FIGURE 2.

DSB repair modification by Cas9 fusion proteins. (A) The traffic light reporter (TLR) construct indicates DSB repair by NHEJ or HDR and was integrated into the AAVS1 locus of HEK cells (HEK^TLR). Upon induction of a DSB in the defective Venus coding region, RFP is expressed upon NHEJ repair resulting into deletions that shift translation by 2 bp. Venus expression occurs upon HDR with a repair template vector that includes the intact Venus coding region (pTLR-repair). (B) Vectors for the expression of Cas9 or fusion proteins between the N- or C-terminal end of Cas9 and Rad52, RPA1, CtIP wildtype (wt), or the T847E mutant (mut), Mre11A or Rad51C, driven by the CBh promoter. pA – polyadenylation signal. (C) For DSB repair assays HEK^TLR reporter cells were cotransfected with expression vectors for Cas9 or Cas9 fusion proteins, sgRosa26, Blasticidine and pTLR-repair. After 4 days of Blasticidine selection the samples were analyzed by FACS for the presence of Venus and RFP positive cells. (D) Bar graph representation of Venus and RFP positive cells, indicating HDR or NHEJ repair of the TLR reporter, upon transfection with an expression vector for Cas9, or Cas9 in C-terminal (Cter) or N-terminal (Nter) fusion with Rad52, RPA1, CtIP wildtype (wt), CtIP mutant T847E (mut), MRE11A, or Rad51C. Bars show mean values of three 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 2–9. n.s., not significant, p > 0.05.