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Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9
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qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging.

Reuben Philip1,2, Amit Sharma1, Laura Matellan1

  • 1Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.

The EMBO Journal
|December 12, 2024
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Summary

The qTAG system simplifies endogenous protein tagging using CRISPR, offering accessible repair cassettes for various applications like live-cell imaging and protein degradation. This open resource enhances native protein studies.

Keywords:
CRISPREndogenous TaggingGene-editing

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biotechnology

Background:

  • Endogenous tagging allows protein study in native cellular environments.
  • CRISPR technology is commonly used for inserting tag sequences into genes.
  • Existing methods can be complex and less accessible for broad research application.

Purpose of the Study:

  • To introduce qTAG, a novel collection of repair cassettes designed to improve the accessibility of endogenous tagging.
  • To provide researchers with a versatile system for various protein analysis applications.
  • To develop and share an open resource for customized endogenous tagging experiments.

Main Methods:

  • Development of repair cassettes (qTAG) for CRISPR-mediated gene editing.
  • Inclusion of N- and C-terminal tagging options with selectable markers and restriction sites.
  • Integration of Lox sites for marker gene removal post-integration.
  • Demonstration using diverse tags for fluorescence imaging, proximity labeling, epitope tagging, and protein degradation.

Main Results:

  • qTAG cassettes facilitate accessible endogenous tagging with selectable markers and customizable features.
  • Demonstrated utility across multiple applications including live-cell imaging with novel tags like mStayGold.
  • Exploration of alternative designs for conditional expression, knockout, and safe-harbor tagging.
  • Availability of a plasmid collection through Addgene for immediate research use.

Conclusions:

  • The qTAG system significantly enhances the accessibility and versatility of endogenous protein tagging.
  • It provides a valuable, adaptable open resource for researchers studying native protein dynamics and functions.
  • qTAG supports a wide range of applications, including advanced live-cell imaging and targeted protein manipulation.