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  • Encapsulating Cas9 into extracellular vesicles by protein myristoylation.

Encapsulating Cas9 into extracellular vesicles by protein myristoylation.

Journal of extracellular vesicles (2022-04-07)
Joseph Andrew Whitley, Sungjin Kim, Lei Lou, Chenming Ye, Omar Awad Alsaidan, Essilvo Sulejmani, Jingwen Cai, Ellison Gerona Desrochers, Zanna Beharry, Catherine Bowes Rickman, Mikael Klingeborn, Yutao Liu, Zhong-Ru Xie, Houjian Cai
ABSTRACT

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.

MATERIALS
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Sigma-Aldrich
Cas9 Protein, from Streptococcus pyogenes, recombinant, expressed in E. coli, 1X NLS