Kunitake Koki, Mizuno Tadahaya, Hattori Kazuki, Oneyama Chitose, Kamiya Mako, Ota Sadao, Urano Yasuteru, Kojima Ryosuke
Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.
Nat Commun. 2024 Nov 19;15(1):9777. doi: 10.1038/s41467-024-53736-x.
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.
小细胞外囊泡（sEVs）在各种生物学环境中都是重要的细胞间信息传递者，但其释放过程仍知之甚少。在此，我们描述了一种高通量检测平台，即CRISPR辅助的基于单个条形码的sEV释放调节剂（CIBER）筛选，用于识别sEV释放中的关键参与者。CIBER筛选通过与sEV标记物融合的gRNA和无活性Cas9的相互作用，利用用CRISPR - gRNA条形码标记的sEVs。条形码定量能够以大规模平行的方式估计从每个细胞释放的sEV数量。在CRISPR混合筛选形式中用不同的sEV标记物对sEVs进行条形码标记，能够以亚群特异性的方式对sEV释放调节剂进行全基因组探索，分别成功识别出先前未知的sEV释放调节剂，并揭示了CD63/CD9 sEVs的外泌体/胞外体性质，以及CD9 sEV释放与细胞周期的同步性。CIBER应该是用于详细研究sEVs的生物发生、释放和异质性的一个有价值的工具。
