a, Activity of wild-type and mutant SpCas9s assessed via U2OS human cell-based EGFP disruption. Frequencies were quantified by flow cytometry; error bars represent s.e.m., n = 3; mean level of background EGFP loss represented by the dashed red line for this and subsequent panels (c, g, h, and j). b, Schematic of the positive selection assay (see also ). c, Combinatorial assembly and human cell testing of mutations obtained from the positive selection for SpCas9 variants that can cleave a target site containing an NGA PAM, using the EGFP disruption assay. d, Schematic of the negative selection assay, adapted to profile Cas9 PAM specificity by generating a library of plasmids that contain a randomized sequence adjacent to the 3’ end of the protospacer (see also ). e, Scatterplot of the post-selection PAM depletion values (PPDVs) of wild-type SpCas9 with two randomized PAM libraries (each with a different protospacer). PAMs are plotted by their 2nd/3rd/4th positions. The red dashed line indicates statistically significant depletion (obtained from a dCas9 control experiment, see ), and the gray dashed line represents five-fold depletion (PPDV of 0.2). f, PPDV scatterplots for the VQR and EQR variants. g, EGFP disruption frequencies for wild-type, VQR, and EQR SpCas9 on sites with NGAN and NGNG PAMs. h, Combinatorial assembly and human cell testing of mutations obtained from the positive selection for SpCas9 variants that can cleave a target site containing an NGC PAM, using the EGFP disruption assay. i, PPDV scatterplot for the VRER variant. j, EGFP disruption frequencies for wild-type and VRER SpCas9 on sites with NGCN and NGNG PAMs.