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 cis -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 CXCL12 (over 500 kb apart). We further uncovered trans -regulatory networks and characterized the DAP3 - YY1AP1 bidirectional promoter, demonstrating a role for DAP3 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.