CRISPR interference-mediated noggin knockdown promotes BMP2-induced osteogenesis and calvarial bone healing

Healing of large calvarial bone defects remains a challenging task in the clinical setting. Although BMP2 (bone morphogenetic protein 2) is a potent growth factor that can induce bone repair, BMP2 provokes the expression of antagonist Noggin that self-restricts its bioactivity. CRISPR interference (CRISPRi) is a technology for programmable gene suppression but its application in regenerative medicine is still in its infancy. We reasoned that Nog inhibition, concurrent with BMP2 overexpression, can promote the osteogenesis of adipose-derived stem cells (ASC) and improve calvarial bone healing. We designed and exploited a hybrid baculovirus (BV) system for the delivery of BMP2 gene and CRISPRi system targeting Nog. After BV-mediated co-delivery into ASC, the system conferred prolonged BMP2 expression and stimulated Nog expression while the CRISPRi system effectively repressed Nog upregulation for at least 14 days. The CRISPRi-mediated Nog knockdown, along with BMP2 overexpression, additively stimulated the osteogenic differentiation of ASC. Implantation of the CRISPRi-engineered ASC into the critical size defects at the calvaria significantly enhanced the calvarial bone healing and matrix mineralization. These data altogether implicate the potentials of CRISPRi-mediated gene knockdown for cell fate regulation and tissue regeneration.