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

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
DOI: 10.1371/journal.pone.0208237 Publication Date: 2019-01-15T18:39:42Z
ABSTRACT
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.
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