A single-copy knock-in system: one plasmid to target all chromosomes in <i>C. elegans</i>

Successful transgenesis in model organisms has significantly helped us understand gene function, regulation, genetic networks, and potential applications. Here, we introduce a single-copy knock-in system that uses 1 plasmid to target all chromosomes in Caenorhabditis elegans (SKI PLACE), designed for inserting a transgene by CRISPR/Cas9. The SKI PLACE system uses the pSKI plasmid to insert a desired transgene at specific harbor loci on each chromosome. The pSKI plasmid contains multiple restriction sites for cloning and serves as a CRISPR/Cas9-based insertion repair template because it has 2 synthetic and long homology arms that recombine with the SKI PLACE cassettes. This system also uses a single crRNA guide, which acts as a Co-CRISPR enrichment marker. Overall, the SKI PLACE system is flexible; with the same SKI PLACE cassette on each chromosome, researchers can select the insertion site, work with 1 plasmid, and streamline tracking using standard primers.