CD34+ cell editing
After 48 hr of pre-stimulation, viable cells were counted by hemocytometer. Prior to nucleofection, cells were washed once with PBS, spun down for 5 min at 300 g, and re-suspended in buffer 1M (Chicaybam et al., 2016) (5 mM KCl; 15 mM MgCl2; 120 mM Na2HPO4/NaH2PO4 pH7.2; 50 mM Manitol) such that each well of the Nucleocuvette strip would contain 20,000–100,000 cells. Assembled RNP, p53 siRNA (20 fmol, Thermo id s605), electroporation enhancer (IDT), and any ssODN donors (IDT) (as specified in each experiment). Overall RNP and other additives were kept at or below 10% of the total 20 µL volume per well. Handling time between wash and nucleofection was kept within a 10 min window. Cells were nucleofected using the Lonza 4D nucleofector device with nucleocuvette strips, Primary P3, and DZ-100 program. Following nucleofection, cells were allowed to rest for 5 min and then added to pre-warmed wells of a 24-well plate containing media (as specified in CD34 + cell culture) supplemented with small molecules as indicated for specific experiments (AZD7648, RS-1, Cayman Chemicals; M-3814, Toronto Research Chemicals). Where AAV donor was used, it was added to the well within 15 min of nucleofection (Charlesworth et al., 2018).