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

Author(s): Chung, Jennifer E; Magis, Wendy; Vu, Jonathan; Heo, Seok-Jin; Wartiovaara, Kirmo; Walters, Mark C; Kurita, Ryo; Nakamura, Yukio; Boffelli, Dario; Martin, David I. K; Corn, Jacob E; DeWitt, Mark A | Abstract: Sickle Cell Disease and s-thalassemia, which are caused by defective or deficient adult s-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Persistent expression of the fetal s-like globin, also known as ?-globin, can ameliorate both disorders by serving in place of the adult s-globin as a part of the fetal hemoglobin tetramer (HbF). Here we use CRISPR-Cas9 gene editing to explore a potential ?-globin silencer region upstream of the δ-globin gene identified by comparison of naturally-occurring deletion mutations associated with up-regulated ?-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 ?-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 ?-globin. These data suggest that the 1.7 kb region is not an autonomous ?-globin silencer, and thus by itself is not a suitable therapeutic target for gene editing treatment of s-hemoglobinopathies.