Figure 2.

Identification and characterization of an optimized high-fidelity SpCas9 variant

(A) On-/off-target ratios obtained with fully matching sgRNA (on target) over two surrogate off targets (sgGFP13-14 and sgGFP18-19 with double mismatches in position 13-14 or 18-19 of the spacer, respectively) with K526 variants with the indicated amino acid substitutions. The editing was measured following transient plasmid transfection in HEK293-GFP reporter cells. The ratios are obtained using raw data from Figure S1. Statistical significance was assessed using one-way ANOVA, comparing each mutant with WT SpCas9 separately for on/off 13-14 and on/off 18-19. (B) Editing activities (percentage of indels) of WT SpCas9, HiFi Cas9, or rCas9HF RNPs were measured through tracking indels by TIDE analysis (n = 2 for K526D activity on HPRT locus). (C and D) Targeted deep sequencing analysis of the on targets (C) and previously validated off targets (D) after electroporation of WT SpCas9, HiFi Cas9, or rCas9HF RNPs in U2OS cells. The off-/on-target ratios calculated from the data in (C) and (D) are reported in Figure S2A. Statistical significance was assessed using paired t test to compare HiFiCas9 and rCas9HF; data reported as mean ± SEM for n ≥ 3 biologically independent replicates. (E) Percentage distribution of GUIDE-seq reads among the on-target and all off-target sites after electroporation of U2OS cells with the WT SpCas9, rCas9HF, and HiFi Cas9 RNPs; GUIDE-seq details are in Figure S3 and Tables S3–S11.