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Modular gene tagging in C. elegans.

Adam Hefel1, Kevin Kruse1, Kaden Wall1

  • 1School of Biological Sciences, University of Utah, Salt Lake City, Utah, USA; Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah, USA.

Biorxiv : the Preprint Server for Biology
|February 6, 2026
PubMed
Summary
This summary is machine-generated.

We introduce PhIT, a recombinase-based method for precise protein tagging in C. elegans. This system overcomes CRISPR limitations, enabling modular and error-free gene tagging for improved protein localization and expression studies.

Keywords:
B3 recombinaseC. elegans genome engineeringDrePhITPhiC31gene tagging

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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.