Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but
therapeutic applications require efficient cargo loading. Here, we developed new methods
for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions
with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F
cells using standard methodology, characterized using nanoparticle tracking analysis,
western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated
functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization
using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation
lipid modification resulted in efficient loading with approximately 25 Cas9 molecules
per EV and high functional delivery with 51% gene editing of the Cre reporter cassette
in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for
targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency
in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions
after size exclusion chromatography, indicative of EV-mediated transfer. Considering
the advantages of EVs over other delivery vectors we envision that this study will
prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated
genome editing.
