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. 2022 Sep 8;13:5295. doi: 10.1038/s41467-022-32743-w

Fig. 2. DSB-Spectrum_V2 is a reporter for both mutagenic repair and HR.

Fig. 2

a Cartoon depicting potential outcomes of a multi-pathway reporter cell-line designed to quantify DSB-repair by mutagenic end-joining and HR. b Diagramatic representation of the genomic DSB-repair reporter construct DSB-Spectrum_V2. Expanded region shows the DNA sequence targeted by Cas9. The sequence of the BFP cDNA is displayed in blue, the PAM sequence of the sgRNA target site is displayed in red. Arrow in the inset and scissors in the cartoon indicate the Cas9 cut site. c, d Flow cytometry plots and quantification of mutagenic repair and HR in DSB-Spectrum_V2 cells (n = 3; mean ± SEM), as in Fig. 1c, d. e DSB-Spectrum_V2 cells were transfected with Cas9 and an sgRNA targeting either a control locus or BFP. At 48 h after Cas9 transfection genome editing was quantified by TIDER analysis of the sequenced target site. At 72 h after Cas9 transfection cells were analyzed by flow cytometry (n = 3; mean ± SEM). f DSB-Spectrum_V2 cells were transfected with Cas9 and an sgRNA targeting BFP, and 72 h later the BFP− population was collected by FACS. InDel frequency was determined by TIDER analysis of the sequenced target site. g, h As in panels c and d, but for U2OS DSB-Spectrum_V2 cells (n = 3; mean ± SEM). i Mutagenic repair and HR was quantified in HEK 293T DSB-Spectrum_V2 cells with or without treatment with NU7441 (2 μM; n = 3; mean ± SEM; ratio paired t-test, two-tailed). j As in panel i, but for U2OS DSB-Spectrum_V2 cells (n = 3; mean ± SEM; ratio paired t-test, two-tailed). Source data for panels d, e, f, h, i, and j are provided as a Source Data file.