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Updated: Jun 28, 2025

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9
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Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies.

Mathieu C Husser1, Nhat P Pham1, Chris Law1,2

  • 1Biology Department, Concordia University, Montreal, Canada.

Elife
|April 23, 2024
PubMed
Summary
This summary is machine-generated.

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Researchers developed a new method for efficiently tagging proteins in human stem cells using a split fluorescent protein. This breakthrough enables better visualization of protein function in healthy cells and various cell types.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biotechnology

Background:

  • Endogenous tags are crucial for visualizing native proteins in live cells.
  • Generating endogenous tags in human cell lines is challenging due to low homology-directed repair efficiency.
  • Previous work demonstrated split fluorescent proteins for large-scale tagging in HEK293 cells.

Purpose of the Study:

  • To establish a large-scale endogenous tag library in human induced pluripotent stem cells (iPSCs) using split mNeonGreen.
  • To enable the study of protein function in healthy iPSCs and their differentiated progeny.
  • To overcome the limitations of homology-directed repair in human stem cell engineering.

Main Methods:

  • Engineered a human iPSC line to express the large fragment of split mNeonGreen (mNG21-10).
Keywords:
cell biologycrisprcytokinesisendogenous tagginggene editinghumanipsclive imaging

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  • Utilized the short fragment (mNG211) for fast and efficient endogenous protein tagging.
  • Employed neural network-based image restoration for live imaging of cellular processes.

    • Main Results:

    • Demonstrated successful and efficient endogenous tagging of proteins in iPSCs.
    • Showcased the ability to perform live imaging of dynamic cellular processes like cytokinesis.
    • Established a foundational iPSC line for future genome-wide tagging efforts.

    Conclusions:

    • This study presents the first genome-wide endogenous tag library in human stem cells.
    • The developed split mNeonGreen system provides a powerful tool for protein studies in iPSCs.
    • This approach facilitates the investigation of protein dynamics in healthy human cells and across differentiation pathways.