Genetically modified primary airway-epithelial cell culture models to study virus-host interactions of emerging respiratory viruses
TL;DR
This chapter describes the generation of knockout (KO) AECs using engineered virus-like particles (eVLPs), which transiently deliver the Cas9–gRNA ribonucleoprotein (RNP) complex into target cells, enabling efficient genome editing of any gene of interest.
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.
