Identification of target genes and regulatory networks for bone mineral density GWAS loci through systematic targeting and inhibition

Osteoporosis is characterized by low bone mineral density (BMD) and elevated fracture risk. Most BMD-associated GWAS variants lie in noncoding regions, complicating efforts to identify causal genes and mechanisms. To overcome this variant-to-function challenge, we previously developed STING-seq, a framework integrating biobank-scale GWAS with single-cell CRISPR inhibition (CRISPRi) screening to directly connect noncoding <i>cis</i> -regulatory elements (CREs) to their target genes. Applied to human fetal osteoblast (hFOB) cells across osteogenic differentiation, STING-seq linked 76 CREs to 75 target genes at BMD loci. Arrayed CRISPRi and activation validated key CRE-gene relationships, including long-range enhancers regulating <i>CXCL12</i> (over 500 kb apart). We further uncovered <i>trans</i> -regulatory networks and characterized the <i>DAP3</i> - <i>YY1AP1</i> bidirectional promoter, demonstrating a role for <i>DAP3</i> in mineralization via mitochondrial pathways. Together, these findings provide mechanistic insight into how noncoding GWAS variants shape osteoblast activity and highlight the genes and pathways that mediate genetic effects on BMD.