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

PLoS One. 2019 Jan 15;14(1):e0208237. doi: 10.1371/journal.pone.0208237. eCollection 2019.

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 tetramer (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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Associated Protein 9 / metabolism*
  • CRISPR-Cas Systems / genetics*
  • Cell Line
  • DNA, Intergenic / genetics
  • Erythroid Cells / metabolism*
  • Fetal Hemoglobin / metabolism*
  • Gene Editing
  • Gene Silencing
  • Genotype
  • Hematopoietic Stem Cells / metabolism
  • Humans
  • Phenotype
  • Repressor Proteins / metabolism*
  • Sequence Deletion / genetics
  • Up-Regulation / genetics
  • gamma-Globins / genetics

Substances

  • DNA, Intergenic
  • Repressor Proteins
  • gamma-Globins
  • Fetal Hemoglobin
  • CRISPR-Associated Protein 9