In vivo genome editing of β⁰/β⁰-thalassemic cells. (A) Experimental procedure for the in vivo genome-editing experiments. Briefly, mobilized CD34⁺ cells from a β0/β0-thalassemic patient were transplanted into nonmyeloablated NBSGW mice (n = 3). Six weeks posttransplantation, the mice were mobilized for 7 days with G-CSF + AMD3100. On the seventh day of mobilization, the mice were injected with the Ad-dualCRISPR (Untr = saline). The mice were euthanized 9 weeks later and their BM was collected for further analysis. (B) Multilineage reconstitution 16 weeks posttransplantation. (C) HbF expression in human engrafted nonerythroid (used as control) and erythroid cells assayed by FACS. (D) γ-/β-globin percentage assayed by quantitative RT-PCR. (E) Engraftment of human erythroid cells (GlyA⁺/CD45⁻) in the mouse BM at the time of euthanization. (F) Reactive oxygen species (ROS) of the thalassemic engrafted cells at the time of euthanization. (G) Adjusted to weight spleen sizes at the time of euthanization. (H) Engraftment of thalassemic hCD45⁺ and hCD2345a⁺ cells in secondary recipients in the BM and spleen as a percentage over the total number or cells. (I) HbF expression in engrafted erythroid cells in the secondary recipients’ BM (left panel) and after ex vivo differentiation of BM cells (right panel).