Fig. 7
The Plk3 inhibitor GW843682X enhances accurate NHEJ in the repair of Cas9-induced DSBs. a, b Percentage of I-SceI- (a) or paired-Cas9- (b) induced GFP+ cells from sGEJ reporter cells transfected with siRNA against CtIP or Luciferase (Luc). Bars represent the mean ± standard deviation (SD) of three independent experiments, each performed in triplicate. In I-SceI-induced HR assays (a), Student’s paired t-test between siLuc and siCtIP **P = 0.0034; In Cas9-induced HR assays (b), Student’s paired t-test between siLuc and siCtIP **P = 0.0006. Depletion of CtIP was determined by western blotting using β-actin as a loading control. c Percentage of paired Cas9-induced GFP+ cells from sGEJ reporter cells treated with the Plk3 inhibitor GW843682X at concentrations of 1 μM, 3 μM, and 5 μM. Bars represent the mean ± SD of three independent experiments, each performed in triplicate. Student’s paired t-test between DMSO and 1 μM *P = 0.016; between DMSO and 3 μM **P = 0.003; between DMSO and 5 μM *P = 0.005. d–f Analysis of NHEJ induced by paired Cas9-gRNAs at the LDHA site in mouse ES cells treated with the Plk3 inhibitor at different concentrations. The normalized editing efficiency (e), the frequency of each group in edited events (f), and the frequency of each category in group I events (g) were calculated. Bars represent the mean ± SD of three independent experiments. For the normalized editing efficiency (d), Student’s paired t-test between DMSO and 1 μM **P = 0.006; between DMSO and 5 μM *P = 0.04. g Deletion length distributions of group I Del events at the LDHA site in mouse ES cells treated with the Plk3 inhibitor. The reads were combined from three independent experiments. Each dot represents 200 reads. The median deletion length is indicated, and deletion length distributions demonstrate a shift towards longer deletions in cells treated with the Plk3 inhibitor (Mann–Whitney test between DMSO and Plk3i ***P < 0.0001). h Frequency of accurate NHEJ among group I (left) and frequency of deletions with different deletion length in Del events of group I NHEJ (right) in mouse ES cells treated with Plk3i at different concentrations as indicated. Del NHEJ events were grouped into 58–63 bp and > 63 bp. The respective reads and frequencies are summarized in the inset and compared by a χ2 test with P values as indicated