Identification of target genes and regulatory networks for bone mineral density GWAS loci through systematic targeting and inhibition [hFOB BMD STING-seq]

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. Overall design: We generated human fetal osteoblast 1.19 (hFOB) cell lines constitutively expressing a CRISPRi system. We then performed a single-cell pooled CRISPRi screen targeting select noncoding candidate cis-regulatory elements. The experiment was performed first as a pilot study on an undifferentiated hFOB (day 0) and then as a full study on hFOB after osteogenic induction, reading out days 0, 2, and 4. We performed single-cell RNA-seq using 10X Genomics Chromium Next GEM Single Cell 5' Reagent Kits v2 (dual index) on cells detached from culture plates. We labeled cells from each state with different hashtag antibodies targeting cell surface proteins (BioLegend TotalSeq 394661, 394663, 394665, 394667) following the manufacturer's recommendations. We then pooled equal numbers of cells from each state into a single sample and aliquoted them into as many reactions as needed to capture approximately 200,000 cells in total. We constructed sequencing libraries for the three different barcoded nucleic acids for each reaction according to 10X Genomics protocols and pooled them disproportionately so that mRNA-derived cDNA libraries represented ~90% of the sample, with gRNA and hashtag libraries comprising the remaining 10%. We sequenced the pooled library to a depth of approximately 20,000 mRNA/cDNA reads per cell on an Illumina NextSeq sequencing platform. We used 2 microfluidics channels for the pilot experiment and 6 channels for the full experiment.