Briefly, CD34+ HSPCs were cultured and pre-stimulated for 72 h after thaw and before genome editing except when specifically indicated. Chemically modified HBB sgRNA was synthesized by Agilent Technologies (Santa Clara, CA, USA). SpyFi Cas9 was purchased from Aldevron (Fargo, ND, USA). HBB donor rAAV6 virus was purchased from Viralgen VC (Donostia, Gipuzkoa, Spain). Electroporation of Cas9-RNP complex was performed using Lonza 4DNucleofector (Lonza Group, Alpharetta, GA, USA) in P3 Primary Cell Solution with program DZ-100 as described previously. Electroporated cells were then plated at 2.5 x 10^5 cells/mL in HSPC culture medium supplemented with cytokines. For RNP/rAAV6 or rAAV6 treatment, HBB donor rAAV6 was immediately dispensed onto cells at an MOI of 2.5 x10^3 vg/cell based on the titer determined by ddPCR except for special indications. After incubation for 24 h, a medium addition or medium exchange was performed to dilute or remove residual HBB donor rAAV6. The CD34+ HSPCs were cultured for an additional 24 h for quantification of genome editing events.