TAG-IN, a swappable strategy for endogenous gene tagging in Caenorhabditis elegans using cassette exchange

Abstract Endogenous gene tagging allows researchers to study genes in their native genomic context, underscoring the need for efficient and scalable tagging approaches. Here, we report TAG-IN, a streamlined and swappable strategy for tagging virtually any gene in the C. elegans genome. A seed allele for the gene of interest is generated by inserting a pair of FRT and FRT3 sites into the desired tagging locus. Fusion or co-expression tags from reusable tag donors can be efficiently swapped in the seed allele using Flp recombinase-mediated cassette exchange (RMCE). Furthermore, the tag-swapping step can be accomplished through simple genetic crossing, making it easy to scale up. We also built a library of plug-and-play tag donor strains that can be crossed with seed alleles to insert the corresponding tags. The TAG-IN strategy is highly efficient: a single seed allele for each tagging locus is needed to insert any tag of interest; a single tag donor strain can be used to swap the tag into any compatible seed allele. Thus, TAG-IN is a modular and combinatorial tool for efficient endogenous gene tagging in C. elegans, accelerating comprehensive functional studies.