A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA.
de Jong OG., Murphy DE., Mäger I., Willms E., Garcia-Guerra A., Gitz-Francois JJ., Lefferts J., Gupta D., Steenbeek SC., van Rheenen J., El Andaloussi S., Schiffelers RM., Wood MJA., Vader P.
Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small non-coding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.