Engineered Cas9 extracellular vesicles as a novel gene editing tool

Area of Science:

• Biotechnology
• Molecular Biology
• Nanomedicine

Background:

• Extracellular vesicles (EVs) are promising biological delivery vehicles.
• Efficient cargo loading into EVs is crucial for therapeutic applications.
• Current methods for loading CRISPR/Cas9 into EVs are often inefficient.

Purpose of the Study:

• To develop and validate novel methods for efficient CRISPR/Cas9 loading into EVs.
• To assess the functional gene editing capacity of CRISPR/Cas9-loaded EVs.
• To explore the therapeutic potential of EV-mediated CRISPR/Cas9 delivery.

Main Methods:

• Engineered Expi293F cells to produce Cas9-fusion proteins.
• Utilized reversible heterodimerization (light-induced dimerization with Cryptochrome 2) and lipid modifications (CD9, MPP) for Cas9 loading.
• Collected and characterized EVs using nanoparticle tracking analysis, western blotting, and transmission electron microscopy.
• Assessed gene editing efficiency in HEK293 and HepG2 cells using a Cre-reporter assay and PCSK9 gene knockdown.

Main Results:

• Achieved efficient loading of approximately 25 Cas9 molecules per EV.
• Demonstrated high functional gene editing with 51% Cre reporter editing in HEK293 cells and 25% in HepG2 cells.
• Successfully targeted PCSK9 gene knockdown with 6% indel efficiency in HEK293 cells.
• Confirmed EV-mediated transfer of Cas9, as indicated by detergent sensitivity and size exclusion chromatography results.

Conclusions:

• Developed a robust and efficient method for loading CRISPR/Cas9 into EVs via reversible heterodimerization.
• Demonstrated the therapeutic potential of these engineered EVs for genome editing and gene knockdown.
• This approach offers a promising platform for advancing EV-based therapeutic delivery systems.