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

Koki Kunitake, Tadahaya Mizuno, Kazuki Hattori, Chitose Oneyama, Mako Kamiya, Sadao Ota, Yasuteru Urano and Ryosuke Kojima ()
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Koki Kunitake: 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
Ryosuke Kojima: The University of Tokyo

Nature Communications, 2024, vol. 15, issue 1, 1-17

Abstract: 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.

Date: 2024
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DOI: 10.1038/s41467-024-53736-x

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