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Related Experiment Video

Updated: Sep 26, 2025

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9
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Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening.

Kinga Vojnits1, Mio Nakanishi1, Deanna Porras1

  • 1Department of Biochemistry and Biomedical Sciences, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON L8N 3Z5, Canada.

Molecules (Basel, Switzerland)
|April 23, 2022
PubMed
Summary

CRISPR/Cas9 gene editing in human pluripotent stem cells (hPSCs) for reporter lines is promising for disease modeling. Targeting the AAVS1 locus provides stable EGFP expression for high-content screening, unlike the OCT4 locus.

Keywords:
AAVS1 locusCRISPR/Cas9EGFP reporter linesOCT4 locusgenome editinghuman pluripotent stem cellsphenotypic screening

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Area of Science:

  • Stem cell biology
  • Genome editing
  • Biotechnology

Background:

  • CRISPR/Cas9 enables fluorescent protein knock-in into human pluripotent stem cells (hPSCs) for disease modeling and drug screening.
  • Evaluating fluorescent reporter hPSC lines is crucial for high-content screening (HCS) applications.

Purpose of the Study:

  • To assess the suitability of EGFP knock-in at the OCT4 and AAVS1 loci in hPSCs for HCS.
  • To determine the stability and consistency of reporter gene expression for long-term screening platforms.

Main Methods:

  • CRISPR/Cas9 mediated EGFP knock-in into the OCT4 and AAVS1 loci of hPSCs.
  • Assessment of EGFP expression, pluripotency markers, and differentiation potential.
  • Evaluation of reporter lines in multi-well formats for HCS.

Main Results:

  • hPSC-OCT4-EGFP lines showed EGFP expression linked to pluripotency but lost expression during long-term culture.
  • hPSC-AAVS1-EGFP lines exhibited stable and consistent EGFP expression throughout in vitro differentiation.
  • The AAVS1 locus proved to be a safe and effective site for stable reporter gene integration without compromising hPSC quality.

Conclusions:

  • The AAVS1 locus is a superior genomic site for generating stable reporter hPSC lines for long-term HCS applications using CRISPR/Cas9.
  • Careful selection of reporter cell lines and genomic loci is essential for successful HCS and drug discovery.
  • CRISPR/Cas9-mediated knock-in at the AAVS1 locus offers a robust platform for high-throughput screening and disease modeling.