Fig. 3

HDR directed by oligonucleotide donors are stimulated by DNA-PKcs inhibitors. a Quantitation of genome editing events from cells transfected with pQCiG-TLR and either ΔeGFP donor (2.5 kbp homology upstream and 1.5 kbp homology downstream of target site) or oligonucleotides (sense or antisense) (110 nucleotides) spanning the sgGFP target site and exposed to vehicle, 2 μM NU7441, or 250 nM KU-0060648. The HDR and NHEJ values are relative to those obtained with Cas9, sgRNA, and ΔeGFP donor in the presence of vehicle (DMSO). N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t-test: *p ≤0.05; **p ≤0.01; ns, not significant. b Representative examples of FACS plot obtained in (a). Note that mCherry⁺ cells are reporting on only one-third of all NHEJ events. c Quantitation of genome editing events from experiments performed as described in (a) except using WT Cas9 or the D10A and H840A nickase variants and the ΔeGFP donor. Values are set relative to editing frequencies observed in 293/TLR cells transfected with WT Cas9 expression vector and propagated in the presence of vehicle. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t-test: *p ≤0.05; **p ≤0.01; ns, not significant