TAG-IN, a modular strategy for swappable endogenous gene tagging in Caenorhabditis elegans

Endogenous gene tagging allows researchers to study genes in their native genomic context, underscoring the need for efficient gene tagging approaches. Here, we report TAG-IN, a streamlined strategy for swappable gene tagging in C. elegans. TAG-IN begins with generating a seed allele for a gene of interest by inserting a small landing pad containing two distinct recombination sites (FRT and FRT3) into the desired tagging locus using CRISPR. Fusion or co-expression tags from reusable tag donors can then be easily swapped into the seed allele using Flp recombinase-mediated cassette exchange (RMCE). Successful TAG-IN alleles can be conveniently identified via drug selection without PCR screening. The tag-swapping step can be accomplished by simple genetic crossing, in addition to traditional microinjection. To facilitate the use of TAG-IN, we also built a library of plug-and-play tag donor strains that can be crossed with seed alleles to insert the corresponding tags and a set of vectors to construct new tag donors. The TAG-IN strategy is both modular and combinatorial: only a single seed allele for each tagging locus is needed to insert any tag; a single tag donor strain can be used to swap the tag into any compatible seed allele. Furthermore, TAG-IN allows iterative tag insertion because the FRT and FRT3 sites are preserved during RMCE, so each TAG-IN allele can serve as a landing pad for further rounds of tag exchange. Thus, TAG-IN provides an efficient, flexible, and scalable platform for endogenous gene tagging in C. elegans, accelerating comprehensive functional studies.