A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA
de Jong, Olivier G.; Murphy, Daniel E.; Mäger, Imre; Willms, Eduard; Garcia-Guerra, Antonio; Gitz-Francois, Jerney J.; Lefferts, Juliet; Gupta, Dhanu; Steenbeek, Sander C.; van Rheenen, Jacco; El Andaloussi, Samir; Schiffelers, Raymond M.; Wood, Matthew J.A.; Vader, Pieter
(2020) Nature Communications, volume 11, issue 1, pp. 1 - 13
(Article)
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
... read more
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.
show less
Download/Full Text
Keywords: Biological Transport, CRISPR-Cas Systems, Cell Communication, Cell Line, Endocytosis/genetics, Extracellular Vesicles/genetics, Fluorescence, Genes, Reporter/genetics, HEK293 Cells, Humans, RNA, Guide/genetics, RNA, Small Untranslated/genetics, Journal Article, Research Support, Non-U.S. Gov't
ISSN: 2041-1723
Publisher: Nature Publishing Group
(Peer reviewed)