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

Autor: Kunitake K; Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.; Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan., Mizuno T; Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan., Hattori K; Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan., Oneyama C; Division of Cancer Cell Regulation, Aichi Cancer Center Research Institute, Nagoya, Japan., Kamiya M; Department of Life Science and Technology, Institute of Science Tokyo, Kanagawa, Japan., Ota S; Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan., Urano Y; Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.; Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan., Kojima R; Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. kojima@m.u-tokyo.ac.jp.; PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan. kojima@m.u-tokyo.ac.jp.; FOREST, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan. kojima@m.u-tokyo.ac.jp.
Jazyk: angličtina
Zdroj: Nature communications [Nat Commun] 2024 Nov 19; Vol. 15 (1), pp. 9777. Date of Electronic Publication: 2024 Nov 19.
DOI: 10.1038/s41467-024-53736-x
Abstrakt: 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.
Competing Interests: Competing interests K.K., Y.U., R.K. have filed a patent for the screening system of sEV release regulators (WO2021095842 (published), application by the University of Tokyo). The other authors report no competing interests.
(© 2024. The Author(s).)
Databáze: MEDLINE
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