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dc.contributor.authorSun, Dawei
dc.contributor.authorEvans, Lewis
dc.contributor.authorPerrone, Francesca
dc.contributor.authorSokleva, Vanesa
dc.contributor.authorLim, Kyungtae
dc.contributor.authorRezakhani, Saba
dc.contributor.authorLutolf, Matthias
dc.contributor.authorZilbauer, Matthias
dc.contributor.authorRawlins, Emma L
dc.date.accessioned2021-10-29T02:13:16Z
dc.date.available2021-10-29T02:13:16Z
dc.date.issued2021-10-06
dc.date.submitted2021-03-03
dc.identifier.other67886
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/330025
dc.descriptionFunder: Alzheimers Research UK Stem Cell Research Centre
dc.description.abstractHuman 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.
dc.languageen
dc.publishereLife Sciences Publications, Ltd
dc.subjectResearch Advance
dc.subjectDevelopmental Biology
dc.subjectorganoids
dc.subjectlung
dc.subjectCRISPR-Cas9
dc.subjecthomologous gene targeting
dc.subjectinducible CRISPRi
dc.subjectCRISPRa
dc.subjectHuman
dc.titleA functional genetic toolbox for human tissue-derived organoids
dc.typeArticle
dc.date.updated2021-10-29T02:13:13Z
prism.publicationNameeLife
prism.volume10
dc.identifier.doi10.17863/CAM.77469
dcterms.dateAccepted2021-10-05
rioxxterms.versionofrecord10.7554/elife.67886
rioxxterms.versionVoR
rioxxterms.licenseref.urihttp://creativecommons.org/licenses/by/4.0/
datacite.contributor.supervisoreditor: Bronner, Marianne E
datacite.contributor.supervisorsenior_editor: Bronner, Marianne E
dc.contributor.orcidSun, Dawei [0000-0003-1551-4349]
dc.contributor.orcidEvans, Lewis [0000-0001-7279-7651]
dc.contributor.orcidLim, Kyungtae [0000-0001-6044-2191]
dc.contributor.orcidRawlins, Emma L [0000-0001-7426-3792]
dc.identifier.eissn2050-084X
pubs.funder-project-idMedical Research Council (MR/P009581/1)
pubs.funder-project-idWellcome Trust (PhD Studentship 109146/Z/15/Z)
pubs.funder-project-idNational Research Foundation of Korea (2018R1A6A3A03012122)
pubs.funder-project-idWellcome Trust (Core Support for Gurdon Institute 203144/Z/16/Z)
pubs.funder-project-idCancer Research UK (Core Support for Gurdon Institute C6946/A24843)
pubs.funder-project-idMedical Research Council (New Investigator Research Grant MR/T001917/1)
pubs.funder-project-idWellcome Trust (PhD studentship 102175/B/13/Z)


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