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Fig. 3 | Genome Biology

Fig. 3

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

Fig. 3

Reduced target binding affinity of Cas9-sgRNA shifts the pathway bias towards c-NHEJ in repair of Cas9-induced DSBs. a Effects of DNA-PKcs inhibition and Xrcc4 deletion on SpCas9-induced NHEJ in mESC transfected with individual SpCas9-sgRNA and its variants as indicated. Left: Frequencies of SpCas9-induced GFP+ cells; Right: Fold change of NHEJ alteration induced by NU7441 or Xrcc4 deletion relative to the SpCas9-20nt control, i.e., the ratio of NHEJ change induced by NU7441 or Xrcc4 deletion for each SpCas9-sgRNA variant to that for the SpCas9-20nt control. b, c Effects of DNA-PKcs inhibition on NHEJ-mediated genome editing at endogenous loci Cola1 (b) and Rosa26 (c) in mESC transfected with individual SpCas9-sgRNA and its variants as indicated. The frequencies of Cas9-induced indels (left) were calculated as ratios of edited reads to total reads from targeted Illumina sequencing and normalized by transfection efficiency. The fold change of NHEJ alteration induced by NU7441 relative to the SpCas9-20nt control (right) was calculated as the ratio of NHEJ change induced by NU7441 for each SpCas9-sgRNA variant to that for the SpCas9-sgRNA control. d, e Effects of DNA-PKcs inhibition and DNA-PKcs, Ku80, or Xrcc4 deficiency on Cas9-induced HDR in mESC transfected with Cas9-gHRC4 (d), Cas9-gHRC2 (e), and its variants as indicated. Left: Frequencies of Cas9-induced GFP+ cells; Right: Fold change of HDR stimulation induced by NU7441 or deletion of DNA-PKcs, Ku80, or Xrcc4 relative to the SpCas9-20nt control. This fold change was calculated as the ratio of HDR stimulation induced by NU7441 or deletion of DNA-PKcs, Ku80, or Xrcc4 for each SpCas9-sgRNA variant to that for the SpCas9-20nt control. Each circle indicates one independent experiment, each in triplicates, and the mean of at least three independent experiments is also indicated. Columns indicate the mean ± S.E.M. Statistical significance was detected by two-tailed Student’s paired t test for frequencies of Cas9-induced GFP+ cells or Cas9-induced indels and by one-way ANOVA followed by post hoc Dunnett’s test for fold changes of NHEJ alteration or HDR stimulation: *P<0.05; **P<0.01; and ***P<0.001

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