Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, Georgia, USA.
School of Electrical and Computer Engineering, College of Engineering, University of Georgia, Athens, Georgia, USA.
J Extracell Vesicles. 2022 Apr;11(4):e12196. doi: 10.1002/jev2.12196.
CRISPR/Cas9 genome editing is a very promising avenue for the treatment of a variety of genetic diseases. However, it is still very challenging to encapsulate CRISPR/Cas9 machinery for delivery. Protein N-myristoylation is an irreversible co/post-translational modification that results in the covalent attachment of the myristoyl-group to the N-terminus of a target protein. It serves as an anchor for a protein to associate with the cell membrane and determines its intracellular trafficking and activity. Extracellular vesicles (EVs) are secreted vesicles that mediate cell-cell communication. In this study, we demonstrate that myristoylated proteins were preferentially encapsulated into EVs. The octapeptide derived from the leading sequence of the N-terminus of Src kinase was a favourable substrate for N-myristoyltransferase 1, the enzyme that catalyzes myristoylation. The fusion of the octapeptide onto the N-terminus of Cas9 promoted the myristoylation and encapsulation of Cas9 into EVs. Encapsulation of Cas9 and sgRNA-eGFP inside EVs was confirmed using protease digestion assays. Additionally, to increase the transfection potential, VSV-G was introduced into the EVs. The encapsulated Cas9 in EVs accounted for 0.7% of total EV protein. Importantly, the EVs coated with VSV-G encapsulating Cas9/sgRNA-eGFP showed up to 42% eGFP knock out efficiency with limited off-target effects in recipient cells. Our study provides a novel approach to encapsulate CRISPR/Cas9 protein and sgRNA into EVs. This strategy may open an effective avenue to utilize EVs as vehicles to deliver CRISPR/Cas9 for genome-editing-based gene therapy.
CRISPR/Cas9 基因组编辑是治疗多种遗传疾病的很有前途的途径。然而，将 CRISPR/Cas9 机制封装用于递送仍然非常具有挑战性。蛋白质 N-豆蔻酰化是一种不可逆的共/翻译后修饰，导致豆蔻酰基团共价连接到靶蛋白的 N 末端。它作为一种锚定蛋白与细胞膜结合，并决定其细胞内运输和活性。细胞外囊泡 (EVs) 是介导细胞间通讯的分泌囊泡。在这项研究中，我们证明了豆蔻酰化蛋白优先被包裹在 EVs 中。Src 激酶 N 末端前导序列衍生的八肽是 N-豆蔻酰转移酶 1（催化豆蔻酰化的酶）的有利底物。该八肽融合到 Cas9 的 N 末端促进了 Cas9 的豆蔻酰化和 EV 包裹。使用蛋白酶消化测定法证实了 Cas9 和 sgRNA-eGFP 被包裹在 EV 内。此外，为了提高转染潜力，将 VSV-G 引入 EVs。EV 中包裹的 Cas9 占总 EV 蛋白的 0.7%。重要的是，用包覆有 VSV-G 的 EV 包裹 Cas9/sgRNA-eGFP 可使受体细胞中的 eGFP 敲除效率高达 42%，且脱靶效应有限。我们的研究提供了一种将 Cas9 蛋白和 sgRNA 封装到 EVs 中的新方法。该策略可能为利用 EV 作为载体将 Cas9/sgRNA 递送至基于基因组编辑的基因治疗开辟有效途径。
