Philip, R.; Sharma, A.; Matellan, L.; Erpf, A. C.; Hsu, W.-H.; Tkach, J. M.; Wyatt, H. D. M.; Pelletier, L.

Endogenous tagging makes it possible to study a protein\'s localization, dynamics, and function within its native regulatory context by genetically inserting a sequence encoding a functional tag into the reading frame of a gene. Here, we introduce the \"quickTAG,\" or qTAG system, a versatile collection of optimized repair cassettes designed to make CRISPR-mediated tagging more accessible. By partitioning the cassette to include both a desired tag sequence linked with a selectable marker, integrations can be quickly isolated post-editing. These constructs also include several key features that enhance flexibility and ease of use, such as: cassette designs for N- and C-terminus tagging; standardized cloning adaptors to simplify the incorporation of homology arms for HDR or MMEJ-based repairs; restriction sites next to each genetic element within the cassette for easy switching of tags and selectable markers; and the inclusion of lox sites flanking the selectable marker to allow marker gene removal following integration. Our library of ~120 plasmids is available on Addgene and includes ready-to-use constructs targeting frequently overexpressed genes as well as ready-to-clone cassettes to tag your own genes of interest. These cassettes were engineered to harbor a variety of tags for fluorescence imaging, proximity-dependent biotinylation, epitope tagging, and targeted protein degradation. Alongside a comprehensive protocol, the qTAG system will provide a framework to streamline endogenous tagging in addition to serving as an open resource for researchers to adapt and tailor for their own purposes.