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

JBMR Plus

Andrew D Wells¹  Matthew E Johnson¹  Alessandra Chesi¹  James A Pippin¹  Matthew C Pahl¹  Struan F A Grant¹  Chun Su¹  Kurt D Hankenson²  Yadav Wagley²  Kenyaita M Hodge³ 

[1] Center for Spatial and Functional Genomics Children's Hospital of Philadelphia Philadelphia PA USA;Department of Orthopedic Surgery University of Michigan Medical School Ann Arbor MI USA;Genetics and Molecular Biology Graduate Program, Laney Graduate School Emory University Atlanta GA USA;

关键词: BONE MINERAL DENSITY (BMD);    CHROMATIN CAPTURE;    CRISPR;    OSTEOBLAST DIFFERENTIATION;    OSTEOBLAST PROGENITOR;   

DOI  :  10.1002/jbm4.10531

来源: DOAJ

【 摘 要 】

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 EPDR1 promoter and multiple BMD‐associated candidate causal variants at the ‘STARD3NL’ locus. RNAi knockdown of EPDR1 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 EPDR1 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 EPDR1 specifically in the edited differentiated osteoblast cells. Consistent with EPDR1 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 EPDR1 in osteoblastogenesis and BMD determination. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

【 授权许可】

Unknown