Prime editing of Nectin-1 functional domains confers knockout-level resistance to pseudorabies virus

BACKGROUND: The continuous evolution and cross-species transmission risk of pseudorabies virus (PRV) poses a substantial threat to the swine industry and public health. Nectin-1, an essential host receptor for PRV entry, represents a promising antiviral target; however, direct gene knockout (KO) raises biosafety concerns, and conventional genome-editing approaches remain inefficient for precise multiplex modification of critical functional residues. RESULTS: Here, we employed prime editing (PE) to systematically re-engineer the PRV entry receptor Nectin-1, introducing single and combinatorial point mutations at four structurally defined amino acids within the gD-binding interface. PE enable efficient homozygous multiplex editing of these functionally critical residues in porcine cells. Functional analyses showed that these targeted mutations specifically impaired viral internalization without affecting attachment and exhibited combinatorial effects, resulting in reductions in viral replication and release. Notably, the quadruple-site mutant conferred a level of resistance comparable to that observed in Nectin-1 KO cells. CONCLUSION: Collectively, this study establishes a framework for "functional knockout," in which precise editing of essential microdomains confers resistance to virus infection while preserving protein integrity. These findings, derived from in vitro cellular models, suggest a potentially safer and more controllable strategy for antiviral genome-edited breeding, pending further validation in vivo.