High Resolution Imaging Screens Thousands of Genes in a Few Days

Researchers at the Broad Institute of MIT and Harvard and MIT have developed a new method that combines large-scale pooled genetic screens with image-based analysis of cellular behavior. The approach, called optical pooled screens, allows researchers to examine how genes affect complex cellular processes with spatial and temporal resolution, in ways that other pooled screens could not.

The authors examined the effects of 952 genes on the signaling activity of an immune-regulating complex called NF-kB by imaging the cellular location of a protein called p65 in millions of cells. They uncovered a new role for two genes, MED12 and MED24, in the relaxation of NF-kB signaling.

Journal Cell – Optical Pooled Screens in Human Cells

Highlights

• 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

Summary

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.

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