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Updated: Oct 17, 2025

Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids
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Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids

Published on: February 18, 2020

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A functional genetic toolbox for human tissue-derived organoids.

Dawei Sun1, Lewis Evans2, Francesca Perrone3

  • 1Wellcome Trust/CRUK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom.

Elife
|October 6, 2021
PubMed
Summary

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Researchers developed a genetic toolbox for human organoid systems, enabling efficient gene editing and manipulation. This innovation significantly advances the study of gene function in organoids for disease modeling.

Area of Science:

  • Developmental Biology
  • Stem Cell Biology
  • Genetic Engineering

Background:

  • Human organoid systems model organ development and disease, but lack efficient tools for studying endogenous gene function.
  • Existing methods struggle with genetic manipulation in tissue-derived organoids, limiting research into gene function.
  • Previous work established a human fetal lung organoid system (Nikolić et al., 2017).

Purpose of the Study:

  • To systematically develop and optimize a comprehensive genetic toolbox for tissue-derived human organoids.
  • To enable efficient gene targeting, knockdown, and overexpression in organoid systems.
  • To facilitate gene perturbation studies for improved human disease modeling.

Main Methods:

  • Developed 'Organoid Easytag', a workflow for CRISPR-mediated homologous recombination and flow cytometry enrichment.
Keywords:
CRISPR-Cas9CRISPRadevelopmental biologyhomologous gene targetinghumaninducible CRISPRilungorganoids

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  • Implemented tightly inducible CRISPR interference (CRISPRi) for conditional gene knockdown.
  • Implemented tightly inducible CRISPR activation (CRISPRa) for conditional gene overexpression.

    • Main Results:

    • Successfully established a complete genetic toolbox for tissue-derived organoids.
    • Demonstrated efficient gene locus targeting using the 'Organoid Easytag' workflow.
    • Achieved the first efficient application of inducible CRISPRi and CRISPRa in tissue-derived organoids.

    Conclusions:

    • The developed genetic toolbox significantly enhances the utility of tissue-derived organoids for functional genomics.
    • These tools will accelerate gene perturbation studies, advancing human disease modeling.
    • This work provides a functional counterpart to large-scale descriptive projects like the Human Cell Atlas Project.