Fig. 5
DNA-PKcs inhibition and Xrcc4 deletion aggravate off-target effect in CRISPR/Cas9 genome editing. Xrcc4+/+ HDR reporter mESC were used for transfection with SpCas9 in complex with gPnpla3 targeting Pnpla3 (a), gMertk targeting Mertk (b), gHRC1 (c), and gHRC2 (d) both targeting the HDR reporter. At 6 h post-transfection, cells were treated with DMSO or NU7441. At 72 h post-transfection, gDNA was isolated and the indel frequency at on-target and selected off-target sites was measured by amplicon deep sequencing and calculated as the ratio of edited reads to total reads normalized by transfection efficiency. In an independent set of experiments, isogenic Xrcc4+/+ and Xrcc4–/– HDR reporter mESC were transfected and the indel frequency at on-target and selected off-target sites was similarly measured. Fold change of off-target effect after treatment of NU7441 or deletion of Xrcc4 was calculated as the ratio of the indel frequency with treatment of NU7441 or in Xrcc4–/– cells to that with DMSO or in Xrcc4+/+ cells at each off-target site, respectively. Each circle indicates one independent experiment, and the mean of these independent experiments is also indicated. Error bars indicate S.E.M. Statistical analysis was performed by two-tailed Student’s paired t test for frequencies of Cas9-induced indels and by one-way ANOVA followed by post hoc Dunnett’s test for fold changes of off-target effect between NU7441 and DMSO, and between Xrcc4+/+ and Xrcc4–/–. *P<0.05 and **P<0.01