A functional genetic toolbox for human tissue-derived organoids

  1. Dawei Sun
  2. Lewis Evans
  3. Francesca Perrone
  4. Vanesa Sokleva
  5. Kyungtae Lim
  6. Saba Rezakhani
  7. Matthias Lutolf
  8. Matthias Zilbauer
  9. Emma L Rawlins  Is a corresponding author
  1. University of Cambridge, United Kingdom
  2. University College London, United Kingdom
  3. École Polytechnique Fédérale de Lausanne (EPFL), Switzerland

Abstract

Human organoid systems recapitulate key features of organs offering platforms for modelling developmental biology and disease. Tissue-derived organoids have been widely used to study the impact of extrinsic niche factors on stem cells. However, they are rarely used to study endogenous gene function due to the lack of efficient gene manipulation tools. Previously, we established a human foetal lung organoid system (Nikolić et al., 2017). Here, using this organoid system as an example we have systematically developed and optimised a complete genetic toolbox for use in tissue-derived organoids. This includes 'Organoid Easytag' our efficient workflow for targeting all types of gene loci through CRISPR-mediated homologous recombination followed by flow cytometry for enriching correctly-targeted cells. Our toolbox also incorporates conditional gene knock-down or overexpression using tightly-inducible CRISPR interference and CRISPR activation which is the first efficient application of these techniques to tissue-derived organoids. These tools will facilitate gene perturbation studies in tissue-derived organoids facilitating human disease modelling and providing a functional counterpart to many on-going descriptive studies, such as the Human Cell Atlas Project.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Dawei Sun

    Wellcome Trust/CRUK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Lewis Evans

    Developmental Biology and Cancer Development, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7279-7651
  3. Francesca Perrone

    Wellcome Trust/CRUK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Vanesa Sokleva

    Wellcome Trust/CRUK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Kyungtae Lim

    Wellcome Trust/CRUK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Saba Rezakhani

    École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  7. Matthias Lutolf

    École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  8. Matthias Zilbauer

    Wellcome Trust/CRUK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Emma L Rawlins

    Wellcome Trust/CRUK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom
    For correspondence
    elr21@cam.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7426-3792

Funding

Medical Research Council (MR/P009581/1)

  • Emma L Rawlins

Wellcome Trust PhD Studentship (109146/Z/15/Z)

  • Dawei Sun

Alzheimers Research UK Stem Cell Research Centre

  • Lewis Evans

National Research Foundation of Korea (2018R1A6A3A03012122)

  • Kyungtae Lim

Wellcome Trust Core Support for Gurdon Institute (203144/Z/16/Z)

  • Emma L Rawlins

Cancer Research UK Core Support for Gurdon Institute (C6946/A24843)

  • Emma L Rawlins

Medical Research Council New Investigator Research Grant (MR/T001917/1)

  • Matthias Zilbauer

Wellcome Trust PhD studentship (102175/B/13/Z)

  • Vanesa Sokleva

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Marianne E Bronner, California Institute of Technology, United States

Publication history

  1. Preprint posted: May 5, 2020 (view preprint)
  2. Received: March 3, 2021
  3. Accepted: October 5, 2021
  4. Accepted Manuscript published: October 6, 2021 (version 1)
  5. Version of Record published: October 28, 2021 (version 2)

Copyright

© 2021, Sun et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Dawei Sun
  2. Lewis Evans
  3. Francesca Perrone
  4. Vanesa Sokleva
  5. Kyungtae Lim
  6. Saba Rezakhani
  7. Matthias Lutolf
  8. Matthias Zilbauer
  9. Emma L Rawlins
(2021)
A functional genetic toolbox for human tissue-derived organoids
eLife 10:e67886.
https://doi.org/10.7554/eLife.67886

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