Highlight: CRISPR-Edited RUNX1 Variants Enhance Osteogenesis

Bone mineral density (BMD) is a complex trait influenced by genetic, behavioural, and environmental factors, with heritability estimates reaching up to 80%. Recent genome-wide association studies (GWAS) have identified over 500 loci associated with BMD, including intronic and intergenic variants near the RUNX1 gene, which is known for its role in skeletal development and repair.

However, how these non-coding variants regulate gene expression and influence osteogenesis remained unclear. The study aimed to uncover the functional roles of these variants in regulating bone formation and repair and pave the way for future therapies targeting bone diseases such as osteoporosis.

To dissect the regulatory roles of BMD-associated variants at the RUNX1 locus, researchers employed CRISPR-Cas9 genome editing to create human induced pluripotent stem cell (iPSC) models. Two deletion lines, Δ1 and Δ2, were engineered to excise regions containing these variants. These iPSCs were subsequently differentiated into mesenchymal stromal cells (iMSCs) and osteoblasts.

The effects of the deletions were evaluated using in vitro assays for osteogenic differentiation and in vivo mouse models for bone regeneration. Circularised chromosome conformation capture sequencing (4C-Seq) was performed to map chromatin interactions, revealing how the deleted regions interacted with RUNX1 and other genes, such as SETD4, involved in chromatin modification.

The experimental results demonstrated that the deletion mutant lines exhibited enhanced osteogenic potential compared to wild-type controls. In vitro, iMSCs derived from Δ1 and Δ2 lines showed increased mineralisation and upregulated expression of osteogenic markers like RUNX2 and SP7. 4C-Seq analyses revealed altered chromatin interactions, with the deletion mutants disrupting a regulatory hub, leading to increased expression of RUNX1 and SETD4.