A functional genetic toolbox for human tissue-derived organoids
- University of Cambridge, United Kingdom
- University College London, United Kingdom
- 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
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.
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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A functional genetic toolbox for human tissue-derived organoids
eLife 10:e67886.
https://doi.org/10.7554/eLife.67886
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