1. A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA
- Author
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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, Afd Pharmaceutics, Pharmaceutics, Afd Pharmaceutics, and Pharmaceutics
- Subjects
0301 basic medicine ,Cell ,General Physics and Astronomy ,Membrane trafficking ,Cell Communication ,0302 clinical medicine ,Genes, Reporter ,CRISPR ,Guide RNA ,lcsh:Science ,Multidisciplinary ,Chemistry ,Extracellular Vesicles/genetics ,Extracellular vesicle ,Non-coding RNA ,Publisher Correction ,Endocytosis ,3. Good health ,Cell biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,RNA, Guide, Kinetoplastida ,Science ,Guide/genetics ,RNA, Guide/genetics ,General Biochemistry, Genetics and Molecular Biology ,Article ,Fluorescence ,Cell Line ,Reporter/genetics ,03 medical and health sciences ,Extracellular Vesicles ,Genes, Reporter/genetics ,medicine ,Humans ,Gene ,RNA, Small Untranslated/genetics ,Assay systems ,Small Untranslated/genetics ,RNA ,Biological Transport ,Endocytosis/genetics ,General Chemistry ,030104 developmental biology ,HEK293 Cells ,Genes ,RNA, Small Untranslated ,Nanoparticles ,lcsh:Q ,CRISPR-Cas Systems - 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., Extracellular vesicles (EV) facilitate intercellular transfer of biological material including RNA, but the regulatory mechanisms for their formation and transfer are incompletely known. Here the authors develop a CRISPR-based reporting system to detect the transfer of guide RNAs and identify genes not previously linked to EV-mediated RNA delivery.
- Published
- 2020