qTAG: An adaptable CRISPR-based endogenous tagging protocol using optimized repair cassettes

Abstract Endogenous tagging makes it possible to study a protein’s localization, dynamics, and function within its native regulatory context. This is typically accomplished via CRISPR, which involves inserting a sequence encoding a functional tag into the reading frame of a gene. However, this process is often inefficient. Here, we introduce the “quickTAG,” or qTAG system, a versatile collection of optimized repair cassettes designed to make CRISPR-mediated tagging more accessible. By including a desired tag sequence linked to a selectable marker in the cassette, integrations can be quickly isolated post-editing. The core sequence scaffold within these constructs incorporates several key features that enhance flexibility and ease of use, such as: specific cassette designs for N- and C-terminus tagging; standardized cloning sequences 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 modification of tags and selectable markers; and the inclusion of lox sites flanking the selectable marker to allow for marker gene removal following integration. We showcase the versatility of these cassettes with a diverse range of tags, demonstrating their applications in fluorescence imaging, proximity-dependent biotinylation, epitope tagging, and targeted protein degradation. The adaptability of this scaffold is also exhibited by incorporating novel tags such as mStayGold, which offer enhanced brightness and photostability, reconciling prolonged live-cell imaging of proteins at their endogenous levels. Finally, by leveraging the restriction sites, entirely distinct cassette structures and editing schemes were developed. These enabled scenarios that included conditional expression tagging, selectable knockout tagging, and safe-harbor expression. Our existing and forthcoming collection of plasmids will be accessible through Addgene. It includes ready-to-use constructs targeting common subcellular marker genes, as well as an assortment of tagging cassettes for the tagging of genes of interest. The qTAG system offers an accessible framework to streamline endogenous tagging and will serve as an open resource for researchers to adapt and tailor for their own experiments.