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Related Experiment Video

Updated: Mar 5, 2026

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation
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Genome Editing in Human Pluripotent Stem Cells.

Jared Carlson-Stevermer1,2, Krishanu Saha3,4

  • 1Department of Biomedical Engineering, University of Wisconsin-Madison, 1550 Engineering Drive, Madison, WI, 53706, USA.

Methods in Molecular Biology (Clifton, N.J.)
|March 30, 2017
PubMed
Summary

We detail a method for genetically modifying human pluripotent stem cells (hPSCs) using transient nuclease expression. This process enables the creation of custom reporter and knockout cell lines for research.

Keywords:
CRISPR/Cas9ElectroporationGenome editingGenomics

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Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

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Area of Science:

  • Stem cell biology
  • Molecular biology
  • Genetics

Background:

  • Human pluripotent stem cells (hPSCs) are crucial for disease modeling and regenerative medicine.
  • Efficient genome editing is essential for creating precise cellular models and therapeutic applications.
  • Existing genome editing tools like zinc finger nucleases, TALENs, and CRISPR/Cas9 offer high efficiency but require optimized delivery methods.

Purpose of the Study:

  • To describe a robust protocol for preparing hPSCs for transient nuclease expression.
  • To outline the subsequent analytical steps for confirming successful genetic modification.
  • To facilitate the generation of genetically engineered hPSC reporter and knockout lines.

Main Methods:

  • Preparation of hPSCs for electroporation.
  • Transient expression of genome editing nucleases (e.g., CRISPR/Cas9) via electroporation.
  • Post-electroporation analysis for assessing gene editing efficiency and cell viability.

Main Results:

  • Successful preparation and electroporation of hPSCs with nuclease-encoding plasmids.
  • Demonstration of transient nuclease expression within hPSCs.
  • Establishment of a workflow for generating genetically modified hPSC lines.

Conclusions:

  • Transient nuclease expression via electroporation is an effective method for genome editing in hPSCs.
  • This protocol supports the creation of custom reporter and knockout hPSC lines.
  • The described methods advance the utility of hPSCs in biological research and therapeutic development.