Gene editing of cell lines and human CD34+ HSPCs
For gene editing experiments, cells were nucleofected with 6 µg Cas9 (Alt-R™ S.p. Cas9 Nuclease V3 or Alt-R™ S.p. HiFi Cas9 Nuclease V3, IDT) and 3.2 µg synthetic sgRNA (Synthego) using a Lonza 4D Nucleofector X (Lonza). Cells were washed in Gibco Opti-MEM Reduced Serum Medium (K562 cells, Thermo Fischer Scientific) or PBS (CD34+ HSPCs) and resuspended in 18-20 µL 1M buffer (50mMDMannitol, 5mM KCL, 120mM Na2HPO4, 15mM MgCl2) prior to nucleofection. When applicable, 100 pmol ssODNs (IDT) were included in the nucleofection. K562 cells were nucleofected using the CM-138 program and the Primary Cell P3 setting. CD34+ HSPCs were nucleofected using the DZ-100 program and the Primary Cell P3 settings. When i53, GSE56, and/or Ad5-E4orf6/7 mRNAs were used, 3 µg of each was included in the nucleofection. Mock nucleofections were carried out the same way, but without adding sgRNA, Cas9, or mRNA. Following nucleofection, cells were seeded at 5×10^5 cells mL-1 and transduced with viral vectors carrying HDR repair templates within 15 min.