Selection and assembly of SaCas9 variants with altered PAM specificities (a) Phylogenetic tree of Cas9 orthologues with SpCas9 and SaCas9 highlighted. (b) Activity of SaCas9 variants with single amino acid substitutions assessed in the bacterial positive selection assay (see also ). Error bars represent s.e.m., n = 3; NS = no survival. (c) Human cell activity of wild-type and R1015H SaCas9. EGFP disruption activity quantified by flow cytometry; error bars represent s.e.m, n = 3, mean level of background EGFP loss represented by dashed red line (for this and panel e). (d) Total number of substitutions observed at each amino acid position when selecting for SaCas9 variants with altered PAM specificities (see also ). Starter mutations at R1015 are not counted. (e) Human cell EGFP disruption activity of variants containing mutations observed when selecting for altered PAM specificities. (f) Mean post-selection PAM depletion value (PPDV) scatterplot of wild-type SaCas9 versus the KKH variant (n = 2, see also ). Two libraries with different protospacers and 8 randomized basepairs in place of the PAM were used to determine which PAMs are targetable by each Cas9. Statistically significant depletion indicated by the red dashed line (relative to a dCas9 control, see ), and 5-fold depletion by the grey dashed line.

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.

Genome-wide specificity profiles of wild-type and KKH SaCas9 (a) and (b) Direct comparison of wild-type and KKH SaCas9 targeted to sites containing NNGRRT PAMs, represented by total number of off-targets (panel a) and mismatches observed at each off-target site (panel b). For panels b and e, GUIDE-seq read counts at each site are indicated; on-target sequences are marked with a black box; mismatched positions within off-target sites are highlighted; sequences have been corrected for cell-type specific SNPs; sites with potential sgRNA or DNA bulge nucleotides are indicated by a small red-bordered base or a dash, respectively. (c) Venn diagram highlighting the differences in off-target site cleavage by wild-type and KKH SaCas9 at VEGFA site 8. (d) and (e) Specificity profile of the KKH variant targeted to sites containing NNHRRT PAMs, represented by total number of off-targets (panel d) and mismatches observed at each off-target site (panel e).