Fig. 5

Variant HeFm2SpCas9 exhibits target selectivity and fidelity between those of SpCas9-HF1 and HeFSpCas9. Disruption and indel formation activities of SpCas9 nuclease variants bearing combinations of eSpCas9 and SpCas9-HF1 mutations. a Tukey-type notched boxplots of ratios of on-target disruption activities of the variants to those of WT nucleases as indicated (data used are from Additional file 1: Figure S9a, b; sites targeted are provided in Additional file 2): center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; notches indicate the 95% confidence intervals for medians; crosses represent sample means; data points are plotted as open circles (17 in case of each variant) and correspond to the targets tested. Statistically different pairs of means at the p < 0.05 level: SpCas9HF1–HeFm1SpCas9 (<0.001), SpCas9HF1–HeFm2SpCas9 (<0.015), SpCas9HF1–HeFSpCas9 (<0.001). b Comparison of specificities of SpCas9-HF1 and HeFm2SpCas9 assessed in disruption assays with partially mismatching sgRNAs on targets where HeFm2SpCas9 reached at least 60% activity of the WT protein as in a. c Left panel: mean percentage modifications by WT SpCas9 and variants at FANCF site 2 as well as off-target site 1 from Fig. 5 in [33], which is readily cleaved by SpCas9-HF1. Percentage modifications were determined by TIDE; error bars represent standard deviation with Gaussian error propagation for n = 3 parallels. Right panel: specificity of WT and mutant variants on the FANCF site 2 plotted as ratio of on-target to off-target activity (calculated from the left panel)