Figure 1.

CRISPR/Cas9-CtIP-dnRNF168 promotes HDR-mediated repair of a designer nuclease-induced DSB. (A) The Traffic Light Reporter (TLR) is integrated into the AAVS1 site of HEK293T cells (HEK-TLR) and includes a fusion of a mutated mVenus with an out-of-frame TagRFP genes. Depending on the engaged DSB repair pathway, the introduction of a CRISPR/Cas9 induced DSB results in the appearance of red or green cells that can be quantified via flow cytometry (left). Representative plots showing the gating strategy for the acquisition of the TagRFP+ (NHEJ) and mVenus+ (HDR) events within the transfected cell population (BFP+) is shown on the right. Δ: mutation abrogating mVenus expression. CAG: CMV enhancer, chicken beta-actin promoter. T2A: 2A self-cleaving peptide. (B, D) The bar graphs show the precision score, computed as the ratio between the HDR and NHEJ events measured as reported in A, for the various Cas9 fusion proteins. The fold change in relation to the unmodified Cas9 is indicated for the best variant. (C, E) The diagrams indicate the percentage of NHEJ or HDR events as determined by flow cytometry on day 6 post transfection. In panels B-E, each dot represents the average of experimental duplicates. Statistically significant differences, as compared to normal Cas9 nuclease, are indicated with asterisks and correspond to p-values calculated with ANOVA (*P< 0.05, **P< 0.01). Error bars indicate SEM.