Induction of fetal hemoglobin synthesis by CRISPR/Cas9-mediated editing of the human b-globin locus

CRISPR/Cas9mediated disruption of the b-globin locus architecture reactivates fetal g-globin expression in adult erythroblasts. l Fetal g-globin reactivation and sickle b-globin downregulation leads to the amelioration of the SCD cell phenotype. Naturally occurring, large deletions in the b-globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of sickle cell disease (SCD) and b-thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the dand b-globin genes and a putative g-d intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in g-globin expression, whereas deletion or inversion of a 13.6-kb region causes a robust reactivation of HbF synthesis in adult erythroblasts that is associated with epigenetic modifications and changes in chromatin contacts within the b-globin locus. In primary SCD patient-derived hematopoietic stem/progenitor cells, targeting the 13.6-kb region results in a high proportion of g-globin expression in erythroblasts, increased HbF synthesis, and amelioration of the sickling cell phenotype. Overall, this study provides clues for a potential CRISPR/Cas9 genome editing approach to the therapy of b-hemoglobinopathies.