Engineered Cas9 extracellular vesicles as a novel gene editing tool

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 E...

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Bibliographic Details
Published in:Journal of extracellular vesicles 2022-05, Vol.11 (5), p.e12225-n/a
Main Authors: Osteikoetxea, Xabier, Silva, Andreia, Lázaro‐Ibáñez, Elisa, Salmond, Nikki, Shatnyeva, Olga, Stein, Josia, Schick, Jan, Wren, Stephen, Lindgren, Julia, Firth, Mike, Madsen, Alexandra, Mayr, Lorenz M., Overman, Ross, Davies, Rick, Dekker, Niek
Format: Article
Language:English
Subjects:
CD9 antigen
CRISPR
CRISPR-Cas Systems - genetics
CRISPR/Cas9 delivery
Dimerization
exosomes
Expression vectors
Extracellular Vesicles
Gene Editing
Genetic engineering
HEK293 Cells
Humans
Ligands
Light
Low density lipoprotein
Myristoylation
Nanoparticles
optogenetics
Palmitoylation
Proprotein Convertase 9 - genetics
Proteins
Therapeutic applications
Transmission electron microscopy
Western blotting
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Summary: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.
ISSN:2001-3078
2001-3078
DOI:10.1002/jev2.12225