The levels and subcellular localizations of proteins regulate critical aspects of many cellular processes and can become targets of therapeutic intervention. However, high-throughput methods for the discovery of proteins that change localization either by shuttling between compartments, by binding larger complexes, or by localizing to distinct membraneless organelles are not available. Here we describe a scalable strategy to characterize effects on protein localizations and levels in response to different perturbations. We use CRISPR-Cas9-based intron tagging to generate cell pools expressing hundreds of GFP-fusion proteins from their endogenous promoters and monitor localization changes by time-lapse microscopy followed by clone identification using in situ sequencing. We show that this strategy can characterize cellular responses to drug treatment and thus identify nonclassical effects such as modulation of protein–protein interactions, condensate formation, and chemical degradation.

中文翻译：

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用于实时监测药物作用的混合蛋白质标记、细胞成像和原位测序

蛋白质的水平和亚细胞定位调节许多细胞过程的关键方面，可以成为治疗干预的目标。然而，用于发现通过在隔室之间穿梭、通过结合更大的复合物或通过定位到不同的无膜细胞器来改变定位的蛋白质的高通量方法是不可用的。在这里，我们描述了一种可扩展的策略来表征对蛋白质定位和水平的影响，以响应不同的扰动。我们使用基于 CRISPR-Cas9 的内含子标记从内源性启动子生成表达数百种 GFP 融合蛋白的细胞库，并通过延时显微镜监测定位变化，然后使用原位测序进行克隆鉴定。