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Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing
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Introducing Point Mutations into Human Pluripotent Stem Cells using Seamless Genome Editing.

Yu Wang1, Andrew J H Smith1, David C Hay2

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Journal of Visualized Experiments : Jove
|May 26, 2020
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Summary

This study details efficient genome editing of the HNF4α locus in human stem cells using CRISPR-Cas9 technology. A two-step selection process with a piggyBac donor plasmid ensured accurate targeting of the HNF4α gene.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Stem Cell Research

Background:

  • CRISPR-Cas9 technology offers precise genome editing capabilities in mammalian cells.
  • Targeting specific genes in human pluripotent stem cells is crucial for disease modeling and regenerative medicine.

Purpose of the Study:

  • To establish a robust protocol for seamless genome editing of the hepatocyte nuclear factor 4 alpha (HNF4α) locus in human pluripotent stem cells.
  • To demonstrate the efficiency and accuracy of the developed genome editing strategy.

Main Methods:

  • Utilized RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nickase mutant for targeted DNA modification.
  • Employed a piggyBac-based donor plasmid for introducing desired genetic modifications.
  • Implemented a two-step genetic selection strategy to isolate correctly edited cells.

Main Results:

  • Successfully achieved seamless genome editing at the HNF4α locus in human pluripotent stem cells.
  • Demonstrated high efficiency and accuracy in targeting the HNF4α locus.
  • Validated the effectiveness of the combined CRISPR-Cas9 nickase and piggyBac donor system with genetic selection.

Conclusions:

  • The described procedure provides a reliable method for precise genome editing of the HNF4α locus in human pluripotent stem cells.
  • This approach facilitates further research into HNF4α function and its role in cellular processes.
  • The study highlights the potential of CRISPR-Cas9 based strategies for targeted gene modification in stem cell applications.