highlighting the potential for SNPs to affect off-target mutagenesis at specific sites. Whole genome sequencing of six individual clones isolated using this protocol from separate genome editing experiments at three loci (TAZ, DNAJC19, JUP) showed that Cas9 does not induce frequent off-target mutagenesis. However, between 1–3 mutations that likely were linked to Cas9 were identified in each of the clones (Fig. 4b). With only one exception, we did not detect significant off-target activity at sites with 3 or more gRNA mismatches. The exception (site B, Fig. 4b) consisted of indel mutation at a genomic site on chromosome 14 that matched the gRNA at bases 8–19 (where position 1 is adjacent to the PAM). However, the “seed” nucleotides 1–7 did not match, and the PAM sequence, GGT, has no predicted activity19. An identical sequence also exists on chromosome 3, and this site also contained an indel mutation in one of the two clones (site C, Fig. 4b), suggesting that mutation at these sites was related to Cas9 activity. In addition, we detected clear off-target activity at a site (site A, Fig. 4b) with a 1 bp gRNA mismatch but a variant PAM sequence, CAG, with known partial activity19. Together these results
