Optical Pooled Screens in Human Cells
Optical Pooled Screens in Human Cells
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DOI：
10.1016/j.cell.2019.09.016
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发表时间：
2019-10-17
期刊：
影响因子：
64.5
通讯作者：
Blainey, Paul C.
中科院分区：
文献类型：
--
作者：
Feldman, David;Singh, Avtar;Blainey, Paul C.
遗传筛选对于系统鉴定细胞表型背后的基因至关重要。汇集基因扰动极大地提高了可扩展性，但与复杂和动态细胞表型的成像不兼容。在这里，我们介绍了一种在哺乳动物细胞中进行光学遗传筛选的混合方法。我们使用靶向原位测序来分离基于图像的表型分析后的遗传扰动库。我们通过对 RelA (p65) 易位至细胞核的过程进行成像，筛选了数百万个细胞中参与核因子 kB (NF-kappa B) 信号转导的一组 952 个基因。在 3 个细胞系的单个时间点进行筛选，恢复了 15 种已知的途径成分，同时通过活细胞成像重复筛选，揭示了介体复合体亚基在调节 p65 核保留持续时间中的作用。这些结果建立了一种高度多重的方法，通过混合文库对空间和时间定义的表型进行基于图像的筛选。
Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor kB (NF-kappa B) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to imagebased screens of spatially and temporally defined phenotypes with pooled libraries.