Genetically modified primary airway-epithelial cell culture models to study virus-host interactions of emerging respiratory viruses

Authors

Stevers, K. M., Jordi, S., Oliveira Esteves, B. I., Dijkman, R.

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.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 18 Oct 2026.

Stats

1-terrible, 9-excellent. How would you rate this preprint? Sign in in to submit your rating.

Post a comment

You need to be signed in to post comments. You can sign in here.

Comments

There are no comments yet.