Genome-Wide CRISPR/Cas9 Screening Identifies the COMMANDER Recycling Complex as a Key Player in EV Uptake

Area of Science:

• Cell Biology
• Molecular Biology
• Biotechnology

Background:

• Extracellular vesicles (EVs) are promising for therapeutic delivery.
• Understanding EV uptake mechanisms is crucial for optimizing their therapeutic potential.

Purpose of the Study:

• To identify key molecular regulators of EV internalization.
• To pinpoint candidate genes influencing EV uptake by target cells.

Main Methods:

• Genome-wide CRISPR (GWC) screen in K562 cells.
• Labeling and incubation of EVs with target cells, followed by fluorescence-activated cell sorting (FACS).
• Next-generation sequencing (NGS) to quantify sgRNA enrichment and identify significant genes.

Main Results:

• Several members of the COMMANDER complex were identified as significant hits in the GWC screen.
• Validation in knockout cell lines confirmed the role of the COMMANDER complex in EV uptake.
• Kinetic analysis indicated the COMMANDER complex influences early EV uptake and cargo fate.

Conclusions:

• The COMMANDER complex is a pivotal regulator of extracellular vesicle uptake.
• This finding advances the understanding of EV-mediated delivery mechanisms.
• Potential implications for developing targeted EV-based therapies.