CRISPR/Cas9-mediated induction of HbF expression improves the RBC sickling phenotype. Bone marrow (donor 1) and mobilized (donor 2) SCD CD34⁺ cells were transfected with plasmids carrying Cas9-GFP and gRNAs targeting the 13.6-kb region. Upon FACS-sorting, GFP⁺ genome-edited (13.6-kb) HSPCs were terminally differentiated in RBCs using a 3-phase liquid erythroid culture system.²⁴  GFP⁺ cells from Cas9-only samples (ctr-1) and GFP⁻ cells from Cas9+gRNAs cultures (ctr-2) were used as controls. (A) FACS analysis of HbF expression in control and genome-edited RBCs. The fraction of F-cells and the MFI of HbF immunostaining (in brackets) are displayed in the histograms. (B) RP-HPLC profiles of control and genome-edited RBCs (donor 1). The ratio of α chains to non–α chains is indicated in brackets. (C-D) Quantification of γ (Aγ+Gγ)-globins and sickle β-globin protein levels by RP-HPLC. Globin chain expression was normalized to α-globin (C). Relative abundance of β-like chains was calculated as fraction of total β-like (β + γ) globins (D). (E) In vitro sickling assay measuring the proportion of sickled RBCs under hypoxic conditions (0% O₂) (left panel). The percentage of sickled cells was calculated as: (sickled RBC count)/(total RBC count). At least 300 enucleated cells and 10 fields per time point were analyzed for each sample. Data are expressed as mean ± SEM. ****P < .0001 (2-way analysis of variance, Tukey’s multiple comparisons test vs ctr-1 and ctr-2). Representative microscopy images of RBCs before (0 minutes) and after (60 minutes) deoxygenation are shown in the right panel. Black and white arrowheads indicate sickled cells and nonsickled cells, respectively, under hypoxic conditions. Original magnification ×40. Scale bars, 50 µm.