Extended Data Fig. 2: Biochemical validation of DNA-PKcs inhibition and CCR5 gene targeting with AZD7648 in PSC.
a. Western blot analysis for phospho DNA-PKcs (pSer2056), DNA-PKcs and phospho AKT (pSer473) in PSCs treated with bleomycin ± AZD for 2 h or gene targeted with and without AZD treatment at 2 h post gene editing. ACTB is used as a loading control. Control denotes sample not treated with bleomycin. Mock denotes control sample nucleofected without RNP. UNT denotes sample either treated with bleomycin or RNP-AAV6 gene editing and AZD denotes AZD7648 treated sample with either bleomycin or RNP-AAV6 gene editing treatment as indicated. b. Allelic gene targeting efficiency of PSCs gene targeted at the CCR5 locus for knock-in of UBC-GFP-bGHpA sequence (a) with and without AZD treatment at 72 h post gene editing as measured by ddPCR (n = 1). c. Percentage of gene targeted cells at the CCR5 locus for knock-in of UBC-GFP-bGHpA sequence with two different concentrations of AZD7648 (0.5 and 0.25 μM) in comparison with the untreated cells (UNT) as measured by flow cytometry for GFP at 5-days post-gene editing (n = 3). Mock and RNP only cells were used as negative controls. d. Allelic distribution of WT, INDEL, HDR frequencies in CCR5 gene edited PSCs (c) (n = 3). HDR frequency was measured by ddPCR analysis, WT and INDEL frequencies were measured using ICE analysis. Mean HDR to INDEL ratio is represented above the bars. All data in c and d are shown as mean ± SEM.
