Authors: Olivier G. de Jong, Daniel E. Murphy, Imre Mäger, Eduard Willms, Antonio Garcia-Guerra, Jerney J. Gitz-Francois, Juliet Lefferts, Dhanu Gupta, Sander C. Steenbeek, Jacco van Rheenen, Samir El Andaloussi, Raymond M. Schiffelers, Matthew J. A. Wood, Pieter Vader
Published: 2020-02-28
DOI: 10.1038/s41467-020-14977-8
Source: Full article
AbstractExtracellular 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.