Fig. 1
Involvement of c-NHEJ varies widely in repair of Cas9-induced DSBs. a Schematic of the HDR reporter with 5 sgRNAs. Repair of Cas9-induced DSBs by HDR between sister chromatids can generate GFP+ cells. Inhibition of c-NHEJ is expected to promote HDR. b Effects of DNA-PKcs inhibition on SpCas9-induced HDR in mESC transfected with individual Cas9-sgRNA. Left: Frequencies of SpCas9-induced GFP+ cells; Right: Relative HDR after normalizing DMSO treatment to 1.0. c Effects of DNA-PKcs, Ku80, or Xrcc4 deficiency on SpCas9-induced HDR in mESC transfected with individual Cas9-sgRNA. Left: Frequencies of SpCas9-induced GFP+ cells; Right: Relative HDR after normalizing both WT cells and Xrcc4+/+ cells to 1.0. d Schematic of the NHEJ reporter with 6 sgRNAs and their target sites indicated. Repair of Cas9-induced DSBs by c-NHEJ or a-EJ generates accurate NHEJ (accNHEJ) products indistinguishable from undamaged targets and mutagenic NHEJ (mutNHEJ) products represented by GFP+ cells. Inhibition of c-NHEJ promotes a-EJ. e Effect of DNA-PKcs inhibition and Xrcc4 deletion on SpCas9-induced NHEJ in mESC transfected with individual Cas9-sgRNA. Left: Frequencies of SpCas9-induced GFP+ cells; Right: Relative NHEJ after normalizing both DMSO treatment and Xrcc4+/+ cells to 1.0. f, g Cells were transfected with SpCas9-sgRNA expression plasmids and treated with DMSO or NU7441. Four different sites of the Cola1 (f) and Rosa26 (g) locus were targeted by 4 sgRNAs indicated. The efficiency of SpCas9-induced genome editing (left) was calculated as ratios of edited reads to total reads from targeted Illumina sequencing and normalized by transfection efficiency. Relative SpCas9-induced NHEJ (right) was calculated by normalizing the editing efficiency with DMSO treatment to 1.0. Each circle indicates one independent experiment, each in triplicates. Columns indicate the mean ± S.E.M of at least three independent experiments. Significance was detected by two-tailed Student’s t test and indicated by * for P<0.05, ** for P<0.01 and *** for P<0.001