Thus, there are two main considerations in the selection of the 20-nt guide sequence for gene targeting: (i) the 5′-NGG PAM for S. pyogenes Cas9 and (ii) the minimization of off-target activity51,52. We provide an online CRISPR Design Tool (http://tools.genome-engineering.org) that takes a genomic sequence of interest and identifies suitable target sites. To experimentally assess off-target genomic modifications for each sgRNA, we also provide computationally predicted off-target sites (for a detailed discussion, see Box 1) for each intended target, ranked according to our quantitative specificity analysis on the effects of base-pairing mismatch identity, position and distribution. For increased targeting specificity, an alternative strategy using the D10A nickase mutant of Cas9 (Cas9n) along with a pair of sgRNAs may be used. The design criteria for orientation and spacing of such sgRNA pairs are described in Box 2.
