Multiplex mutagenesis in CD34⁺ cells from patients with β-thalassemia major. (A) Frequency of CD34⁺/CD38⁻/CD90⁺ cells 2 days post–Ad-dualCRISPR transduction. (B) Number of total, erythroid (BFU-E), and myeloid (CFU-GM) colonies per 2000 CD34⁺ cells. (C) Percentage of erythroid cells (GlyA⁺) at the end point of in vitro differentiation. (D) Morphology of unedited normal (wt), unedited thalassemic, and edited thalassemic cells on days 11 and 18 of the differentiation. Green arrows point to late-stage maturing erythroid cells . Hematoxylin & eosin stain; original magnification ×40. (E) Percentage of enucleated RBCs at the end of differentiation. (F) Indel percentage at different time points during differentiation. (G) Composition of the thalassemic progenitor cells (CFCs) in terms of single BCL11A (solid blue), single HBG-115 (solid green), or double BCL11A⁺HBG-115 (striped) mutations. Unedited colonies are represented with gray. (H) HbF expression in enucleated and nucleated cells. (I) ROS levels of edited and unedited thalassemic cells on day 14 of the differentiation. (J) Representative FACS plots of ROS expression in thalassemic erythroid cells before and after dual editing. Values are represented as means ± SEM. **P ≤ .001, *P ≤ .05 vs untreated (C,E,I) or day 3 (F) (unpaired Student t test).