A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA

摘要

Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer ofbiological cargo, including RNA, that plays a pivotal role in physiological and pathologicalprocesses. Unfortunately, whereas biological effects of EV-mediated RNA transfer areabundantly studied, regulatory pathways and mechanisms remain poorly defined due to alack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-basedreporter system that allows direct functional study of EV-mediated transfer of small noncodingRNA molecules at single-cell resolution. Using this CRISPR operated stoplight systemfor functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involvedin EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identifymultiple genes involved in endocytosis and intracellular membrane trafficking that stronglyregulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidationof regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis,endocytosis, intracellular trafficking, and RNA delivery.