Editing a -globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype

Sickle cell disease (SCD) is caused by a single amino acid change in the adult hemoglobin (Hb) beta chain that causes Hb polymerization and red blood cell (RBC) sickling. The co-inheritance of mutations causing fetal gamma-globin production in adult life hereditary persistence of fetal Hb (HPFH) reduces the clinical severity of SCD. HPFH mutations in the HBG gamma-globin promoters disrupt binding sites for the repressors BCL1 1A and LRF. We used CRISPR-Cas9 to mimic HPFH mutations in the HBG promoters by generating insertions and deletions, leading to disruption of known and putative repressor binding sites. Editing of the LRF-binding site in patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in gamma-globin derepression and correction of the sickling phenotype. Xenotransplantation of HSPCs treated with gRNAs targeting the LRF-binding site showed a high editing efficiency in repopulating HSPCs. This study identifies the LRF-binding site as a potent target for genome-editing treatment of SCD.