Robust HbF upregulation in HSPC-derived erythroblasts upon genome editing of the 13.6-kb region. (A) Assessment of deletion and inversion efficiency by ddPCR in mature erythroblasts and erythroid progenitors (erythroid burst-forming units [BFU-E]) derived from GFP⁺ genome-edited healthy donor and SCD HSPCs. Data represent the mean ± SEM of 7 independent experiments. (B) Genotype of single colonies derived from GFP⁺ HSPCs. The occurrence of deletion and inversion events was assessed in randomly picked BFU-E by PCR (2 healthy donors; n = 100). (C) Sanger sequencing of deletion and inversion junctions in single BFU-E (n = 40). Top rows indicate the predicted junction sequences (in bold). The expected deletion and inversion junctions with small InDels were observed in the majority of genome-edited alleles. In two alleles, we detected the insertion of 354 bp (*) and 373 bp (^). The frequency of each event is calculated as: (number of alleles harboring an identical deletion or inversion junction)/(total number of deleted or inverted alleles). Arrows indicate the predicted junction sites. Dashes represent deleted nucleotides. Inserted nucleotides are displayed in lowercase. (D) qRT-PCR analysis of γ (Aγ+Gγ)-, δ-, and β-globin transcripts in mature erythroblasts derived from 13.6-kb genome-edited HSPCs. mRNA levels were expressed as fold change vs control cells (ctr-1). γ-Globin expression levels were significantly increased in genome-edited compared with control samples (**P < .01; unpaired Student t test, 2 tailed). A significant β-globin downregulation was detected in edited cells in comparison with control (*P < .05; unpaired Student t test, 2 tailed). δ-Globin expression was decreased in genome-edited samples. Data represent the mean ± SEM of 7 independent experiments. (E) Representative FACS histograms showing the percentage of F-cells and the MFI of HbF immunostaining (in brackets) in mature erythroblasts derived from GFP⁺ genome-edited (13.6-kb) HSPCs of 2 healthy donors. GFP⁺ cells from Cas9-only samples (ctr-1) and GFP⁻ cells from Cas9+gRNAs cultures (ctr-2) served as controls. (F) RP-HPLC chromatograms showing peaks corresponding to α-globin and β-like globins in genome edited and control samples. The expression of a common AγT chain variant was detected in samples derived from healthy donor 2. The ratio of α chains to non–α chains (in brackets) was unchanged in CRISPR/Cas9-modified samples. (G-H) Quantification of γ- (Aγ+Gγ), β-, and δ- globin protein levels. β-Like globin expression was normalized to α-globin (G). Relative abundance of β-like chains was calculated as percentage of total β-like (β + γ + δ) globins (H). Targeting the 13.6-kb region increased γ-globin chain expression and decreased β-globin protein levels. δ-Globin protein levels were unaffected, suggesting an increased translation of the residual δ-globin transcripts in the absence of β-globin mRNA. Alternatively, the reduced β-chain synthesis favors the incorporation of the δ-globin chain in the Hb tetramers.