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A functional genetic toolbox for human tissue-derived organoids
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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8553336/ https://www.ncbi.nlm.nih.gov/pubmed/34612202 http://dx.doi.org/10.7554/eLife.67886 |
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author | Sun, Dawei Evans, Lewis Perrone, Francesca Sokleva, Vanesa Lim, Kyungtae Rezakhani, Saba Lutolf, Matthias Zilbauer, Matthias Rawlins, Emma L |
author_facet | Sun, Dawei Evans, Lewis Perrone, Francesca Sokleva, Vanesa Lim, Kyungtae Rezakhani, Saba Lutolf, Matthias Zilbauer, Matthias Rawlins, Emma L |
author_sort | Sun, Dawei |
collection | PubMed |
description | 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 knockdown 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 ongoing descriptive studies, such as the Human Cell Atlas Project. |
format | Online Article Text |
id | pubmed-8553336 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-85533362021-10-29 A functional genetic toolbox for human tissue-derived organoids Sun, Dawei Evans, Lewis Perrone, Francesca Sokleva, Vanesa Lim, Kyungtae Rezakhani, Saba Lutolf, Matthias Zilbauer, Matthias Rawlins, Emma L eLife Developmental Biology 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 knockdown 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 ongoing descriptive studies, such as the Human Cell Atlas Project. eLife Sciences Publications, Ltd 2021-10-06 /pmc/articles/PMC8553336/ /pubmed/34612202 http://dx.doi.org/10.7554/eLife.67886 Text en © 2021, Sun et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited. |
spellingShingle | Developmental Biology Sun, Dawei Evans, Lewis Perrone, Francesca Sokleva, Vanesa Lim, Kyungtae Rezakhani, Saba Lutolf, Matthias Zilbauer, Matthias Rawlins, Emma L A functional genetic toolbox for human tissue-derived organoids |
title | A functional genetic toolbox for human tissue-derived organoids |
title_full | A functional genetic toolbox for human tissue-derived organoids |
title_fullStr | A functional genetic toolbox for human tissue-derived organoids |
title_full_unstemmed | A functional genetic toolbox for human tissue-derived organoids |
title_short | A functional genetic toolbox for human tissue-derived organoids |
title_sort | functional genetic toolbox for human tissue-derived organoids |
topic | Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8553336/ https://www.ncbi.nlm.nih.gov/pubmed/34612202 http://dx.doi.org/10.7554/eLife.67886 |
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