CRISPR activation (CRISPRa) is a burgeoning technology for programmable gene activation, but its potential for tissue regeneration has yet to be fully explored. Bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into osteogenic or adipogenic pathways, which are governed by the Wnt (Wingless-related integration site) signaling cascade. To promote BMSC differentiation toward osteogenesis and improve calvarial bone healing by BMSCs, we harnessed a highly efficient hybrid baculovirus vector for gene delivery and exploited a synergistic activation mediator (SAM)-based CRISPRa system to activate Wnt10b (that triggers the canonical Wnt pathway) and forkhead c2 (Foxc2) (that elicits the noncanonical Wnt pathway) in BMSCs. We constructed a Bac-CRISPRa vector to deliver the SAM-based CRISPRa system into rat BMSCs. We showed that Bac-CRISPRa enabled CRISPRa delivery and potently activated endogenous Wnt10b and Foxc2 expression in BMSCs for >14 days. Activation of Wnt10b or Foxc2 alone was sufficient to promote osteogenesis and repress adipogenesis in vitro. Furthermore, the robust and prolonged coactivation of both Wnt10b and Foxc2 additively enhanced osteogenic differentiation while inhibiting adipogenic differentiation of BMSCs. The CRISPRa-engineered BMSCs with activated Wnt10b and Foxc2 remarkably improved the calvarial bone healing after implantation into the critical-sized calvarial defects in rats. These data implicate the potentials of CRISPRa technology for bone tissue regeneration.