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...

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Autores principales:	Dawei Sun, Lewis Evans, Francesca Perrone, Vanesa Sokleva, Kyungtae Lim, Saba Rezakhani, Matthias Lutolf, Matthias Zilbauer, Emma L Rawlins
Formato:	article
Lenguaje:	EN
Publicado:	eLife Sciences Publications Ltd 2021
Materias:	organoids lung CRISPR-Cas9 homologous gene targeting inducible CRISPRi CRISPRa Medicine R Science Q Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/94a700ff207d48a3bc0ed257b016de58
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spelling	oai:doaj.org-article:94a700ff207d48a3bc0ed257b016de582021-11-15T06:06:55ZA functional genetic toolbox for human tissue-derived organoids10.7554/eLife.678862050-084Xe67886https://doaj.org/article/94a700ff207d48a3bc0ed257b016de582021-10-01T00:00:00Zhttps://elifesciences.org/articles/67886https://doaj.org/toc/2050-084XHuman 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.Dawei SunLewis EvansFrancesca PerroneVanesa SoklevaKyungtae LimSaba RezakhaniMatthias LutolfMatthias ZilbauerEmma L RawlinseLife Sciences Publications LtdarticleorganoidslungCRISPR-Cas9homologous gene targetinginducible CRISPRiCRISPRaMedicineRScienceQBiology (General)QH301-705.5ENeLife, Vol 10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	organoids lung CRISPR-Cas9 homologous gene targeting inducible CRISPRi CRISPRa Medicine R Science Q Biology (General) QH301-705.5
spellingShingle	organoids lung CRISPR-Cas9 homologous gene targeting inducible CRISPRi CRISPRa Medicine R Science Q Biology (General) QH301-705.5 Dawei Sun Lewis Evans Francesca Perrone Vanesa Sokleva Kyungtae Lim Saba Rezakhani Matthias Lutolf Matthias Zilbauer Emma L Rawlins A functional genetic toolbox for human tissue-derived organoids
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	article
author	Dawei Sun Lewis Evans Francesca Perrone Vanesa Sokleva Kyungtae Lim Saba Rezakhani Matthias Lutolf Matthias Zilbauer Emma L Rawlins
author_facet	Dawei Sun Lewis Evans Francesca Perrone Vanesa Sokleva Kyungtae Lim Saba Rezakhani Matthias Lutolf Matthias Zilbauer Emma L Rawlins
author_sort	Dawei Sun
title	A functional genetic toolbox for human tissue-derived organoids
title_short	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_sort	functional genetic toolbox for human tissue-derived organoids
publisher	eLife Sciences Publications Ltd
publishDate	2021
url	https://doaj.org/article/94a700ff207d48a3bc0ed257b016de58
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A functional genetic toolbox for human tissue-derived organoids

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