Fig. 4: DSB-Spectrum_V3 is a reporter for mutagenic end-joining, SSA, and HR. | Nature Communications

Fig. 4: DSB-Spectrum_V3 is a reporter for mutagenic end-joining, SSA, and HR.

From: Multi-pathway DNA-repair reporters reveal competition between end-joining, single-strand annealing and homologous recombination at Cas9-induced DNA double-strand breaks

Fig. 4: DSB-Spectrum_V3 is a reporter for mutagenic end-joining, SSA, and HR.The alternative text for this image may have been generated using AI.

a Cartoon depicting potential outcomes of a multi-pathway reporter cell-line designed to quantify DSB-repair by mutagenic end-joining (mut-EJ), SSA, and HR. b Diagramatic representation of the genomic DSB-repair reporter construct DSB-Spectrum_V3. Scissors indicate the Cas9-target site, orange pacman indicates endogenous nucleases. c, d DSB-Spectrum_V3 cells were transfected with Cas9 and an sgRNA targeting either AAVS1 or BFP, followed by treatment with NU7441 (2 µM). At 72 h after Cas9 transfection cells were analyzed by flow cytometry. Panel c shows representative flow plots. Panel d shows quantification of the three repair pathways from multiple experiments (n = 3; mean ± SEM; ratio paired t-test, two-tailed). e DSB-Spectrum_V3 cells were transfected with indicated siRNAs, followed by flow cytometric analysis of mut-EJ, SSA, and HR (n = 3; mean ± SEM; One-way ANOVA, post-hoc Dunnett’s). f Western blot of lysates from cells analyzed in panel e. Tubulin is used as a loading control. Red arrow indicates Rad52 band, asterisk indicates nonspecific background band. g Mut-EJ, SSA, and HR was analyzed as in panel e, following siRNA-mediated knockdown of the indicated repair factors (n = 4; mean ± SEM; One-way ANOVA, post-hoc Dunnett’s). h Western blot of lysates from cells analyzed in panel g. Source data for panels d, e, and g are provided as a Source Data file.

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