Modular gene tagging in C. elegans

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• Endogenous gene tagging is crucial for studying protein expression and localization.
• CRISPR technology, while widely used, presents challenges due to errors and reagent specificity.
• Recombinase systems offer a potential solution for error-free and modular DNA insertion.

Purpose of the Study:

• To evaluate germline recombinase function in C. elegans.
• To develop and introduce PhIT, a novel recombinase-based protein tagging system.
• To establish a versatile platform for modular gene and protein tagging.

Main Methods:

• Screened eight recombinases for germline activity in C. elegans.
• Utilized CRISPR to insert a PhiC31 attB landing pad into the target gene locus.
• Employed PhiC31 integrase for tag insertion and a tyrosine recombinase for backbone removal.

Main Results:

• Demonstrated the efficacy of PhIT for error-free, modular protein tagging in C. elegans.
• Developed a resource strain for versatile tag insertion via CRISPR.
• Successfully implemented various modular tags, including fluorescent proteins, FLP-regulated constructs, and degron tags.

Conclusions:

• PhIT provides a robust and modular alternative to CRISPR for endogenous gene tagging.
• The system facilitates tag insertion through genetic crosses, simplifying the experimental workflow.
• PhIT enhances the study of gene expression and protein localization in C. elegans.