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Fig. 2 | Genome Biology

Fig. 2

From: Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing

Fig. 2

Cas9 recleavage increases c-NHEJ-mediated mutations. a Model for enrichment of mutNHEJ products promoted by frequent SpCas9 recleavage with increased amount of Cas9-sgRNA transfected. With sufficient amount of SpCas9-sgRNA, accNHEJ products could be recleaved until mutNHEJ products are generated, resulting in enrichment of mutNHEJ products. b,c Effect of DNA-PKcs inhibition on NHEJ induced by varying amount of SpCas9-sgRNA. NHEJ reporter mESC were transfected with varying amounts of expression plasmids for SpCas9-gEJW6 (b) or SpCas9-gEJW7 (c) as indicated and treated with DMSO or NU7441. Frequencies of SpCas9-induced GFP+ cells were measured by FACS at 3 days post-transfection and relative SpCas9-induced NHEJ was calculated by normalizing DMSO treatment to 1.0. d Frequencies of GFP+ cells (left) and relative NHEJ (right) induced by SpCas9-sgRNA at 0.001 μg each, 1/250 of the regular amount (0.25 μg each) transfected into mESC. Each circle indicates one independent experiment, each in triplicates, and the mean of at least three independent experiments is also indicated. e,f Effect of DNA-PKcs inhibition on HDR induced by varying amount of SpCas9-sgRNA. HDR reporter mESC were transfected with varying amounts of expression plasmids for SpCas9-gHRC2 (e) or SpCas9-gHRC4 (f) as indicated and treated with DMSO or NU7441. Frequencies of SpCas9-induced GFP+ cells were measured by FACS at 3 days post-transfection and relative SpCas9-induced NHEJ was calculated by normalizing DMSO treatment to 1.0. For NHEJ and HDR assays in b–f, more than 200,000 cells were usually harvested to ensure at least 100 GFP+ cells counted for reliable calculation. Columns indicate the mean ± S.E.M. Statistical significance was detected by two-tailed Student’s t test: *P<0.05; **P<0.01; and ***P<0.001

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