Encapsulating Cas9 into extracellular vesicles by protein myristoylation.

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Whitley, JA; Kim, S; Lou, L; Ye, C; Alsaidan, OA; Sulejmani, E; Cai, J; Desrochers, EG; Beharry, Z; Rickman, CB; Klingeborn, M; Liu, Y; Xie, Z-R; Cai, H

Published Date

  • April 2022

Published In

Volume / Issue

  • 11 / 4

Start / End Page

  • e12196 -

PubMed ID

  • 35384352

Pubmed Central ID

  • PMC8982324

Electronic International Standard Serial Number (EISSN)

  • 2001-3078

Digital Object Identifier (DOI)

  • 10.1002/jev2.12196


  • eng

Conference Location

  • United States