Figure 1. Genome editing of the HBG1 and HBG2 promoters increases erythroid fetal hemoglobin (HbF) levels.

(a) Extended β-globin locus showing β-like genes as boxes. Small arrows mark DNase hypersensitive sites within the locus control region, an upstream enhancer. A region of the HBG1 promoter is shown numbered according to position upstream of the transcription start with the 13-nt HPFH deletion boxed. Guide RNA spacer sequences are blue and PAM motifs (NGG) are orange; gRNA-1 and gRNA-2 are complementary to the sense and antisense strands, respectively. Large arrows show predicted Cas9 cleavage sites.

(b) Representative flow cytometry plots showing HbF⁺ immunostaining HUDEP-2 cells 5d after transduction with Cas9 ± gRNA-1 or gRNA-2 lentivirus. Numbers indicate mean ± standard error (SE) from four independent experiments.

(c) Normal human CD34⁺ cells transduced with lentivirus encoding Cas9 ± gRNA-1 or gRNA-2 were cultured for 21d in erythroid cytokines, then analyzed for hemoglobin (Hb) protein by HPLC. %HbF = [HbF/(HbA + HbF) × 100]. Each dot represents a separate experiment performed with CD34⁺ cells from the same donor. On-target editing rates of HBG1/HBG2 in three experiments were 56%, 65% and 77%.

(d) HbF⁺ erythroblasts, derived as described for panel (c). Numbers indicate mean ± SE from three experiments.

(e) CD34⁺ cells from an SCD (HbSS) patient were transduced with lentivirus expressing Cas9 ± gRNA-1, differentiated into RBCs, and cultured in 2% O₂.Red arrows denote cells with sickle-like morphology. Original magnification 200×. Size bars indicate 20 µm.

(f) Quantification of hypoxia-induced sickled cells depicted in panel (e). Mean ± SE from three experiments using CD34⁺ cells from three different SCD donors (> 1,000 cells scored per experiment).

The unpaired t-test was used to analyze data in panels b-d and f. **** P < 0.0001, ** P < 0.01.