Universal Single Copy Knock-In System in Caenorhabditis elegans : One Plasmid to Target All Chromosomes

Successful transgenesis in model organisms has dramatically helped us understand gene function, regulation, genetic networks, and potential applications. Here, we introduce the universal single-copy knock-in system (Universal SKI System or U-SKI), designed for inserting any transgene by CRISPR/Cas9 in the Caenorhabditis elegans genome. The Universal SKI System takes advantage of a plasmid (pSKI), which can also be used for extrachromosomal arrays, to facilitate the insertion of a transgene at specific safe harbor loci on each autosomal chromosome. The pSKI plasmid contains multiple restriction sites for easy cloning and serves as a CRISPR/Cas9-based insertion repair template because it has two synthetic and long homology arms that recombine with the SKI cassettes. This system also uses a single crRNA guide, which acts as a Co-CRISPR enrichment marker. Overall, the Universal SKI System is highly flexible; with the same Universal SKI cassette on each autosome, researchers can select the insertion site and streamline tracking while reducing the complexity of expressing single-copy transgenes in C. elegans .