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

Author

Listed:
  • Olivier G. de Jong

    (University Medical Center Utrecht
    University of Oxford)

  • Daniel E. Murphy

    (University Medical Center Utrecht)

  • Imre Mäger

    (University of Oxford)

  • Eduard Willms

    (University of Oxford
    University of Oxford)

  • Antonio Garcia-Guerra

    (University of Oxford
    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
    University Medical Center Utrecht)

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.

Suggested Citation

  • 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 E, 2020. "A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA," Nature Communications, Nature, vol. 11(1), pages 1-13, December.
  • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-14977-8
    DOI: 10.1038/s41467-020-14977-8
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