Fig. 4

Fluorescence-based DSB repair assay using Rad18^UBD and RNF169^UBD fusion proteins in HEK^TLR6 reporter cells. Fusion constructs for BRCA1 or TetR with Rad18^UBD or RNF169 ^UBD were cotransfected with the TLR HDR repair template (TLR-donor-tetO), sgRNA and Cas9 into HEK^TLR6 cells. The frequency of Venus⁺ cells (green bars) and RFP⁺ cells (red bars) within the population of BFP⁺ cells was measured by FACS analysis 72 h after transfection and used to calculate the ratio of Venus/RFP positive cells. The X-axis shows the transfected samples and the selection of cotransfected plasmids below. Samples 1 and 2 are controls showing the basic frequency of Venus⁺ and RFP⁺ cells upon transfection with Cas9 and sgRNA or in combination with TLR-donor-tetO as repair template. Data from four independent experiments, each with three replicates per sample, are represented as mean values ± SD. Statistical significance of samples 3–16 in comparison to the control sample 2 was determined by two-way ANOVA and Dunnett’s multiple comparison tests with **P < 0.01, ***P < 0.001 (HDR) and ##P < 0.01, ###P < 0.001 (NHEJ). Raw data are shown in the Supplementary data file