CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells

Abstract

Sickle Cell Disease and ß-thalassemia, which are caused by defective or deficient adult ß-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Persistent expression of the fetal ß-like globin, also known as γ-globin, can ameliorate both disorders by serving in place of the adult ß-globin as a part of the fetal hemoglobin tet-ramer (HbF). Here we use CRISPR-Cas9 gene editing to explore a potential γ-globin silencer region upstream of the δ-globin gene identified by comparison of naturally-occurring deletion mutations associated with up-regulated γ-globin. We find that deletion of a 1.7 kb consensus element or select 350 bp sub-regions from bulk populations of cells increases levels of HbF. Screening of individual sgRNAs in one sub-region revealed three single guides that caused increases in γ-globin expression. Deletion of the 1.7 kb region in HUDEP-2 clonal sublines, and in colonies derived from CD34+ hematopoietic stem/progenitor cells (HSPCs), does not cause significant up-regulation of γ-globin. These data suggest that the 1.7 kb region is not an autonomous γ-globin silencer, and thus by itself is not a suitable therapeutic target for gene editing treatment of ß-hemoglobinopathies.