Fig. 2

Accurate NHEJ is influenced by frequent + 1 templated insertions (TI), not directly by the orientations of paired Cas9 or the distance between target sites of paired Cas9. a Correlation between the frequency of accurate NHEJ and the frequency of + 1 TI (blue dot) or + 2 TI (red rhombus). TI template insertions with no additional mutations at repair junctions. b Comparisons between accurate NHEJ and accurate NHEJ combined with + 1 and + 2 template insertions (i.e., Accurate+TI). The difference indicates the underestimation of accurate NHEJ due to + 1 and + 2 TI. Student’s paired t-test P < 0.0001. c Correlation between the frequency of group I and the frequency of accurate NHEJ (blue dot) or the frequency of accurate NHEJ+TI (red rhombus). d Four different Cas9-gRNA orientations guided by paired PAMs. W/W, W/C, C/C, and C/W orientations were defined by the position of paired PAMs on either the Watson strand (W) or the Crick strand (C). e The frequency of Accurate or Accurate+TI from four orientations of 71 endogenous genome sites were calculated and summarized. Bars represent the mean ± standard deviation (SD). In the Accurate group, one-way ANOVA P < 0.0001; post LSD pairwise comparisons: **P < 0.01 for W/W vs W/C; ***P < 0.0005 for W/C vs C/W; *P < 0.05 for W/C vs C/C; not significant (NS) for the others. In the Accurate+TI group, one-way ANOVA NS. f The frequency of + 1 TI from four orientations of 71 endogenous genome sites were calculated and summarized. Bars represent the mean ± SD. One-way ANOVA P < 0.0001; post LSD pairwise comparison test ***P < 0.0005 for W/W vs W/C, ***P < 0.0005 for W/C vs C/W, ***P < 0.0005 for W/C vs C/C; not significant (NS) for the others. g, h Correlation between the frequency of accurate NHEJ (blue circle) or the frequency of Accurate+TI (red rhombus) and the distance between two cleavage sites at 71 genome sites (g) or 29 genome sites with the same W/W orientation (h)