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Fig. 5 | Genome Biology

Fig. 5

From: Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing

Fig. 5

Precise out-of-frame deletion mediated by accurate NHEJ promotes gene knockout. a Classification of the Common, Ideal, and Paired approaches. With paired Cas9-gRNAs, Common, Ideal, and Paired represent genome editing that depends, respectively, upon NHEJ without group I events, NHEJ for ideally all simultaneous Cas9 cleavage, and NHEJ including group I events. b Correlation between accurate NHEJ and out-of-frame mutations derived from the Common approach (black squares), the Ideal approach (blue squares), or the Paired approach (red circle). c The frequency of out-of-frame mutations between different groups as indicated. Bars represent the mean ± standard deviation (SD) of the out-of-frame frequencies. Data were analyzed by one-way ANNOVA followed by post-hoc LSD pairwise comparisons (**P < 0.005, ***P ≤ 0.0005, NS not significant). d Testing of paired Cas9-gRNA at exon 2 of mMDC1. Mouse ES cells were transfected with expression plasmids for single or paired gRNAs and Cas9 and genomic DNA were purified 72 h post-transfection and amplified by primers flanking the cutting sites. The PCR amplicons were subjected to agarose gel electrophoresis. The distance between paired Cas9-gRNA target sites is 52 bp as indicated. e The frequency of out-of-frame (i.e., knockouts) and in-frame MDC1 editing events induced by single or paired Cas9-gRNA. For knockout efficiency, Student’s paired t-test between g2 and g2 + 3 *P = 0.029; between g3 and g2 + 3 *P = 0.013. f The frequency of group I, group II, group III, and group IV events among those edited by Cas9 guided by paired gRNAs as indicated. g The frequency of accurate, deletion, insertion, and indel events among group I events induced by paired Cas9-gRNAs. h The HR reporter. Repair of an I-SceI- or Cas9-induced DSB by HR between sister chromatids generates wild-type GFP. i, j Percentage of I-SceI- (i) or Cas9- (j) induced GFP+ cells from HR reporter mouse ES cells transiently transfected with expression plasmids for single or paired gRNA-guided Cas9. Values are the mean ± SD of three independent experiments, each in triplicate. In I-SceI-induced HR (i), Student’s paired t-test between g2 and g2 + 3 **P = 0.0048; between g3 and g2 + 3 *P = 0.0154. In Cas9-induced HR assays (j), Student’s paired t-test between g2 and g2 + 3 ***P = 0.0004; between g3 and g2 + 3 *P = 0.0449. k Generation of MDC1 mutant clones by paired Cas9-gRNAs. Clones were picked, grown, and identified by Sanger sequencing. The frequencies of specific MDC1 mutations are indicated in parentheses. l MDC1 knockout was confirmed by western blotting using β-actin as a loading control. m Percentage of I-SceI-induced GFP+ cells from one MDC1+/+ and three isogenic MDC1−/− HR reporter clones (clones #4, #26, and #32). Bars represent the mean ± SD of three independent experiments, each in triplicate. Student’s unpaired t-test: ***P < 0.0001 between MDC1+/+ and MDC1−/−. WT wild type

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