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

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 and Pieter Vader ()
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Olivier G. de Jong: University Medical Center Utrecht
Daniel E. Murphy: University Medical Center Utrecht
Imre Mäger: University of Oxford
Eduard Willms: University of Oxford
Antonio Garcia-Guerra: University of Oxford
Jerney J. Gitz-Francois: University Medical Center Utrecht
Juliet Lefferts: University Medical Center Utrecht
Dhanu Gupta: Karolinska Institutet
Sander C. Steenbeek: Netherlands Cancer Institute
Jacco van Rheenen: Netherlands Cancer Institute
Samir El Andaloussi: Karolinska Institutet
Raymond M. Schiffelers: University Medical Center Utrecht
Matthew J. A. Wood: University of Oxford
Pieter Vader: University Medical Center Utrecht

Nature Communications, 2020, vol. 11, issue 1, 1-13

Abstract: Abstract 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.

Date: 2020
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DOI: 10.1038/s41467-020-14977-8

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