A functional genetic toolbox for human tissue-derived organoids.
CRISPR-Cas9
CRISPRa
developmental biology
homologous gene targeting
human
inducible CRISPRi
lung
organoids
Journal
eLife
ISSN: 2050-084X
Titre abrégé: Elife
Pays: England
ID NLM: 101579614
Informations de publication
Date de publication:
06 10 2021
06 10 2021
Historique:
received:
03
03
2021
accepted:
05
10
2021
pubmed:
7
10
2021
medline:
15
12
2021
entrez:
6
10
2021
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 34612202
doi: 10.7554/eLife.67886
pii: 67886
pmc: PMC8553336
doi:
pii:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : Wellcome Trust
ID : 102175/B/13/Z
Pays : United Kingdom
Organisme : Cancer Research UK
ID : C6946/A24843
Pays : United Kingdom
Organisme : Wellcome Trust
ID : 109146/Z/15/Z
Pays : United Kingdom
Organisme : Wellcome Trust
ID : PhD studentship 102175/B/13/Z
Pays : United Kingdom
Organisme : Medical Research Council
ID : MR/P009581/1
Pays : United Kingdom
Organisme : Wellcome Trust
Pays : United Kingdom
Organisme : Medical Research Council
ID : MR/T001917/1
Pays : United Kingdom
Organisme : Wellcome Trust
ID : 203144/Z/16/Z
Pays : United Kingdom
Informations de copyright
© 2021, Sun et al.
Déclaration de conflit d'intérêts
DS, LE, FP, VS, KL, SR, ML, MZ, ER No competing interests declared
Références
Proc Natl Acad Sci U S A. 2009 Aug 4;106(31):12771-5
pubmed: 19625615
Dev Cell. 2018 May 21;45(4):526-536.e5
pubmed: 29787711
Nat Commun. 2016 Jan 28;7:10548
pubmed: 26817820
Cell Stem Cell. 2015 Feb 5;16(2):142-7
pubmed: 25658371
Nat Protoc. 2017 Nov;12(11):2263-2274
pubmed: 28981121
Genome Res. 2014 Jun;24(6):1012-9
pubmed: 24696461
Cell. 2013 Jul 18;154(2):442-51
pubmed: 23849981
Elife. 2014 Dec 15;3:e04766
pubmed: 25497837
Nature. 2016 Nov 24;539(7630):560-564
pubmed: 27851739
Nat Cell Biol. 2020 Mar;22(3):321-331
pubmed: 32123335
Nature. 2016 May 5;533(7601):125-9
pubmed: 27120160
Chem Biol. 2010 Sep 24;17(9):981-8
pubmed: 20851347
Cell Stem Cell. 2016 Apr 7;18(4):541-53
pubmed: 26971820
Nature. 2018 Apr 5;556(7699):57-63
pubmed: 29512652
Elife. 2020 May 22;9:
pubmed: 32441252
Nat Methods. 2019 Nov;16(11):1087-1093
pubmed: 31659326
Dev Cell. 2020 Dec 21;55(6):771-783.e5
pubmed: 33290721
Methods Mol Biol. 2019;1961:153-183
pubmed: 30912046
Mol Biol Cell. 2017 Oct 15;28(21):2854-2874
pubmed: 28814507
Elife. 2016 Sep 23;5:
pubmed: 27661255
Cell. 2021 Apr 29;184(9):2503-2519.e17
pubmed: 33838111
Neuron. 2019 Oct 23;104(2):239-255.e12
pubmed: 31422865
Elife. 2017 Jun 30;6:
pubmed: 28665271
STAR Protoc. 2021 Jun 12;2(2):100597
pubmed: 34169291
Stem Cell Reports. 2019 May 14;12(5):1145-1158
pubmed: 30956114
Nat Biotechnol. 2015 May;33(5):538-42
pubmed: 25798939
Dis Model Mech. 2009 Sep-Oct;2(9-10):508-15
pubmed: 19692579
Nat Commun. 2019 Sep 6;10(1):4056
pubmed: 31492834
J Clin Invest. 2013 Jul;123(7):3025-36
pubmed: 23921127
Stem Cells. 2008 Feb;26(2):496-504
pubmed: 18024421