Fig. 1

Repair of Cas9-induced DSBs by NHEJ is inherently accurate. a The sGEJ reporter. NHEJ repair of paired DSBs induced by I-SceI or Cas9-gRNAs are indicated. Repair products are divided into four groups based on DSBs induced at either or both target sites by I-SceI or Cas9-gRNA in the reporter. Groups I, II, III, and IV represent, respectively, NHEJ for DNA cleavage simultaneously at both target sites, only at the first target site, only at the second target site, and individually at two target sites as indicated. b–d Analysis of I-SceI- or paired gRNA-guided Cas9-induced NHEJ in the sGEJ reporter. The normalized editing efficiency (b) was calculated as ratios of edited events to total reads and normalized by transfection efficiency. The frequency of group I, group II, group III, and group IV (c) was calculated as ratios of reads from each group to total edited reads. The frequency of accurate NHEJ, deletion, insertion, and InDel (d) was calculated as ratios of reads from each category in group I to total group I reads. Bars represent the mean ± standard deviation (SD) of three independent experiments. For normalized editing efficiency (b), Student’s paired t-test between I-SceI and gsGEJ2 + 2a *P = 0.025. e Analysis of NHEJ induced by paired Cas9-gRNAs at 70 endogenous genome sites from mouse and human cells. The editing efficiency without transfection efficiency, the frequency of group I in edited events, and the frequency of accurate NHEJ in group I events from each site were analyzed. Bars represent the mean ± SD. f–h Analysis of NHEJ induced by paired Cas9-gRNA at mPIF1, mLDHA, and two hp53 sites. The normalized editing efficiency (f), the frequency of each group in edited events (g), and the frequency of each category in group I events (h) were calculated. Bars represent the mean ± SD of three independent experiments