FIGURE 3.

Cas9 fusion proteins in 53BP1 knockout HEK^TLR cells. (A) For DSB repair assays in HEK^TLR and HEK^TLR cells harboring 53BP1 knockout alleles (HEK^TLR/Δ53BP1), reporter cells were cotransfected in duplicate with expression vectors for Cas9 or Cas9 fusion proteins, sgRosa26, BFP and pTLR-repair. After 4 days the samples were analyzed by flow cytometry (FACS) for the presence of Venus and RFP positive cells indicating repair of the TLR construct by HDR or NHEJ. (B) Bar graph representation of Venus and RFP positive cells, indicating HDR or NHEJ repair of the reporter in HEK^TLR wildtype (53BP1-WT) cells or (C) HEK^TLR/Δ53BP1 (53BP1-KO) cells, upon transfection with an expression vector for Cas9, or Cas9 in C-terminal fusion with MRE11A, Rad52, CtIP wildtype (wt), or the CtIP mutant T847E (mut). Bars show mean values of four (samples 1–3) or three (samples 4, 5) independent samples with standard deviation. These values were used to calculate the ratio of Venus⁺ to RFP⁺ cells and p-values (T-test) to determine the significance in levels of Venus⁺ or RFP⁺ cells between samples 1 and 2–5. n.s., not significant, p > 0.05.