Targeting of a 13.6-kb genomic region in the β-globin locus reactivates γ-globin expression in the adult HUDEP-2 erythroid cell line. HUDEP-2 cells were transfected with plasmids carrying Cas9-GFP and gRNA pairs targeting the 13.6-kb, 7.2-kb, and 3.5-kb regions. Cells treated only with Cas9-GFP plasmid were used as control (ctr). GFP⁺ cells were FACS-sorted and differentiated into mature erythroblasts. (A) Representative images of May-Grünwald-Giemsa–stained undifferentiated (day 0) and differentiated (day 9) cultures. Original magnification ×20. Scale bars, 50 µm (left). Bars indicate the percentage cell number for each erythroblast population after differential counting (right). Similar proportions of the different erythroid precursors were observed in control and genome-edited cultures. (B) qRT-PCR analysis of γ (Aγ+Gγ)-, δ-, and β-globin mRNA levels in differentiated samples. Results were normalized to α-globin. Error bars denote standard deviation (SD). (C) Representative FACS analyses of HbF⁺ cells (F-cells). Data are expressed as mean ± standard error of the mean (SEM) of 3 experiments. (D) RP-HPLC chromatograms showing peaks corresponding to α-globin and β-like globins in differentiated HUDEP-2 samples. The ratio of α chains to non–α chains is indicated in brackets. (E) Quantification of γ (Aγ+Gγ)-globin and β-globin protein levels, as assessed by RP-HPLC. β-Like globin expression was normalized to α-globin. Targeting the 13.6-kb region, but not the 3.5-kb putative HbF silencer and the 7.2-kb region, reduced β-globin chain levels and strongly increased γ-globin chain expression. δ-Globin protein levels were decreased only in 7.2-kb and 3.5-kb genome-edited samples.