Fig. 1: On- and off-target activities of the SuperFi-Cas9 nuclease in mammalian cells and in vitro.

a, b On-target activity of various SpCas9 variants as measured a in EGFP disruption assay on 24 or b by NGS on 26 endogenous target sites presented on a scatter dot plot. Data are also presented in Supplementary Fig. 1b and c, respectively. c Off-target activity of various SpCas9 variants as measured in EGFP disruption assay presented on a scatter dot plot. Only those data points are presented here for which the on-target activity exceeded 70%. d Off-target activity of various SpCas9 variants as measured by NGS on endogenous target sites presented on a heatmap. The heatmap shows the mean on- and off-target modifications (indels) of three parallel transfections. Data related to endogenous on-target sites where editing was low (<5%) with SuperFi-Cas9 are not shown on this figure, but data are available in Supplementary Data 3. On- and off-target editing was measured from the same samples. The mismatching nucleotides at DNA off-target sites are indicated as red letters. c, d Data are also presented in Supplementary Fig. 2a and b, respectively. e, f Bar charts of e the total number of off-target sites and f the on-target cleavage specificity, expressed as the percentages of the on-target reads from all reads, as measured by GUIDE-seq. Data are also presented in Supplementary Fig. 3. g In vitro cleavage activities of the variants employing 8 targets of panel a. Data are also presented in Supplementary Fig. 4. The median and interquartile range are shown; data points represent the mean of the fitted k value triplicates. a–c The median and interquartile ranges are shown; data points are plotted as open circles representing the mean of triplicates. a–g Summary of target and primer sequences, EGFP disruption, NGS, GUIDE-seq and in vitro data are reported in Supplementary Data 1–5. Statistical significance was assessed by RM one-way ANOVA, statistical details and p-values are available in Methods and in Supplementary Data 6 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).