Combination of CRISPR-Cas9-RNP and Single-Cell RNAseq to Identify Cell State-Specific FOXJ1 Functions in the Human Airway Epithelium.
Air-Liquid Interface cell culture
CRISPR-Cas9 RiboNucleoParticles
FOXJ1
Gene invalidation
Nanopore sequencing
Nasal airway epithelial cells
Single-cell RNA sequencing
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2024
2024
Historique:
medline:
31
10
2023
pubmed:
19
10
2023
entrez:
19
10
2023
Statut:
ppublish
Résumé
The study of the airway epithelium in vitro is routinely performed using air-liquid culture (ALI) models from nasal or bronchial basal cells. These 3D experimental models allow to follow the regeneration steps of fully differentiated mucociliary epithelium and to study gene function by performing gene invalidation. Recent progress made with CRISPR-based techniques has overcome the experimental difficulty of this approach, by a direct transfection of ribonucleoprotein complexes combining a mix of synthetic small guide RNAs (sgRNAs) and recombinant Cas9. The approach shows more than 95% efficiency and does not require any selection step. A limitation of this approach is that it generates cell populations that contain heterogeneous deletions, which makes the evaluation of invalidation efficiency difficult. We have successfully used Flongle sequencing (Nanopore) to quantify the number of distinct deletions. We describe the use of CRISPR-Cas9 RNP in combination with single-cell RNA sequencing to functionally characterize the impact of gene invalidation in ALI cultures. The complex ecosystem of the airway epithelium, composed of many cell types, makes single-cell approaches particularly relevant to study cell type, or cell state-specific events. This protocol describes the invalidation of FOXJ1 in ALI cultures through the following steps: (1) Establishment of basal cell cultures from nasal turbinates, (2) CRISPR-Cas9 RNP invalidation of FOXJ1, (3) Quantification of FOXJ1 invalidation efficiency by Nanopore sequencing, (4) Dissociation of ALI cultures and single-cell RNAseq, (5) Analysis of single-cell RNAseq data from FOXJ1-invalidated cells.We confirm here that FOXJ1 invalidation impairs the final differentiation step of multiciliated cells and provides a framework to explore other gene functions.
Identifiants
pubmed: 37856015
doi: 10.1007/978-1-0716-3507-0_1
doi:
Substances chimiques
RNA, Guide, CRISPR-Cas Systems
0
FOXJ1 protein, human
0
Forkhead Transcription Factors
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1-25Informations de copyright
© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Références
Kotton DN, Morrisey EE (2014) Lung regeneration: mechanisms, applications and emerging stem cell populations. Nat Med 20:822–832. https://doi.org/10.1038/nm.3642
doi: 10.1038/nm.3642
pubmed: 25100528
pmcid: 4229034
Deprez M, Zaragosi L-E, Truchi M et al (2020) A single-cell atlas of the human healthy airways. Am J Respir Crit Care Med 202:1636–1645. https://doi.org/10.1164/rccm.201911-2199OC
doi: 10.1164/rccm.201911-2199OC
pubmed: 32726565
Pezzulo AA, Starner TDT, Scheetz TET et al (2011) The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia. Am J Physiol Lung Cell Mol Physiol 300:L25–L31. https://doi.org/10.1152/ajplung.00256.2010
doi: 10.1152/ajplung.00256.2010
pubmed: 20971803
Gras D, Bourdin A, Vachier I et al (2012) An ex vivo model of severe asthma using reconstituted human bronchial epithelium. J allergy Clin Immunol 129:1259-1266.e1. https://doi.org/10.1016/j.jaci.2012.01.073
Ruiz Garcia S, Deprez M, Lebrigand K et al (2019) Novel dynamics of human mucociliary differentiation revealed by single-cell RNA sequencing of nasal epithelial cultures Development:177428. https://doi.org/10.1242/dev.177428
Recordon-pinson P, Esteves P, Faure M (2022) Bronchial epithelia from adults and children : SARS-CoV-2 spread via syncytia formation and type III interferon infectivity restriction. 1–12. https://doi.org/10.1073/pnas.2202370119/-/DCSupplemental.Published
Mulay A, Konda B, Garcia G et al (2021) SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery. Cell Rep 35:109055. https://doi.org/10.1016/j.celrep.2021.109055
doi: 10.1016/j.celrep.2021.109055
pubmed: 33905739
pmcid: 8043574
Everman JL, Rios C, Seibold MA (2018) Primary airway epithelial cell gene editing using CRISPR-Cas9. Methods Mol Biol 1706:267–292. https://doi.org/10.1007/978-1-4939-7471-9_15
doi: 10.1007/978-1-4939-7471-9_15
pubmed: 29423804
Koh KD, Siddiqui S, Cheng D et al (2020) Efficient RNP-directed human gene targeting reveals SPDEF is required for IL-13-induced mucostasis. Am J Respir Cell Mol Biol 62:373–381. https://doi.org/10.1165/rcmb.2019-0266OC
doi: 10.1165/rcmb.2019-0266OC
pubmed: 31596609
pmcid: 7055692
Satija R, Farrell JA, Gennert D et al (2015) Spatial reconstruction of single-cell gene expression data. Nat Biotechnol 33:495–502. https://doi.org/10.1038/nbt.3192
doi: 10.1038/nbt.3192
pubmed: 25867923
pmcid: 4430369
Butler A, Hoffman P, Smibert P et al (2018) Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 36:411–420. https://doi.org/10.1038/nbt.4096
doi: 10.1038/nbt.4096
pubmed: 29608179
pmcid: 6700744
Lun ATL, Riesenfeld S, Andrews T et al (2019) EmptyDrops: distinguishing cells from empty droplets in droplet-based single-cell RNA sequencing data. Genome Biol 20:1–9. https://doi.org/10.1186/s13059-019-1662-y
doi: 10.1186/s13059-019-1662-y
Islam S, Zeisel A, Joost S et al (2014) Quantitative single-cell RNA-seq with unique molecular identifiers. Nat Methods 11:163–166. https://doi.org/10.1038/nmeth.2772
doi: 10.1038/nmeth.2772
pubmed: 24363023
Stoeckius M, Zheng S, Houck-Loomis B et al (2018) Cell hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics. Genome Biol 19:224. https://doi.org/10.1186/s13059-018-1603-1
doi: 10.1186/s13059-018-1603-1
pubmed: 30567574
pmcid: 6300015