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

Dinneen, E.; Silva-Garcia, C. G.

doi:10.1101/2024.12.06.627295

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bioRxiv · 10.1101/2024.12.06.627295

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

Dinneen, E.·Silva-Garcia, C. G.

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Abstract

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.

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Dinneen, E., Silva-Garcia, C. G.. 2024-12-10. Universal Single Copy Knock-In System in Caenorhabditis elegans: One Plasmid to Target All Chromosomes. https://doi.org/10.1101/2024.12.06.627295

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