A concern of Cas9-based genome-editing strategies has been off-target mutagenesis. Recently several studies used whole genome sequencing to demonstrate that Cas9 genome editing does not significantly impact the mutation burden of iPSCs4,17,18. We confirmed that our strategy is not substantially mutagenic by deep sequencing of 31 potential off-target sites (Fig. 4a). At each site we sequenced a minimum of 100,000 amplicons, which we previously showed yields a detection sensitivity of 0.07%4. At the 31 computationally predicted potential off-target sites, 30 sites had 3 nucleotide mismatches to the reference genome. Significant off-target activity was not detected at these sites. The final site, site 28, was designed to also have 3 nucleotide mismatches to the reference sequence, but as we reported previously4 a single nucleotide polymorphism in the PGP1 genome sequence eliminated one mismatch. This site with two mismatches was frequently mutated, 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
