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Fig. 6 | Genome Biology

Fig. 6

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

Fig. 6

Transcription has no effect on c-NHEJ in repair of Cas9-induced DSBs. a dSpCas9-mediated transcriptional silencing in mESC containing pPGK-GFP expression cassette. The mean fluorescence intensity of GFP indicates relative transcription. b Involvement of c-NHEJ in SpCas9-induced DSBs in mESC containing pPGK-GFP expression cassette. Cells were transfected with a low amount of individual SpCas9-sgRNA expression plasmids (0.001 μg SpCas9, 0.001 μg sgRNA, 1/500 of total DNA each) as shown and treated with NU7441 at 6 h post-transfection. Relative NHEJ (n=3) was calculated by normalizing DMSO treatment to 1.0. c Analysis of strand bias in transcriptional silencing (top) and c-NHEJ involvement (bottom) between transcription template strand and transcription non-template strand targeted by dSpCas9-sgRNA or SpCas9-sgRNA. Transcriptional silencing and c-NHEJ involvement were defined as the percentage of GFP fluorescence intensity reduced by dSpCas9-sgRNA and the percentage of SpCas9-induced NHEJ stimulated by NU7441, respectively. d Correlation between dSpCas9-mediated transcriptional silencing and c-NHEJ involvement in repair of SpCas9-induced DSBs. Each circle indicates the level of dSpCas9-mediated transcriptional silencing and stimulation of SpCas9-induced NHEJ by NU7441 at the same target. Two sgRNAs gGW5 and gGC4 are indicated by arrows for their strong effect on transcriptional silencing. e dSaCas9-mediated transcriptional silencing in mESC containing pPGK-GFP expression cassette. The mean fluorescence intensity of GFP indicates relative transcription. Transcription blockage by dSaCas9-gSaGW1 and dSaCas9-gSaGW2 induced significant transcription silencing. Positions of the targets by dSaCas9-sgRNAs are indicated in the reporter. f Little effect of transcription blockage by dSaCas9-gSaGW1 and dSaCas9-gSaGW2 on DNA-PKcs involvement in SpCas9-mediated GFP gene editing. GFP+ cells were co-transfected with SpCas9-sgRNA (SpCas9 and sgRNA at 0.125 μg and 0.0005 μg respectively) and dSaCas9-sgRNA (0.125 μg each), and frequencies of SpCas9-induced GFP– cells measured by FACS at 4 days post-transfection. Each circle indicates one independent experiment, and the mean of these independent experiments is also indicated. Error bars indicate S.E.M. Two-tailed Student’s paired or unpaired t test is indicated by * for P<0.05, ** for P<0.01, *** for P<0.001, and n.s. for not significant. Correlation between transcription silencing and the NHEJ increase was determined by linear regression analysis

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