HbF reactivation occurs predominantly in HUDEP-2 clones harboring biallelic rearrangements of the 13.6-kb target region. Control and genome-edited bulk populations were cloned by limiting dilution to isolate cells harboring biallelic or monoallelic modifications of the 13.6-kb target region. We selected and differentiated 11 control (ctr), 4 biallelic deleted (del/del), 4 mono-allelic deleted (del/wt), 5 biallelic inverted (inv/inv), and 5 monoallelic inverted (inv/wt) clones. (A) Sanger sequencing of deletion and inversion junctions in genome-edited clones. Top rows show 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 2 alleles, we detected larger deletions (14.1 and 14.9 kb) removing 527 and 1319 bp upstream and downstream of the 13.6-kb region, respectively. In 1 allele, we detected a shorter deletion (11.6 kb), leaving 2001 bp at the 3′ end of the 13.6-kb region (*). No difference in HbF expression was observed among these clones regardless of the deletion. 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 and dots represent deleted and hidden nucleotides, respectively. Inserted nucleotides are displayed in lowercase. (B) qRT-PCR analysis of γ (Aγ+Gγ)-, δ-, and β-globin transcripts in differentiated clones. All samples were normalized to α-globin. Error bars represent SEM. ****P < .0001, ***P < .001, **P < .01, *P < .05 (unpaired 2-tailed Student t test vs control). (C) Representative FACS plots showing the percentage of F-cells in differentiated HUDEP-2 clones. The proportion of HbF⁺ cells was significantly higher in genome-edited vs control clones (P < .0001). Data are displayed as mean ± SEM. Clones harboring a biallelic inversion of the 13.6-kb regions tend to show higher γ-globin expression at both mRNA and protein levels than clones harboring a biallelic deletion. (D) 3C analysis of chromatin interactions between the LCR and the γ-globin promoters in differentiated clones (2 controls, 2 del/del, and 2 inv/inv clones). A higher interaction frequency was observed in clones harboring a biallelic rearrangement, as compared with control clones. The interaction frequencies were normalized to the cross-linking frequency in the ERCC3 locus. The hypersensitive sites of the LCR are indicated (HS1, HS2, HS3, and HS4). We used as anchor a genomic fragment containing HS3 (black rectangle). HindIII restriction sites are depicted as black triangles. Black circles indicate the β-like globin promoters. Distances on x-axis are in kilobases counting from the transcription start site (TSS) of the HBE1 gene. (E) Analysis of H3K27 acetylation at γ-globin promoters in differentiated clones (2 controls, 2 del/del, and 2 inv/inv clones). γ-Globin promoters were highly enriched in H3K27ac in del/del and inv/inv clones. The DEFB122 genomic region served as a negative control (Neg. ctr). Error bars indicate SD.