CRISPR/Cas9 Delivery Using Extracellular Vesicles

Extracellular vesicles (EVs), particles released from cells, have the potential to become an important in vivo delivery vehicle for CRISPR machinery. Unlike viral vectors such as Adeno-associated virus (AAV), EVs reduce risks associated with immunogenicity, long-term expression, and potential off-target effects. EVs can efficiently deliver CRISPR/Cas9 ribonucleoprotein (RNP) complexes, which provide transient, ready-to-function editing machinery with reduced off-target risk compared with plasmid DNA or mRNA delivery. RNP loading into EVs can occur without specific targeting signals, although strategies such as membrane anchoring, inducible dimerization systems, or fusion with EV-associated proteins can enhance cargo enrichment. EVs are typically produced by transfecting producer cells with plasmids encoding Cas9 and sgRNA, followed by vesicle release into culture media. Purification requires removal of cellular debris and enrichment of vesicles using methods such as ultracentrifugation, ultrafiltration, chromatography, or precipitation. As no single gold-standard purification approach exists, method selection should balance yield and purity, and characterization using vesicle markers and size distribution profiling is recommended.