A Single-Copy Knock-In System: One Plasmid to Target All Chromosomes in C. elegans

Erica Dinneen; Purbasha Dasgupta; Avinash Sharma; Khairun Nisaa; Carlos G Silva-García

doi:10.1093/g3journal/jkaf220

A Single-Copy Knock-In System: One Plasmid to Target All Chromosomes in C. elegans

G3 (Bethesda). 2025 Sep 19:jkaf220. doi: 10.1093/g3journal/jkaf220. Online ahead of print.

Authors

Erica Dinneen^{1

2}, Purbasha Dasgupta^{1

2}, Avinash Sharma^{1

2}, Khairun Nisaa^{1

2}, Carlos G Silva-García^{1

2}

Affiliations

¹ Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02903, U.S.A.
² Center on the Biology of Aging, Brown University, Providence, RI 02903, U.S.A.

PMID: 40973646
DOI: 10.1093/g3journal/jkaf220

Abstract

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 one 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 two 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 one plasmid, and streamline tracking using standard primers.

Keywords: C. elegans methods; CRISPR/Cas9; genome engineering; single-copy knock-in; transgenesis.