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. Author manuscript; available in PMC: 2018 Jul 31.
Published in final edited form as: Nat Biotechnol. 2018 Jan 29;36(3):265–271. doi: 10.1038/nbt.4066

Figure 2. Selection of highly specific SpCas9 variants in mammalian cells.

Figure 2

(a) Combination of selected mutations obtained from the yeast screening. Mean EGFP knockout levels in 293multiEGFP following transfection with single, double and triple mutants generated by the combination of mutations obtained from the best performing yeast-isolated variants together with the on-target sgRNA (sgGFPon) or each of the indicated mismatched guides (sgGFP1819 and sgGFP18). (b) Side-by-side comparison of the best-generated variants with previously published mutants. Wild-type SpCas9 (WT) and the indicated variants were tested in 293multiEGFP cells to evaluate their mean on-target efficiency (sgGFPon) and specificity (sgGFP1314, sgGFP1819 and sgGFP18 with mismatches in position 13-14, 18-19 and 18, respectively, from the PAM). Dotted lines indicate on/off target ratios calculated for the indicated on/off couples.(c) Ratio of the on-target activity of evoCas9 (VNEQ) and the VNEL variant to wild-type SpCas9 calculated on EGFP targets and endogenous loci in 293T cells (knockout levels are reported in Supplementary Fig. 2b,c). The median and interquartile range are shown. On-target activity above 70% of the wild-type protein is indicated in green. (d) evoCas9 activity on endogenous loci. Wild-type SpCas9, evoCas9, SpCas9-HF1 and eSpCas9(1.1) were transfected in 293T cells to measure the mean indel formation towards endogenous loci 7 days post-transfection using the TIDE tool. (e) Ratio of the mean on-target activity of evoCas9, SpCas9-HF1 and eSpCas9(1.1) over wild-type SpCas9 relative to endogenous loci in (d). The median and interquartile range are shown. On-target activity above 70% of the wild-type protein is indicated in green. In panels (a-b) loss of EGFP fluorescence was measured by FACS analysis 7 days post-transfection; dashed lines indicate the background loss of EGFP fluorescence. In panels (c) and (e) statistical significance was assessed using the two-sided paired Wilcoxon signed-rank test, sample size n=10 in (c) and n=19 in (e). Summary of data distributions and statistical details are reported in Supplementary Table 5; ns, not significant.