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

• Published in: PloS one Vol. 14; no. 1; p. e0208237
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 15.01.2019; Public Library of Science (PLoS)
• Subjects: Analysis; Binding sites; Biology and Life Sciences; CD34 antigen; Cell Line; Cells (biology); Chung, Wendy; Clonal deletion; CRISPR; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems - genetics; CRISPR-Cas technology; Disease; DNA, Intergenic - genetics; Erythroid cells; Erythroid Cells - metabolism; Fetal Hemoglobin - metabolism; Fetuses; gamma-Globins - genetics; Gene deletion; Gene Editing; Gene mutation; Gene Silencing; Genes; Genetic engineering; Genetic modification; Genome editing; Genomes; Genomics; Genotype; Globin genes; Hematological diseases; Hematopoietic stem cells; Hematopoietic Stem Cells - metabolism; Hemoglobin; Hospitals; Humans; Interrogation; Methods; Mutation; Phenotype; Progenitor cells; Red blood cells; Regulatory sequences; Repressor Proteins - metabolism; Research and Analysis Methods; Sequence Deletion - genetics; Sickle cell disease; Stem cells; Thalassemia; Therapeutic applications; Transcription factors; Transplants & implants; Up-Regulation - genetics; California; United States > US; Japan; Finland
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0208237

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.