RT Journal Article SR Electronic T1 Engineering of the Endogenous HBD promoter increases HbA2 JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.12.19.521003 DO 10.1101/2022.12.19.521003 A1 Boontanrart, Mandy Y. A1 Mächler, Elia A1 Ponta, Simone A1 Nelis, Jan C. A1 Preiano, Viviana G. A1 Corn, Jacob E. YR 2022 UL http://biorxiv.org/content/early/2022/12/20/2022.12.19.521003.abstract AB The β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of β-globin subunits in adult hemoglobin. Many gene editing efforts to treat the β-hemoglobinopathies attempt to correct β-globin mutations or increase γ-globin for fetal hemoglobin production. δ-globin, the subunit of adult hemoglobin A2, has high homology to β-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of δ-globin is a relatively unexplored avenue to increase the amount of functional hemoglobin. Here, we use CRISPR-Cas9 to repair non-functional transcriptional elements in the endogenous promoter region of δ-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate δ-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of δ-globin from the endogenous locus. Promoter edited HUDEP-2 immortalized erythroid progenitor cells exhibit striking increases of HBD transcript, from less than 5% to over 20% of total β-like globins. Edited CD34+ hematopoietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 35% HBD. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat β-hemoglobinopathies.Competing Interest StatementThe authors have declared no competing interest.