High dosages of Cas9 RNP reduced cell viability but had a negligible effect on off-target editing. (A) Cell viability and viable CD96⁻ cells at increasing dosages of Cas9 RNP were analyzed by Precision beads assay. Representative flow cytometry histograms were from 40 pmol Cas9 RNP. (B) The results of SIGLEC7 KO. (C) Analysis of CD96 and SIGLEC7 on- and off-target editing by amplicon-based NGS. Mismatches between the on- and off-target sequences are labeled in red. Sequence variations were determined by CRISPResso2 and are presented as indel percentage. The parental cells contained a single-nucleotide polymorphism in the SIGLEC7 on-target site that gave rise to 5% of background indel. Chr, chromosome; PAM, protospacer adjacent motif. (D and E) Influence of different sgRNA preparations on cell viability in the KO of CD96 (D) and SIGLEC7 (E). Data are shown as mean ± SD of three donors (n = 3). The off-target analysis was performed in one donor (n = 1). Two-tailed Welch’s unequal variances t test was used to test for statistical significance. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. IVT, in vitro transcription; ns, not significant; std, standard for comparison.