CRISPR delivery with extracellular vesicles: Promises and challenges

The CRISPR gene editing tool holds great potential for curing genetic disorders. However, the safe, efficient, and specific delivery of the CRISPR/Cas9 components into cells and tissues remains a challenge. While many currently available delivery methods achieve high levels of gene editing effects in vivo, they often result in genotoxicity and immunogenicity. Extracellular vesicles (EVs), which are cell-derived lipid nanoparticles, are capable of transferring protein and nucleic acid cargoes between cells, making them a promising endogenous alternative to synthetic delivery methods.

Researchers from Aarhus University provide a comprehensive analysis of the currently available strategies for EV-mediated delivery of CRISPR/Cas9. These strategies include cell-based, passive loading obtained by overexpression of CRISPR/Cas9, active loading involving protein or RNA dimerization, and loading into already purified EVs. All these approaches suggest that EV-based CRISPR/Cas9 delivery is useful for achieving both in vitro and in vivo gene editing. Despite that, substantial variations in cellular uptake and gene editing efficiencies indicate that further improvement and standardization are required for the therapeutic use of EVs as a CRISPR/Cas9 delivery vehicle. These improvements include, but is not limited to, the high-yield purification of EVs, increased loading and release efficiencies, as well as improved tissue- or cell-specific targeting specificities.

Cell-based loading of CRISPR/Cas9 RNP (upper) and non-cell-based loading of CRISPR/Cas9 plasmid, RNA or RNP post-EV-purification (lower). Endogenous EVs are formed through inward budding of the multivesicular endosome (MVE), which are released by merging of the MVE with the cell membrane, or direct outward budding of the cell membrane. 

Berggreen AH, Petersen L, Lin L, Benabdellah K, Luo Y. (2023) CRISPR delivery with extracellular vesicles: Promises and challenges. J Extracell Biol [Epub ahead of print]. [article]

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