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） 是各种生物学环境中重要的细胞间信息传递者，但对其释放过程仍然知之甚少。在此，我们描述了一个高通量检测平台，CRISPR 辅助 indididually barcoded sEV based release regulator （CIBER） 筛选，用于识别 sEV 释放中的关键参与者。CIBER 筛选采用通过 gRNA 和与 sEV 标记融合的死亡 Cas9 的相互作用，用 CRISPR-gRNA 标记的 sEV 进行编码。条形码定量能够以大规模并行方式估计从每个细胞释放的 sEV 量。在 CRISPR 混合筛选格式中，用不同的 sEV 标记对 sEV 进行条形码，可以以亚群特异性方式对 sEV 释放调节因子进行全基因组探索，成功识别以前未知的 sEV 释放调节因子，并分别揭示 CD63 ⁺ / CD9 ⁺ sEVs 的外泌体/外泌体性质，以及 CD9 ^的同步sEV 随细胞周期释放。CIBER 应该是详细研究 sEVs 的生物发生、释放和异质性的有价值的工具。