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bioRxiv · 10.1101/2023.09.28.559700

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

Kunitake, K.·Mizuno, T.·Hattori, K.·Oneyama, C.·Kamiya, M.·Ota, S.·Urano, Y.·Kojima, R.
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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.

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Kunitake, K., Mizuno, T., Hattori, K., Oneyama, C., Kamiya, M., Ota, S., Urano, Y., Kojima, R.. 2023-09-28. Barcoding of small extracellular vesicles with CRISPR-gRNA enables comprehensive, subpopulation-specific analysis of their biogenesis/release regulators. https://doi.org/10.1101/2023.09.28.559700

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