Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Sun, Dawei, Evans, Lewis, Perrone, Francesca, Sokleva, Vanesa, Lim, Kyungtae, Rezakhani, Saba, Lutolf, Matthias, Zilbauer, Matthias, Rawlins, Emma L
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2021
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
_version_ 1784591558774882304
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
work_keys_str_mv AT sundawei afunctionalgenetictoolboxforhumantissuederivedorganoids
AT evanslewis afunctionalgenetictoolboxforhumantissuederivedorganoids
AT perronefrancesca afunctionalgenetictoolboxforhumantissuederivedorganoids
AT soklevavanesa afunctionalgenetictoolboxforhumantissuederivedorganoids
AT limkyungtae afunctionalgenetictoolboxforhumantissuederivedorganoids
AT rezakhanisaba afunctionalgenetictoolboxforhumantissuederivedorganoids
AT lutolfmatthias afunctionalgenetictoolboxforhumantissuederivedorganoids
AT zilbauermatthias afunctionalgenetictoolboxforhumantissuederivedorganoids
AT rawlinsemmal afunctionalgenetictoolboxforhumantissuederivedorganoids
AT sundawei functionalgenetictoolboxforhumantissuederivedorganoids
AT evanslewis functionalgenetictoolboxforhumantissuederivedorganoids
AT perronefrancesca functionalgenetictoolboxforhumantissuederivedorganoids
AT soklevavanesa functionalgenetictoolboxforhumantissuederivedorganoids
AT limkyungtae functionalgenetictoolboxforhumantissuederivedorganoids
AT rezakhanisaba functionalgenetictoolboxforhumantissuederivedorganoids
AT lutolfmatthias functionalgenetictoolboxforhumantissuederivedorganoids
AT zilbauermatthias functionalgenetictoolboxforhumantissuederivedorganoids
AT rawlinsemmal functionalgenetictoolboxforhumantissuederivedorganoids