Pooled protein tagging, cellular imaging and in situ sequencing for monitoring drug action in real time

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 unbiased 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 non-classical effects such as modulation of protein-protein interactions, condensate formation and chemical degradation.

Reicher A, Koren A, Kubicek S

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idr0097-reicher-proteintag ()

idr0097-reicher-proteintag/experimentB ()

idr0097-reicher-proteintag/experimentC ()

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