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Barcoding of small extracellular vesicles with CRISPR-gRNA enables comprehensive, subpopulation-specific analysis of their biogenesis and release regulators

Author

Listed:
  • Koki Kunitake

    (The University of Tokyo
    The University of Tokyo)

  • Tadahaya Mizuno

    (The University of Tokyo)

  • Kazuki Hattori

    (The University of Tokyo)

  • Chitose Oneyama

    (Aichi Cancer Center Research Institute)

  • Mako Kamiya

    (Institute of Science Tokyo)

  • Sadao Ota

    (The University of Tokyo)

  • Yasuteru Urano

    (The University of Tokyo
    The University of Tokyo)

  • Ryosuke Kojima

    (The University of Tokyo
    Japan Science and Technology Agency
    Japan Science and Technology Agency)

Abstract

Small extracellular vesicles (sEVs) are important intercellular information transmitters in various biological contexts, but their release processes remain poorly understood. Herein, we describe a high-throughput assay platform, CRISPR-assisted individually barcoded sEV-based release regulator (CIBER) screening, for identifying key players in sEV release. CIBER screening employs sEVs barcoded with CRISPR-gRNA through the interaction of gRNA and dead Cas9 fused with an sEV marker. Barcode quantification enables the estimation of the sEV amount released from each cell in a massively parallel manner. Barcoding sEVs with different sEV markers in a CRISPR pooled-screening format allows genome-wide exploration of sEV release regulators in a subpopulation-specific manner, successfully identifying previously unknown sEV release regulators and uncovering the exosomal/ectosomal nature of CD63+/CD9+ sEVs, respectively, as well as the synchronization of CD9+ sEV release with the cell cycle. CIBER should be a valuable tool for detailed studies on the biogenesis, release, and heterogeneity of sEVs.

Suggested Citation

  • Koki Kunitake & Tadahaya Mizuno & Kazuki Hattori & Chitose Oneyama & Mako Kamiya & Sadao Ota & Yasuteru Urano & Ryosuke Kojima, 2024. "Barcoding of small extracellular vesicles with CRISPR-gRNA enables comprehensive, subpopulation-specific analysis of their biogenesis and release regulators," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-53736-x
    DOI: 10.1038/s41467-024-53736-x
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