Fig. 1

DNA-PKcs inhibitors stimulate HDR following Cas9 induction of a DSB. a Schematic outline of the Traffic Light Reporter assay. The eGFP and mCherry open reading frames (ORF) are fused by a ribosome skipping sequence (T2A) where translating ribosomes skip from a Gly to a Pro codon without forming a peptide bond while maintaining the reading frame to produce two distinct polypeptides [39, 40]. In the TLR, the eGFP and mCherry ORFs are positioned in different frames and NHEJ repair of a DSB directed to the GFP ORF will, in one out of three events, place ΔeGFP in frame with mCherry - leading to mCherry production. Since the eGFP ORF contains an insertion harboring a premature termination codon (and lacks a start codon), its expression can only be recovered upon HDR-mediated repair following delivery of an appropriate homologous template. The reading frame for each fluorescent protein is indicated in parentheses. b Outline of siRNA and drug treatment protocol. In one set of experiments, the 293/TLR cell line was transfected with the indicated siRNAs (yellow box) and 24 h later expression constructs driving synthesis of Cas9 and sgRNAs and an eGFP donor template were introduced into the cells. In a second set of experiments, the 293/TLR cell line was first transfected with expression constructs driving synthesis of Cas9 and sgRNAs and an eGFP donor template. Compounds were added 16 h later (orange box). In both cases, cells were allowed to propagate for 5 days before FACS analysis. c Knockdown of DNA-PK, DNA Ligase IV, and PI3K-p110α in 293/TLR cells transfected with the indicated siRNAs. Western blots were probed with antibodies to the indicated proteins. The dashed line separates two different sets of membranes. d Quantitation of genome editing events in 293/TLR cells transfected with the indicated siRNAs. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to scrambled siRNAs) was calculated using the Student’s t-test: *P ≤0.05; **P ≤0.01; ns, not significant. e DNA-PK inhibitors prevent H2AX phosphorylation upon γ-irradiation. Cells were pre-incubated with the indicated small molecules for 1 h followed by 4 GY of γ-irradiation (IR). Extracts for western blotting were prepared from non-irradiated (IR) cells (lane 1) or IR-exposed cells incubated in the presence of vehicle (lane 2), 2 μM NU77441 (lane 3), and 250 nM KU-0060648 (lane 4). Western blots were probed with antibodies directed to the proteins indicated to the left of the blot. The ratio of p-H2AX and eEF2 band intensities is indicated below the blots. f Flow cytometry analysis of 293/TLR cells transfected with pQCX vectors driving synthesis of Cas9 and sgRNAs targeting Rosa26 or eGFP. When indicated, the GFP repair template, pRRL SFFV d20GFP.T2A.mTagBFP (ΔeGFP Donor) was also included. NU7441 and KU-0060648 were used at a final concentration of 2 μM and 250 nM, respectively. Cells were gated for the live population as determined by PI staining. Transfection efficiencies were greater than 70 %. g Quantitation of genome editing events from cells propagated in the presence of the indicated concentrations of DNA-PKcs inhibitors. 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