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

Extracellular vesicles (EVs) hold immense potential in therapeutic delivery, warranting a comprehensive investigation of the mechanisms that regulate their uptake by target cells. To identify key molecular regulators of EV internalization, we conducted a genome-wide CRISPR (GWC) screen aimed to pinpoint candidate genes that influence EV uptake. We employed a GWC library spanning the entire human genome in K562 cells. 3.6 × 10¹² EVs isolated from the SKMEL147 human melanoma cell line were labelled with Alexa633-C5-Maleimide and incubated for 2 h with 500 × 10⁶ K562 cells, providing a 2000× coverage of the library. The top 5% of high and low fluorescence populations were sorted. Next-generation sequencing (NGS) was performed to quantify sgRNA enrichment in the sorted populations compared to the unsorted control. Remarkably, among other genes, several members of the COMMANDER complex emerged as significant hits in our screen. We validated the hits in knockout (KO) cell lines of both K562 and HeLa cells using EVs derived either from melanoma or breast cancer cell lines. Kinetic follow-up of EV cargo, including surface or luminal proteins, suggests that the COMMANDER complex plays a pivotal role in the early stages of EV uptake but also in the final fate of EV components in the target cell.