Pooled tagging and hydrophobic targeting of endogenous proteins for unbiased mapping of unfolded protein responses

Abstract

System-level understanding of proteome organization and function requires methods for direct visualization and manipulation of proteins at scale. We developed an approach enabled by high-throughput gene tagging for the generation and analysis of complex cell pools with endogenously tagged proteins. Proteins are tagged with HaloTag to enable visualization or direct perturbation. Fluorescent labeling followed by in situ sequencing and deep learning-based image analysis identifies the localization pattern of each tag, providing a birds-eye-view of cellular organization. Next, we use a hydrophobic HaloTag ligand to unfold tagged proteins, inducing spatially restricted proteotoxic stress that is read out by single cell RNA sequencing. By integrating optical and perturbation data, we map compartment-specific responses to protein misfolding, revealing inter-compartment organization and direct cross-talk, and assigning proteostasis functions to uncharacterized genes. Altogether, we present a powerful and efficient method for large-scale studies of proteome dynamics, function, and homeostasis.

Pooled endogenous protein tagging and recruitment for scalable discovery of effectors for induced proximity therapeutics

“…To survey effector function at a proteome scale, we set to design a flexible approach to generically induce protein-protein interactions between a target of interest and many endogenously tagged potential effector proteins simultaneously. This approach is based on our recent method, Scalable POoled Targeting with a LIgandable Tag at Endogenous Sites (SPOTLITES) 30 , in which we construct a complex cell pool where endogenous proteins are tagged with HaloTag (HT), a generic ligand-binding domain 35 . Each cell in the pool has a single protein site tagged with HT that can be recruited to a target with an orthogonal ligand binding domain by treatment with a generic heterobifunctional molecule ( Fig.…”

“…2A ). We constructed a SPOTLITES cell pool using an sgRNA library for endogenous tagging 30 ( www.pooledtagging.org ). Much like for CRISPR screens 39 , the sgRNA library is transduced at low multiplicity of infection followed by antibiotic selection.…”

“…Induced protein proximity is poised to transform small molecule therapeutics, yet its full potential will not be realized without scalable methods to profile the effector landscape. Here, we apply our SPOTLITES platform 30 for pooled endogenous gene tagging with an optimized generic recruitment system to screen for effectors for TPD. Two orthogonal screens reveal multiple potential effectors from different pathways, with members of the CTLH complex, WDR26 and RANBP9, most frequently enriched in this first test of the technology using a focused library.…”

“…In this study, we present a novel platform for the systematic and scalable profiling of effector function by endogenous protein recruitment. We utilized our recently developed method, Scalable POoled Targeting with a LIgandable Tag at Endogenous Sites (SPOTLITES), to generate a cell library with hundreds of potential effector proteins that are tagged with HaloTag at multiple locations 30 . We then used optimized heterobifunctional small molecules for endogenous protein recruitment to systematically assess the degradation potential of effector proteins in two parallel approaches: fluorescence-based monitoring of target protein expression and a growthbased approach capitalizing on the cellular toxicity induced by BRD4 degradation [31][32][33] .…”

Proteome-scale tagging and functional screening in mammalian cells by ORFtag

“…However, current systematic methods hinge on the delivery and expression of open reading frame (ORF) libraries 4,5 , which are not only costly and challenging to maintain but also tend to favor shorter ORFs (<5kb) due to limitations in DNA synthesis, cloning, viral packaging, and delivery into cells 2 . While targeted engineering of native gene locations can bypass these limitations, recent CRISPR-Cas9 techniques for systematic gene tagging have only scaled to several hundred genes [6][7][8][9][10] .Here, we present ORFtag, a versatile approach that allows for the massive, parallel, and proteome-scale tagging of endogenous ORFs, overcoming critical limitations of current methods. ORFtag is based on insertional elements such as retroviral vectors containing a constitutively active promoter, a selection gene, and a functional tag of interest followed by a splice donor sequence (Fig.…”