Fig. 2. Ube2T and FANCL promote HR-mediated repair of Cas9-induced DSBs.

a HEK 293T + DSB-Spectrum_V3 Ube2T^KO clones 4.1, 4.16, and 4.21, as well as the parental control (Con.) were treated with Mitomycin C (MMC, 300 nM) for 24 h. Next, Ube2T expression levels and FANCD2 ubiquitination status were analyzed by western blotting. b Indicated HEK 293T + DSB-Spectrum_V3 cell lines were transfected to express S. pyogenes Cas9 and either a control sgRNA or the sgRNA targeting the BFP gene in the reporter locus. Next, cells were analyzed by flow cytometry to determine the frequency of repair by each of the three indicated pathways (n = 6 independent biological replicates; mean ± SEM; one-way ANOVA with post-hoc Dunnett’s). HR homologous recombination, mut-EJ mutagenic end-joining, SSA single-strand annealing. c DNA sequence alignment of the FANCL sgRNA target site in unedited parental control cells and the HEK 293T + DSB-Spectrum_V3 FANCL^KO clones. Depicted are representative sequence chromatograms, red shaded boxes indicate deviations in the DNA sequence of the FANCL^KO clone compared to control. d As in panel (b), now analyzing FANCL^KO cells (n = 7 independent biological replicates; mean ± SEM; one-way ANOVA with post-hoc Dunnett’s). e HEK 293T + DSB-Spectrum_V3 FANCL^KO clones were transduced with an empty vector (EV), FANCL wild-type cDNA (WT), or FANCL Ligase-Dead cDNA (LD), and treated with Mitomycin C (MMC, 1 μM) for 24 h. Next, FANCD2 ubiquitination was analyzed by western blot. f As in panel b, now analyzing the HEK 293T + DSB-Spectrum_V3 FANCL^KO cells described in panel (e) (n = 3 independent biological replicates; mean ± SEM; one-way ANOVA with post-hoc Dunnett’s). Source data are provided as a Source Data file.