Fig. 5: Comparison of VRKG with Wt Cas9 and other NAG-permissive variants in human cell culture targeting assays.

a Disruption of a chromosomally integrated constitutive EGFP cassette in the U2OS background, using catalytically active wild-type or engineered variants with NGG- or NAG-targeting sgRNAs as indicated. EGFP knockdown activity is plotted as the percentage of EGFP-negative cells, determined from transiently transfected populations of single cells analyzed by flow cytometry. sgRNA sequences and nomenclature were designed previously⁷. p values calculated from two-tailed t tests comparing Wt Cas9 and VRKG with each NAG-targeting sgRNA are shown. Error bars, mean ± s.d. (n = 4, biological replicates). Black dashed line denotes the EGFP-negative background level determined from experiments with a non-targeting sgRNA. b Quantification of GUIDE-seq off-target sites detected for Wt Cas9, VRKG, and SpRY with the ‘HEK site 4’ sgRNA in U2OS.EGFP cells. Bars quantify all off-target sites (black bars) or only those off-targets that contributed >0.50% of summed GUIDE-seq read counts (gray bars) detected in each experiment. c Percent on-target activity, determined for each variant from the fraction of their on-target GUIDE-seq read counts out of summed GUIDE-seq reads detected overall. Source data are available in the Source Data file.