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.

中文翻译：

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CRISPR激活共激活内源性Wnt10b和Foxc2可增强BMSC成骨作用并促进颅骨再生。

CRISPR激活（CRISPRa）是一种用于可编程基因激活的新兴技术，但其组织再生潜力尚未得到充分开发。骨髓来源的间充质干细胞（BMSC）可以分化为成骨或成脂途径，由Wnt（无翅相关整合位点）信号级联控制。为了促进BMSC向成骨的分化并通过BMSC改善颅骨愈合，我们利用高效的混合杆状病毒载体进行基因传递，并利用基于协同激活介体（SAM）的CRISPRa系统激活Wnt10b（触发经典Wnt途径）并BMSC中的叉头c2（Foxc2）（引发非经典Wnt途径）。我们构建了一个Bac-CRISPRa载体，以将基于SAM的CRISPRa系统传递到大鼠BMSC中。我们显示，Bac-CRISPRa使CRISPRa递送并有效激活BMSCs中的内源性Wnt10b和Foxc2表达超过14天。单独激活Wnt10b或Foxc2足以在体外促进成骨和抑制脂肪形成。此外，Wnt10b和Foxc2的强大而持久的共激活可增加成骨分化，同时抑制BMSCs的成脂分化。CRISPRa工程化的BMSC具有激活的Wnt10b和Foxc2，可显着改善大鼠临界大小的颅骨缺损植入后的颅骨愈合。这些数据暗示了CRISPRa技术在骨组织再生中的潜力。单独激活Wnt10b或Foxc2足以在体外促进成骨和抑制脂肪形成。此外，Wnt10b和Foxc2的强大而持久的共激活可增加成骨分化，同时抑制BMSCs的成脂分化。CRISPRa工程化的BMSC具有激活的Wnt10b和Foxc2，可显着改善大鼠临界大小的颅骨缺损植入后的颅骨愈合。这些数据暗示了CRISPRa技术在骨组织再生中的潜力。单独激活Wnt10b或Foxc2足以在体外促进成骨和抑制脂肪形成。此外，Wnt10b和Foxc2的强大而持久的共激活可增加成骨分化，同时抑制BMSCs的成脂分化。CRISPRa工程化的BMSC具有激活的Wnt10b和Foxc2，可显着改善大鼠临界大小的颅骨缺损植入后的颅骨愈合。这些数据暗示了CRISPRa技术在骨组织再生中的潜力。CRISPRa工程化的BMSC具有激活的Wnt10b和Foxc2，可显着改善大鼠临界大小的颅骨缺损植入后的颅骨愈合。这些数据暗示了CRISPRa技术在骨组织再生中的潜力。CRISPRa工程化的BMSC具有激活的Wnt10b和Foxc2，可显着改善大鼠临界大小的颅骨缺损植入后的颅骨愈合。这些数据暗示了CRISPRa技术在骨组织再生中的潜力。