Abstract
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.
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References
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
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
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
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
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
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
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
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
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
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
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
Acknowledgments
The authors thank the UCAGenomiX platform for fruitful discussions and technical help on single-cell RNA sequencing. Supported by grants from Fondation pour la Recherche Médicale (DEQ20180339158), 020, INSERM (Human Developmental Cell Atlas program), the association Vaincre la Mucoviscidose (RF20180502280), the Chan Zuckerberg Initiative (Silicon Valley Foundation, 2017-175159-5022), ANR SAHARRA (ANR-19-CE14–0027), and H2020-SC1-BHC-2018-2020 DiscovAIR (grant agreement 874656). The UCAGenomiX platform, a partner of the National Infrastructure France Génomique, is supported by Commissariat aux Grands Investissements (ANR-10-INBS-09-03, ANR-10-INBS-09-02), Conseil Départemental des Alpes Maritimes (2016-294DGADSH-CV).
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Zaragosi, LE. et al. (2024). Combination of CRISPR-Cas9-RNP and Single-Cell RNAseq to Identify Cell State-Specific FOXJ1 Functions in the Human Airway Epithelium. In: Mennella, V. (eds) Cilia. Methods in Molecular Biology, vol 2725. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3507-0_1
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DOI: https://doi.org/10.1007/978-1-0716-3507-0_1
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