Selection of therapeutic targets for γ-globin reactivation in erythroblasts derived from clinically relevant HSPCs. HSPCs derived from healthy donors were transfected with plasmids carrying Cas9-GFP and gRNAs targeting the 13.6-, 7.2-, and 3.5-kb regions. (A) Flow cytometry sorting strategy of control and genome-edited cells. GFP⁺ HSPCs were FACS-sorted from Cas9-only (ctr-1) and Cas9+gRNAs samples (13.6, 7.2, and 3.5 kb). Data are expressed as mean ± SEM of 7 independent experiments. FSC, forward scatter. (B) Assessment of deletion and inversion efficiency by ddPCR. FACS-sorted control and genome-edited (13.6-kb, 7.2-kb, and 3.5-kb) cells were differentiated in liquid culture toward the erythroid lineage. Data represent the mean ± SD of at least 2 independent experiments. (C) qRT-PCR analysis of γ (Aγ+Gγ)-, δ-, and β-globin transcripts in mature erythroblasts derived from GFP⁺ HSPCs. mRNA levels were normalized to α-globin. γ-Globin expression levels were significantly increased in 13.6-kb genome-edited cells compared with the control sample. A modest increase in γ-globin mRNA was detected in 7.2-kb and 3.5-kb genome-edited samples. A robust and significant β-globin downregulation was observed in 13.6-kb genome-edited erythroblasts compared with control cells. *P < .05, **P < .01; ***P < .001 (2-way analysis of variance, Bonferroni’s multiple comparisons test vs ctr-1). Data represent the mean ± SD of at least 2 independent experiments. (D) Representative FACS histograms showing the increase in both the percentage of F-cells and the median fluorescence intensity (MFI) (in brackets) in mature erythroblasts derived from GFP⁺ genome-edited HSPCs in comparison with the control sample (Cas9-only cells; ctr-1).