Linear recruitment of the LCR reactivates fetal globin in HUDEP-2 cells. (A) A schematic representation of the HBB locus on chromosome 11 depicting the hypersensitive sites of the LCR (light blue), HBE (brown), HBG2 (pink), HBG1 (green), HBD (light purple), and HBB (dark purple) genes. Below (left) is a zoom-in on HS2 showing the most relevant transcription factor binding site (TF-BS) (gray) and the binding site of gRNA-HS2. On the right side, a zoom-in on the HBG2 promoter with the most relevant TF-BS (gray) and position of gRNA_500. At the bottom, the 25-kb deletion (Del2Rec) is shown that was generated by the combination of gRNA_500 and gRNA-HS2. (B) Frequency of indels as measured by the TIDE analysis for Δ115, 500, and HS2 gRNAs in single-gRNA transfected samples. (C) Frequency of the 25-kb deletion (blue), inversion (lime green), and the unmodified iteration (gray) as measured by ddPCR in the Del2Rec condition. (D) RT-qPCR analysis of the HBB and HBG messenger RNA (mRNA) levels in proliferating (day 0) cells. The HBB and HBG mRNA expression was normalized to actin mRNA and is displayed as a percentage of HBB plus HBG. (E) Same as in panel D but for cells differentiated for 10 days. (F) Flow cytometry plots showing the percentage of HbF-positive cells in proliferation (top row) and after 10 days of differentiation (bottom row). AU, arbitrary units; Ctrl., control; LRF, leukemia/lymphoma-related factor; Neg., negative; NFI, nuclear factor I.