Fig. 4: Structure-guided engineering improves the editing efficiency of activity-enhanced KKH-SaCas9 variants.

a Molecular modeling of N888Q and N888R/A889Q mutations on WED domain of SaCas9 depicts their increased interactions with residues on its PI domain and the DNA backbone. b KKH-SaCas9 variants carrying mutations on residues 888 and/or 889 were individually constructed and characterized using GFP disruption assays with three independent sgRNAs at 5 days post infection. The mean editing efficiency +/− SD (error bar) of the KKH-SaCas9 variants (n = 4 biologically independent samples) was measured as the percentage of cells with depleted GFP fluorescence using flow cytometry. Statistical significance was analyzed by one-way ANOVA with Tukey’s test. The P-values of 0.016–0.024 indicate the comparisons with the wild-type variant's activity. c, d Assessment of KKH-SaCas9 variants’ on-target editing with sgRNAs targeting endogenous loci. The percentage of sites with indels was measured using a T7 endonuclease-I (T7E1) assay in (c) and deep-sequencing assay in (d). Mean and standard deviation are shown for the loci tested. Each locus was measured in n = 3 biological independent samples. Statistical significance was analyzed by one-way ANOVA with Tukey’s test. The P-values of <0.0001–0.048 indicate the comparisons with the wild-type variant’s activity. The ratios of the on-target activity of KKH-SaCas9 variants with N888Q and N888R/A889Q mutations to the activity of KKH-SaCas9 were determined, and mean for the normalized activity is shown and highlighted by a red line (n = 7, one-sample t-test). n.s. indicates not significant. e Western blot analysis on protein expression of the KKH-SaCas9 variants. This experiment was performed once. The source data for figures (b–e) are provided as a Source Data file.