<scp>CRISPR‐Cas9</scp> –Mediated Genome Editing Confirms <scp> <i>EPDR1</i> </scp> as an Effector Gene at the <scp>BMD GWAS</scp> ‐Implicated ‘ <scp> <i>STARD3NL</i> </scp> ’ Locus

Abstract

Genome-wide-association studies (GWASs) have discovered genetic signals robustly associated with BMD, but typically not the precise localization of effector genes. By intersecting genome-wide promoter-focused Capture C and assay for transposase-accessible chromatin using sequencing (ATAC-seq) data generated in human mesenchymal progenitor cell (hMSC)-derived osteoblasts, consistent contacts were previously predicted between the <i>EPDR1</i> promoter and multiple BMD-associated candidate causal variants at the '<i>STARD3NL</i>' locus. RNAi knockdown of <i>EPDR1</i> expression in hMSC-derived osteoblasts was shown to lead to inhibition of osteoblastogenesis. To fully characterize the physical connection between these putative noncoding causal variants at this locus and the <i>EPDR1</i> gene, clustered regularly interspaced short-palindromic repeat Cas9 endonuclease (CRISPR-Cas9) genome editing was conducted in hFOB1.19 cells across the single open-chromatin region harboring candidates for the underlying causal variant, rs1524068, rs6975644, and rs940347, all in close proximity to each other. RT-qPCR and immunoblotting revealed dramatic and consistent downregulation of <i>EPDR1</i> specifically in the edited differentiated osteoblast cells. Consistent with <i>EPDR1</i> expression changes, alkaline phosphatase staining was also markedly reduced in the edited differentiated cells. Collectively, CRISPR-Cas9 genome editing in the hFOB1.19 cell model supports previous observations, where this regulatory region harboring GWAS-implicated variation operates through direct long-distance physical contact, further implicating a key role for <i>EPDR1</i> in osteoblastogenesis and BMD determination. © 2021 The Authors. <i>JBMR Plus</i> published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.