgRNAs to produce null alleles of their target gene, we discovered sequence features that improved activity, including a further optimization of the proto-spacer adjacent motif (PAM) for Cas9 from Streptococcus pyogenes. These results establish a predictive model of sgRNA activity beyond the NGG PAM sequence to improve sgRNA design for gene editing and genetic screens.