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

• DOI: 10.1371/journal.pone.0208237
• Publication Date: 2019-01-15T18:39:42Z
• Authors: Jennifer E. Chung; Wendy Magis; Jonathan Vu; Seok-Jin Heo; Kirmo Wartiovaara; Mark C. Walters; Ryo Kurita; Yukio Nakamura; Dario Boffelli; David I. K. Martin; Jacob E. Corn; Mark A. DeWitt
• Keywords: 0301 basic medicine; Genotype; Science; DISEASE; Cell Line; 03 medical and health sciences; Erythroid Cells; CRISPR-Associated Protein 9; Humans; gamma-Globins; Gene Silencing; HEMOGLOBIN; Fetal Hemoglobin; Sequence Deletion; Gene Editing; 0303 health sciences; Q; R; Hematopoietic Stem Cells; 16. Peace & justice; GENE; Up-Regulation; 3. Good health; LIFE; Repressor Proteins; Biomedicine; Phenotype; Medicine; BRII recipient: DeWitt; DNA, Intergenic; CRISPR-Cas Systems; Research Article

AbstractSickle 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. 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. Here we use CRISPR-Cas9 gene editing to explore a putative γ-globin silencer region 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 fetal hemoglobin (HbF) or γ-globin. Screening of individual sgRNAs in one sub-region revealed three single guides that caused mild increases in γ-globin expression. However, clonal cell lines with the 1.7 kb region deleted did not up-regulate γ-globin and neither did lines with either of two of sub-regions identified in the screen deleted. These data suggest that the region is not an autonomous γ-globin silencer, and thus by itself is not a suitable therapeutic target in the ß-hemoglobinopathies.