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

Abstract

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.

In vivo Pooled Screening: A Scalable Tool to Study the Complexity of Aging and Age-Related Disease

“…In situ readout of pooled screening inside intact tissues could have key advantages, beyond just restoring cells to their spatial contexts. For example, some in-situ readouts are able to detect subtler properties of cells, such as the subcellular localization of specific proteins ( Reicher et al, 2020 ) or nucleic acids ( Alon et al, 2021 ) within specific organelles or compartments, or morphological features relevant to aging, like cell membrane shape or nuclear membrane integrity ( Faulkner et al, 2021 ). Such techniques could be used to identify, for example, cytoplasmic DNAs that have been linked to inflammatory processes in aging ( Simon et al, 2019 ).…”

Identification and selectivity profiling of small-molecule degraders via multi-omics approaches

“…As a consequence, we will need more elaborate tools to discern the underpinning molecular mechanism of action. Among others, we believe that CRISPR screens with a single-cell transcriptome readout will be an important component of that toolbox, similar to optical pooled CRISPR screens or functional genomic approaches that enable engineering of endogenous alleles in a multiplexed fashion (Datlinger et al, 2017;Dixit et al, 2016;Feldman et al, 2019;Reicher et al, 2020). Similarly, we believe that the field will benefit tremendously from recent advances in single-cell proteomics, or scalable chemoproteomics platforms that identify ligandable proteins at unprecedented scale (Kelly, 2020;Kuljanin et al, 2021).…”

High-throughput tagging of endogenous loci for rapid characterization of protein function

“…Here we report the development of High-throughput Insertion of Tags Across the Genome (HITAG), a fast and accessible method for generating libraries of endogenously tagged proteins. Although numerous methods exist for performing targeted knock-ins into the mammalian genome, most allow only a single protein at a time to be targeted, are inefficient, or place tags in between coding exons which can disrupt protein folding and function 7,8,[20][21][22][23] . By increasing the throughput of performing C-terminal tagging, HITAG will help facilitate the scalable interrogation of protein function and dynamics.…”