Genome editing tools have simplified the generation of knock-in gene fusions, yet the prevalent use of gene-specific homology-directed repair (HDR) templates still hinders scalability. Consequently, realization of large-scale gene tagging requires further development of approaches to generate knock-in protein fusions via generic donors that do not require locus-specific homology sequences. Here, we combine intron-based protein trapping with homology-independent repair-based integration of a generic donor and demonstrate precise, scalable, and efficient gene tagging. Because editing is performed in introns using a synthetic exon, this approach tolerates mutations in the unedited allele, indels at the integration site, and the addition of resistance genes that do not disrupt the target gene coding sequence, resulting in easy and flexible gene tagging.

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.

Genome editing tools have simplified the generation of knock-in gene fusions, yet the requirement for gene-specific homology directed repair (HDR) templates still hinders the scalability of most approaches. Here, we combine intron-based protein trapping with homology independent repairbased editing and demonstrate precise and efficient gene tagging that can be easily scaled due to use of a generic donor. As editing is done in introns, this approach tolerates mutations in the unedited allele, disruptive indels, and allows for flexible donor and sgRNA design. MainFusing endogenous proteins with fluorescence or epitope tags is a widely used and essential approach for studying proteins within their natural regulatory context. The advent of CRISPR tools for modifying the genome 1-3 has made this easier and even more accessible, yet scalability is still very limited. The need for a gene-specific homology directed repair (HDR) template requires costly synthesis or labor-intensive molecular cloning, and since precise targeting must be achieved in frame with the coding sequence, it necessitates careful design of reagents and screening of clonal cell lines to avoid disruptive editing at the non-tagged allele. A recent development of split fluorescent proteins has simplified the generation of fluorescent fusions, since only a minimal tag is required for genomic knock-in 4-6 . Nevertheless, these endogenous tagging methods still require individual HDR donors. Several approaches to develop

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