TY - JOUR
T1 - A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA
AU - de Jong, Olivier G
AU - Murphy, Daniel E
AU - Mäger, Imre
AU - Willms, Eduard
AU - Garcia-Guerra, Antonio
AU - Gitz-Francois, Jerney J
AU - Lefferts, Juliet
AU - Gupta, Dhanu
AU - Steenbeek, Sander C
AU - van Rheenen, Jacco
AU - El Andaloussi, Samir
AU - Schiffelers, Raymond M
AU - Wood, Matthew J A
AU - Vader, Pieter
PY - 2020/2/28
Y1 - 2020/2/28
N2 - 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.
AB - 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.
KW - Biological Transport
KW - CRISPR-Cas Systems
KW - Cell Communication
KW - Cell Line
KW - Endocytosis/genetics
KW - Extracellular Vesicles/genetics
KW - Fluorescence
KW - Genes, Reporter/genetics
KW - HEK293 Cells
KW - Humans
KW - RNA, Guide/genetics
KW - RNA, Small Untranslated/genetics
U2 - 10.1038/s41467-020-14977-8
DO - 10.1038/s41467-020-14977-8
M3 - Article
C2 - 32111843
SN - 2041-1723
VL - 11
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 1113
ER -