清脆的
Cas9
生物
细胞生物学
计算生物学
细胞
核糖核蛋白
呼吸上皮
电池类型
上皮
基因
核糖核酸
遗传学
作者
Laure‐Emmanuelle Zaragosi,Alizé Gouleau,Margot Delin,Kévin Lebrigand,Marie-Jeanne Arguel,Cédric Girard-Riboulleau,Géraldine Rios,Elisa Redman,Magali Plaisant,Rainer Waldmann,Virginie Magnone,Brice Marcet,Pascal Barbry,Gilles Ponzio
出处
期刊:Methods in molecular biology
日期:2023-10-20
卷期号:: 1-25
标识
DOI:10.1007/978-1-0716-3507-0_1
摘要
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 cellsMulticiliated cells (MCCs) and provides a framework to explore other gene functions.
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