Genetically modified primary airway-epithelial cell culture models to study virus-host interactions of emerging respiratory viruses
- 1Institute for infectious diseases, University of Bern, Bern, Switzerland
Abstract
Airway epithelial cell (AEC) cultures such as air-liquid interface cultures or airway organoids are widely used to study emerging respiratory virus infection in a physiologically representative model system of the human respiratory epithelium. Loss-of-function studies in AEC culture systems offer a unique opportunity to better understand viral disease mechanisms of emerging viruses in the human host, may reveal biologically relevant host-factor dependencies that can be therapeutically targeted, or identify human host barriers of cross-species transmission of emerging viruses that can inform risk assessment. In this chapter, we describe the generation of knockout (KO) AECs using engineered virus-like particles (eVLPs). These particles transiently deliver the Cas9-gRNA ribonucleoprotein (RNP) complex into target cells, enabling efficient genome editing of any gene of interest. We detail the production, quantification, and application of eVLPs for gene editing in AECs, alongside an optimized protocol for AEC expansion and differentiation at the air-liquid interface that is compatible with genetically modified cells. Finally, we describe procedures for infecting KO AECs with emerging respiratory viruses. Together, these protocols provide a robust workflow for generating renewable KO AEC cultures in less than two months, facilitating the functional characterization of host factors that influence viral infection of the airway epithelium.
Related
- Rapid recombinase polymerase amplification-based detection of the bar gene for environmental monitoring of genetically modified soybean and rice.
- Genetically modified crops in Africa: progress, challenges, and future prospects.
- The Fate and Degradation of Genetically Modified Soybean DNA Along the Soybean Oil Processing Chain: Quantitative Tracking and Comparison of Degumming Methods.
- SIM2s coordinates programmed mitophagy with respiratory chain supercomplex remodeling during mammary epithelial differentiation
- Small extracellular vesicle-enriched preparations derived from genetically modified multipotent mesenchymal stromal cells promote fibroblast proliferation and extracellular matrix remodeling in vitro
BibTeX
@misc{stevers2026genetically,
title = {{Genetically modified primary airway-epithelial cell culture models to study virus-host interactions of emerging respiratory viruses}},
author = {K. M. Stevers and S. Jordi and B. I. Oliveira Esteves and R. Dijkman},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.16.758770},
url = {https://www.biorxiv.org/content/10.64898/2026.10.16.758770v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Genetically modified primary airway-epithelial cell culture models to study virus-host interactions of emerging respiratory viruses
AU - K. M. Stevers
AU - S. Jordi
AU - B. I. Oliveira Esteves
AU - R. Dijkman
PY - 2026
DA - 2026/10/16
PB - bioRxiv
DO - 10.64898/2026.10.16.758770
UR - https://www.biorxiv.org/content/10.64898/2026.10.16.758770v1
AB - Airway epithelial cell (AEC) cultures such as air-liquid interface cultures or airway organoids are widely used to study emerging respiratory virus infection in a physiologically representative model system of the human respiratory epithelium. Loss-of-function studies in AEC culture systems offer a unique opportunity to better understand viral disease mechanisms of emerging viruses in the human host, may reveal biologically relevant host-factor dependencies that can be therapeutically targeted, or identify human host barriers of cross-species transmission of emerging viruses that can inform risk assessment. In this chapter, we describe the generation of knockout (KO) AECs using engineered virus-like particles (eVLPs). These particles transiently deliver the Cas9-gRNA ribonucleoprotein (RNP) complex into target cells, enabling efficient genome editing of any gene of interest. We detail the production, quantification, and application of eVLPs for gene editing in AECs, alongside an optimized protocol for AEC expansion and differentiation at the air-liquid interface that is compatible with genetically modified cells. Finally, we describe procedures for infecting KO AECs with emerging respiratory viruses. Together, these protocols provide a robust workflow for generating renewable KO AEC cultures in less than two months, facilitating the functional characterization of host factors that influence viral infection of the airway epithelium.
ER -