Activity of the SaCas9 KKH variant targeted to endogenous sites in human cells (a) Mutagenesis frequencies across 55 different sites bearing NNNRRT PAMs induced by KKH SaCas9, determined by T7E1 assay. Error bars represent s.e.m., n = 3, ND, not detectable by T7E1 assay. (b) KKH variant preference for the third position of the PAM. Mean activities from data in panel a are shown for this and panels b and c. (c) KKH variant preference for the fourth and fifth positions of the PAM. (d) Spacer length preference of the KKH SaCas9 variant. (e) Comparison of the human cell EGFP disruption activity of wild-type and KKH SaCas9 targeted to various sites containing NNNRRT PAMs. EGFP disruption quantified by flow cytometry; error bars represent s.e.m, n = 3, mean level of background EGFP loss represented by dashed red line. (f) Mutagenesis frequencies of wild-type SaCas9 against one site for each of the 16 possible NNNRRT sites from panel a (sites with the highest KKH activity were selected). Error bars represent s.e.m., n = 3, ND, not detectable by T7E1 assay.