Fig. 5: Increased-fidelity SpCas9 variants in the higher fidelity ranks with in-between activity/fidelity.

a, c The results of an on-target EGFP disruption assay for WT and different SpCas9 variants on various target sites shown on a column graph. Means and SD are shown; n = 3 biologically independent samples (overlaid as white circles). a, Reverting three mutations of xCas9 back to the WT residues, suggested by Guo et al.⁴⁸ as being responsible for its increased fidelity and target-selectivity, did not increase its activity the way we expected. Statistical significance was assessed by RM one-way ANOVA and shown in Supplementary Data file 9. b Normalized on-target activities of various SpCas9 variants presented on a scatter dot plot. The sample points correspond to data presented in (c); n = 14. Continuous red line indicates 0.20 normalized disruption activity, under which we consider the IFNs not to be active on a given target. The median and interquartile range are shown; data points are plotted as open circles representing the mean of biologically independent triplicates. Differences between SpCas9 variants were tested by using either two-tailed paired-samples Student’s t-test or by using two-tailed Wilcoxon Signed Ranks test in the cases where differences did not meet the assumptions of Paired t-test. Statistical details and p-values are available in Methods and in Supplementary Data file 9. d, f Heatmaps show the normalized EGFP disruption activities of SpCas9 nucleases with perfectly matching 20G-sgRNAs. The bold line indicates the dividing line defined by the cleavage rule between the classes of cleaved and not-cleaved values. d SpCas9-NG and xCas9 do not strictly obey the cleavage rule when fitted on the heatmap of Fig. 2a (only 42 EGFP target sites were tested here), even though some of the targets for which the order were not determined by the 19 IFNs were reordered (compared to Fig. 2a heatmap) to favor the accommodation of SpCas9-NG and xCas9 into the cleavage map. These results explain the failure to develop an IFN series with a looser PAM requirement and emphasize that the cleavage rule identified here is non-trivial and does not apply to all other SpCas9 variants with reduced activity or increased fidelity. e The ROC curves demonstrate that the order of the target sequences, determined by the cleavage rule, competently separates the classes of the cleaved and not-cleaved targets in case of the IFN variants, but xCas9 (AUC: 0.68) and SpCas9-NG (AUC:0.70) do not appear to strictly follow the cleavage rule, emphasizing that the rule is not self-evident. f Additional IFNs provide a finer resolution within the higher fidelity region of the IFN ranking between evo- and HeFSpCas9, and their activities on these targets also strictly follow the cleavage rule. For these experiments, we selected targets from the higher cleavability region of the target ranking in Fig. 2, as these were expected to be the point of distinction between the additional variants, and therefore facilitate the ordering of these IFNs and their fitting into the already existing ranking. This is the reason for these high fidelity IFNs showing much higher normalized disruption activities, than they would on randomly selected targets. a–f Target sequences, raw, processed, heatmap disruption data and statistical details are reported in Supplementary Data files 1–4, 9.