Deletion of HBA2 reduces α-globin precipitates in HUDEP-2 β⁰cells. (A) Schematic representation of the α-globin locus. HBA1 and HBA2 genes are located in the subtelomeric region of chromosome (Chr) 16. Expression is regulated by 4 erythroid-specific enhancers (multispecies conserved elements [MCS-R]) located 10 to 50 kb upstream. Common occurring deletions are shown as gray bars and thin lines indicate regions of uncertainty of the breakpoints (adapted from Harteveld and Higgs⁹⁸ ). gRNA cutting sites are indicated as scissors. gRNA nucleotide sequence is indicated on top (in bold/underlined the protospacer adjacent motif sequence). (B) Editing efficiency in HUDEP-2 cells is expressed as percentage of modified HBA alleles. Bars represent mean ± SD (n = 3). (C) HBA2 copies in edited cells in panel B. Red dashed line indicates the number of expected HBA2 alleles in normal cells. (D) Reverse transcription-qPCR quantification of α/β-like globin mRNA ratios in HUDEP-2 β⁰ (n = 3-5) and in single-cell clones with monoallelic (α-/αα; n = 3) or biallelic (α-/α-; n = 3) HBA2 deletions. β⁰ + RNP = HUDEP2 β⁰ bulk population edited with gRNA HBA15/Cas9. β⁰ KOα = HUDEP2 β⁰ bulk population edited with gRNA HBB/Cas9 to induce α-globin KO. Red dashed line indicates the physiological α/β-like ratio of 1. Bars represent mean ± SD (**P < .01, ANOVA, Tukey test). (E) Representative HPLC chromatograms of globin tetramer analysis in edited HUDEP-2 β⁰. γ₄, Hb Bart; acHbF, acetylated fetal hemoglobin; HbF, fetal hemoglobin (α₂γ₂); α-p, α-precipitates; HbA, adult hemoglobin (α₂β₂). (F) Quantification of α-precipitates. Every tetramer is reported as percent of total hemoglobins; bars show mean ± SD (n = 3; **P < .01; ANOVA, Tukey test).