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Updated: Jan 27, 2026

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CRISPR/Cas9-Mediated Gene Tagging: A Step-by-Step Protocol.

Xi Xiang1,2, Conghui Li3, Xi Chen4

  • 1BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, China. xiangxi@genomics.cn.

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|March 27, 2019
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Summary
This summary is machine-generated.

CRISPR/Cas9 gene editing successfully tagged the porcine GAPDH gene with GFP. This allows for easier tracking of protein location and expression, overcoming technical challenges in creating tagged cell lines.

Keywords:
CRISPR/Cas9GAPDHGene taggingHomologous recombinationReporter genecopGFP

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR/Cas9 is a versatile tool for DNA modification.
  • Gene tagging with markers like GFP aids in studying protein localization and dynamics.
  • Existing methods like immunostaining have limitations.

Purpose of the Study:

  • To demonstrate the generation of a GFP-tagged porcine GAPDH gene using CRISPR/Cas9.
  • To overcome technical challenges in creating gene-tagged cell lines.
  • To enable real-time monitoring of pGAPDH expression and localization.

Main Methods:

  • Utilized CRISPR/Cas9 gene editing technology.
  • Employed homology-directed repair for precise gene insertion.
  • Targeted the porcine GAPDH (pGAPDH) gene for tagging with Green Fluorescent Protein (GFP).

Main Results:

  • Successfully generated a porcine cell line with the pGAPDH gene tagged by GFP.
  • Demonstrated the feasibility of using CRISPR/Cas9 for endogenous gene tagging.
  • Established a method for visualizing pGAPDH in real-time within the cell.

Conclusions:

  • CRISPR/Cas9-based homology-directed repair is an effective method for generating fluorescence-tagged genes.
  • This technique facilitates the study of protein dynamics and subcellular localization.
  • The developed method provides a valuable tool for molecular and cell biology research.