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

Area of Science:

• Genetics
• Molecular Biology
• Developmental Biology

Background:

• Transgenesis in model organisms is crucial for understanding gene function and regulation.
• CRISPR/Cas9 technology has revolutionized genome engineering.
• Efficient methods for single-copy transgene insertion are needed in *C. elegans*.

Purpose of the Study:

• To introduce a universal system for inserting any transgene into the *Caenorhabditis elegans* genome using CRISPR/Cas9.
• To facilitate the insertion of transgenes at specific safe harbor loci on autosomal chromosomes.
• To provide a flexible and streamlined method for single-copy transgene expression.

Main Methods:

• Development of the Universal SKI System (U-SKI) utilizing a pSKI plasmid.
• Employing CRISPR/Cas9 for targeted transgene insertion.
• Using synthetic homology arms for recombination with SKI cassettes and a single crRNA guide as a Co-CRISPR enrichment marker.

Main Results:

• The U-SKI system enables transgene insertion at specific safe harbor loci on each autosomal chromosome.
• The pSKI plasmid facilitates easy cloning and acts as a CRISPR/Cas9-based insertion repair template.
• The system allows for selection of insertion sites and streamlined tracking of single-copy transgenes.

Conclusions:

• The Universal SKI System offers a highly flexible approach for expressing single-copy transgenes in *C. elegans*.
• This system reduces the complexity associated with transgene integration and tracking.
• U-SKI provides a valuable tool for genetic research in *C. elegans*.