HEK293 cells carrying biallelic copies of the eTLR system in AAVS1 were transfected with a promoterless repair template for mScarlet-I and an all-in-one CRISPR–Cas9 plasmid targeting eTLR. At 16 h after transfection, the indicated compounds were added to the cells. At 3 days after transfection, cells were analyzed for red and green fluorescence by flow cytometry to quantify the effect of the compounds on the repair outcome. Colored bars quantify the fraction of cells with green, red, or both (orange) signals representing the different editing outcomes (left y axis), whereas the gray bars (right y axis) display the ratio of HDR over mutagenic end-joining events as a measure of editing precision. Selected results of a Bonferroni MCT after one-way ANOVA were shown for the HDR events as well as editing precision (HDR/mutEJ) and are indicated by asterisks; * P < 0.05, **** P < 0.0001. Bars, mean ± s.d. ( n = 3 biological replicates). Please note that only very few red or green events were recorded by FACS for the condition without donor and Cas9, and the non-targeting control (NTC) such that the corresponding HDR/mutEJ ratios are not informative (shaded bars).
