CRISPR/Cas9-mediated Endogenous Fluorescent Tagging of Germline-specific Genes in Caenorhabditis elegans.

Protein localization in the Caenorhabditis elegans (C. elegans) germline is central to interpreting gene function during gametogenesis, yet conventional transgene approaches often yield variable expression and can be silenced in germ cells. Here, a practical CRISPR/Cas9 workflow inserting a fluorescent tag into an endogenous locus is described, enabling the generation of stable knock-in alleles that report protein distribution under native regulation. The protocol covers key stages of the procedure: selecting a tagging strategy appropriate for the target protein, delivering CRISPR reagents by gonadal microinjection into young adult hermaphrodites, and recovering injected animals for screening. Knock-in candidates are identified through PCR-based genotyping across two generations to isolate homozygous worms and verify the edited allele. Finally, confocal microscopy is used to verify germline fluorescence and assess subcellular localization in vivo. The workflow is designed to be reproducible and broadly applicable to germline-enriched genes, providing a straightforward route to establish homozygous tagged strains for developmental and cell-biological analyses.