Multiplex mutagenesis in CD34⁺ cells to enhance endogenous HbF reactivation. (A) Schematic depiction of the nuclease combinations tested. (B) Editing levels presented as indel percentage in cells edited with 1 (SKO) or 2 (DKO) RNPs. (C) Histogram plots of HbF expression assayed by flow cytometry. (D) γ-globin chain expression presented as fold difference over the untransfected samples (HPLC). (E) Clonogenic capacity of the edited cells with the different RNP combinations. (F) Immunofluorescence imaging of the abundance of 53BP1 to assess DNA damage response and toxicity after introducing 2 mutations in different loci in CD34⁺ cells 6 and 24 hours postelectroporation. 4′,6-diamidino-2-phenylindole (DAPI; blue)/ 53BP1 (red) stain; scale bar, 5 μm. Values are represented as means ± SEM (n ≥ 3). *P ≤ .05 vs untreated (untr), †P ≤ .05 vs HBG-197, ‡P ≤ .05 vs HBG-115 (2-way analysis of variance [ANOVA]).