由於自癒能力差，頭蓋骨修復在臨床醫學上仍是一大難題。間葉幹細胞，包括脂肪間葉幹細胞 (ASC) 以及骨髓間葉幹細胞 (BMSC)，已被廣泛運用於硬骨及軟骨修復。在實驗室先前的研究已證實，誘導細胞進行軟骨分化而非硬骨分化，可以將修復途徑由膜內成骨轉換至軟骨內成骨，進而促進頭骨修復。
CRISPRa及CRISPRi 為新穎的工具，其利用dCas9蛋白以及單股嚮導RNA進行基因調控。在此篇研究，我們發展了兩個可同時進行基因抑制及活化的系統。第一篇主題中，我們設計了單一桿狀病毒載體搭載CRIPRai系統，此系統包含dSpCas9蛋白用以結合DNA、MCP-p65-HSF用以基因活化、COM-KRAB用以抑制基因表現，以達到同時抑制及提升基因表現。此外，為了將轉錄活化/抑制蛋白，正確且有區別性地標靶至目標基因座，我們改造了嚮導RNA的結構，使其具有MS2 hairpin 或com hairpin區域，可與轉錄活化/抑制蛋白前端的MCP/COM區域進行結合。在第二主題中，我們利用來自不同菌種的dCas9系統，結合助於鬆散染色質以促進轉錄的p300core (dSpCasp-p300core)、以及結合使染色體結構聚集以抑制基因轉錄的DNMT3A (dSaCas9-DNMT3A)，搭配單股嚮導RNA標靶目標基因座，同時達到基因抑制及活化，且兩者不互相干擾。
我們證實了，我們的設計可在不同細胞株中，可達到不同程度的基因表現提升(Soc5, Sox6, Sox9)以及基因表現抑制(C/ebpα and Pparγ)。此外，在2D培養下，我們透過Alcian Blue及Oil Red O染色實驗，證實改質後的ASC及BMSC可有效地進行軟骨分化，亦可在脂肪誘導培養基的培養下減少脂肪分化。將細胞植入明膠支架進行3D培養時，透過組織化學染色法以及分析GAG及collagen type 2表現量，我們可以發現軟骨分化有受到顯著的提升。最後，在大鼠模型中，透過micro CT照影技術及組織免疫染色分析，將含有改質間葉幹細胞的明膠支架植入大鼠的頭骨缺陷處，可達到良好的骨修復情形。
綜觀我們的實驗結果，我們證實了透過CRISPR技術改質的間葉幹細胞可提升軟骨分化的能力以及進一步有效促進頭骨修復。

Calvarial bone regeneration remains a hurdle in clinical settings due to poor spontaneous healing. Mesenchymal stem cell (MSC), namely adipose-derived stem cell (ASC) and bone marrow stem cell (BMSC), has been widely utilized for bone/cartilage regeneration. Our previous studies showed that stimulating chondrogenic instead of osteogenic differentiation may switch the repair pathway from the native intramembranous to endochondral ossification pathway and boost calvarial bone healing.
CRISPR activation (CRISPRa) and CRISPR inhibition (CRISPRi) are newly developed technology that exploits dCas9 protein and single guide RNA (sgRNA) for programmable gene manipulation. In this study, we developed two systems facilitating simultaneous gene activation and repression. First of all, we designed a single baculovirus (Bac-CRISPRai) harboring the DNA-binding effector (SpdCas9), activation effector (MCP-p65-HSF), and repression effector (COM-KRAB) modules for simultaneous gene activation and repression. In addition, to differentially guide appropriate module (activator and/or repressor) to the locus of interest, either or each set of sgRNA, whose structure was engineered to carry MS2 hairpin (sgRNAa) or com hairpin (sgRNAi) structure that can specifically bind to MCP or COM on the activator or repressor, respectively, was incorporated upstream of the effectors. Secondly, we utilized two dCas9 orthologs fusing with ether chromatin relaxer p300core (dSpCas9-p300core) or condenser DNMT3A (dSaCas9-DNMT3A), enabling orthogonal recruitment of the effectors to the locus of interest.
We showed that transduction of mesenchymal stem cells with our designs conferred modest to significant gene upregulation (Sox5, Sox6, and Sox9) and downregulation (C/ebpa and Pparg), depending on the targeted genes and cells. Furthermore, we revealed that the engineered ASC or BMSC was able not only to robustly undergo chondrogenesis under normal culture condition but also defer from adipogenesis under adipogenic induction condition at 2D culture format via Alcian Blue and Oil Red O staining. Furthermore, we demonstrated that seeding the transduced cells onto gelatin scaffold significantly enhanced the expression of chondrogenic phenotype evidenced by histological analysis as well as GAG and collagen type 2 content quantification. Finally, implantation of the engineered cell/gelatin constructs into critical-size calvarial defect created on SD rats effectively promoted fracture healing as judged by micro CT imaging and immunohistological analysis. These data altogether affirmed that CRISPR-based programmed MSC is feasible and able to improve chondrogenic differentiation and subsequently augment calvarial bone healing.

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