Fig. 4: Sequence contributions are evident in vitro, supporting that it directly affects the enzymatic activity of IFNs.

The effect of increased-fidelity mutations and sequence contributions seen in cellulo also manifests in the rate constants of in vitro cleavage activities of the variants employing 21 targets of Fig. 2. a Representative agarose gel showing the activity of B-evoSpCas9 variant in a plasmid-cleaving in vitro assay at different timepoints in triplicates. b Plot showing values representing the consumption of the intact circular plasmid (not-cleaved) derived from the intensity of bands from the representative agarose gel in (a). Exponential curves were fitted to the timepoints of each replicate separately. c The average k values of three individual fits are shown for 21 EGFP target sites, separated into categories based on the in cellulo cleavage results. The median and interquartile range are shown; data points represent the mean of the fitted k value triplicates; n = 21, 16, 5, 9, 12, respectively. Differences between groups were tested by using either two-tailed unpaired Student’s t-test with Welch’s correction (B-SpCas9-HF1) or by using two-tailed Mann–Whitney test (B-evoSpCas9) in the cases where differences did not meet the assumptions of unpaired t-test. Statistical details and exact p-values are available in Materials and Methods section and in Supplementary Data file 9. a–c Data related to Supplementary Fig. 6. Target and primer sequences, in vitro data and statistical details are reported in Supplementary Data files 1, 8, 9.