Disorders resulting from HBB (β-globin) gene mutations, mainly sickle cell disease (SCD) and β-thalassemia, become symptomatic postnatally as fetal γ-globin expression from two parologous genes HBG1 and HBG2 falls and adult β-globin increases, thereby shifting red blood cell (RBC) hemoglobin from the fetal (HbF, α2γ2) to adult form (HbA, α2β2). These disorders are alleviated when postnatal expression of fetal γ-globin is maintained. For example, in hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition, mutations attenuate γ-to-β switching, causing high-level HbF expression throughout life. Co-inheritance of HPFH with β-thalassemia or SCD mutations alleviates their clinical manifestations. Here we performed CRISPR-Cas9-mediated genome editing of human blood progenitors to mutate a 13-nucleotide HBG1/HBG2 promoter sequence, thereby recapitulating a naturally occurring HPFH-associated mutation. Edited progenitors produced RBCs with increased HbF levels that were sufficient to inhibit pathological hypoxia-induced RBC morphology of SCD. Our findings identify a potential DNA target for genome editing-mediated therapy of β–hemoglobinopathies.

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Key Points Cas9 editing of the γ-globin gene promoters in hematopoietic stem cells (HSCs) increases red cell HbF by ≤40%. No deleterious effects on hematopoiesis or off-target mutations were detected 16 weeks after xenotransplantation of edited HSCs.

We characterized the human β-like globin transgenes in two mouse models of sickle cell disease (SCD) and tested a genome-editing strategy to induce red blood cell fetal hemoglobin (HbF; α2γ2). Berkeley SCD mice contain four to 22 randomly arranged, fragmented copies of three human transgenes (HBA1, HBG2-HBG1-HBD-HBBS and a mini-locus control region) integrated into a single site of mouse chromosome 1. Cas9 disruption of the BCL11A repressor binding motif in the γ-globin gene (HBG1 and HBG2; HBG) promoters of Berkeley mouse hematopoietic stem cells (HSCs) caused extensive death from multiple double-strand DNA breaks. Long-range sequencing of Townes SCD mice verified that the endogenous Hbb genes were replaced by single-copy segments of human HBG1 and HBBS including proximal but not some distal gene-regulatory elements. Townes mouse HSCs were viable after Cas9 disruption of the HBG1 BCL11A binding motif but failed to induce HbF to therapeutic levels, contrasting with human HSCs. Our findings provide practical information on the genomic structures of two common mouse SCD models, illustrate their limitations for analyzing therapies to induce HbF and confirm the importance of distal DNA elements in human globin regulation. This article has an associated First Person interview with the first author of the paper.