Engineered Cas9 extracellular vesicles as a novel gene editing tool

Osteikoetxea, Xabier [Osteikoetxea, Xabier (immunológia, gene...), szerző] Genetikai, Sejt- és Immunbiológiai Intézet (SE / AOK / I); HCEMM-SE Extracelluláris Vezikula Kutatócsoport (SE / AOK / I / GSII); Silva, Andreia; Lazaro-Ibanez, 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 ✉

Angol nyelvű Szakcikk (Folyóiratcikk) Tudományos
Megjelent: JOURNAL OF EXTRACELLULAR VESICLES 2001-3078 11 (5) Paper: e12225 , 17 p. 2022
  • SJR Scopus - Cell Biology: D1
Azonosítók
Támogatások:
  • (739593) Támogató: Horizon 2020
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.
Hivatkozás stílusok: IEEEACMAPAChicagoHarvardCSLMásolásNyomtatás
2024-07-25 01:56