Optical Pooled Screens in Human Cells

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 κB (NF-κB) 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 image-based screens of spatially and temporally defined phenotypes with pooled libraries. [Display omitted] •In situ sequencing of perturbations or barcodes enables image-based pooled screens•p65 translocation is assayed by imaging in fixed and live cell pools•Pooled live-cell screen identifies MED12 and MED24 as negative regulators of NF-κB A screening approach that combines high-content imaging with in situ sequencing can identify genes that affect spatially and temporally defined phenotypes like morphology and subcellular localization, expanding the list of scientific questions that can be asked with genetic tools.