An integration-free CRISPR platform for genetic dissection of respiratory virus host dependencies in human nasal epithelium

Respiratory viruses, including coronaviruses, generally initiate infection in the upper respiratory tract, where mechanistic studies of host-virus interactions require models amenable to genetic manipulation. Human nasal air-liquid interface (ALI) cultures are realistic models of the nasal epithelium, but robust CRISPR/Cas9 engineering workflows for this primary cell system have been lacking. Here, we established an integration-free CRISPR/Cas9 workflow for genetic engineering of human nasal basal cells, which are subsequently differentiated into gene-edited ALI cultures. GFP-based sorting of RNP-transfected nasal basal cells substantially increases editing, achieving knockout efficiencies of 70-90% across gene targets (B2M, SLC35A1, ACE2, and TMPRSS2). Genetic disruption of ACE2 markedly reduces infection by SARS-CoV-2 and hCoV-NL63, confirming its important role for both viruses in the human nasal epithelium. TMPRSS2 knockout substantially reduces replication of both viruses, demonstrating SARS-CoV-2 depending on TMPRSS2 in the human nasal epithelium and providing genetic evidence in primary human cell cultures that hCoV-NL63 relies on TMPRSS2. This platform enables systematic genetic perturbation in a physiologically relevant model of the primary site of respiratory virus infection, validates ACE2 and TMPRSS2 as targets for medical countermeasures against SARS-CoV-2 and hCoV-NL63, and provides a versatile approach to identify host dependency factors for both circulating and emerging respiratory viruses.