CRISPRa genome-wide screen identifies novel gene targets for osteogenic cell engineering

Bone tissue regeneration and fracture healing remain a significant challenge for physicians, with nonunion failures occurring in an estimated 5%-10% of bone-healing treatments. The autologous bone graft has long been the gold standard of treatment. However, these procedures suffer from persistent donor-site morbidity and extended surgery times, while still having high revision and nonunion failure rates. Cell therapies and tissue engineering strategies utilizing stem cells have been considered as promising alternatives to autologous bone grafts. Here, we explore the concept of using CRISPR-activation (CRISPRa) as a cell-engineering tool to drive osteogenesis without exogenous growth factors. We present a genome-wide CRISPRa screen in adipose-derived stem cells (ASCs) to identify upregulation targets that drive osteogenesis. Top targets from the screen, SPRED2 and ATXN7L3B, demonstrated significant increases in alkaline phosphatase activity and mineralization in monolayer and 3D culture. These results are the first evidence of these genes as osteogenic targets in ASCs.