Open AccessEngineered Cas9 extracellular vesicles as a novel gene editing tool
Author(s) -
Osteikoetxea Xabier,
Silva Andreia,
LázaroIbáñez 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
Publication year - 2022
Publication title -
journal of extracellular vesicles
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.94
H-Index - 68
ISSN - 2001-3078
DOI - 10.1002/jev2.12225
Subject(s) - crispr , cas9 , genome editing , hek 293 cells , palmitoylation , computational biology , extracellular vesicles , gene delivery , reporter gene , biology , microbiology and biotechnology , gene , chemistry , genetic enhancement , genetics , biochemistry , gene expression , cysteine , enzyme
Abstract 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.