Physiological breakdown following complete host-receptor removal and bottlenecks in designing genetically antiviral breeds Aujeszky’s disease virus (PRV), a threat to the economic productivity of the pig industry, is an enveloped double-stranded DNA virus belonging to the alphaherpesvirus subfamily. This pathogen recognizes nectin-1, a cell-surface adhesion molecule, as a key host receptor and activates pathways for endocytosis induction and membrane fusion. Conventional genetic engineering guidelines have relied on a traditional knockout (KO) strategy that disrupts the entire nectin-1 coding gene to block viral infection at its source. However, this simple-cut approach has a critical blind spot: it disables nectin-1’s normal physiological and developmental mechanisms, including cell–cell adhesion and epithelial tissue formation, causing developmental lethality or a spectrum of false-positive malformations in animals. The absence of reversible microdomain engineering that preserves the protein’s beneficial endogenous functions while selectively filtering viral entry has long impeded the development of sustainable pipelines for breeding disease-resistant livestock.

Prime editing-driven precision reprogramming of four amino acids at the nectin-1 gD-binding site Recent genome engineering research fully deployed next-generation prime editing (PE), which can precisely substitute, insert, or delete targeted bases without DNA double-strand breaks (DSBs), to eliminate developmental cellular toxicity associated with complete receptor loss. The team targeted nectin-1 residues N77, I80, M85, and F129, which previous structural and biochemical studies had identified as participating in binding PRV glycoprotein D (gD). By designing prime editor guide RNAs (pegRNAs) for these four key amino-acid positions, the researchers successfully introduced small sequence variants with high efficiency in both monoallelic and homozygous configurations.

Demonstration of a topological stalling mechanism in viral entry and establishment of a functional knockout Tracking the kinetics of PRV infection susceptibility in the established prime-edited pig cell lines fully validated a distinctive “functional knockout” phenotype. The modified nectin-1 receptor architecture allowed the initial adsorption kinetics, in which external PRV particles physically attach to the cell surface, to proceed normally. However, it rejected allosteric conformational matching with gD, strongly constraining the downstream rate-limiting steps through which the viral genome crosses the cell membrane and is internalized. In particular, X4, a model with mutations at all four targeted residues, was obtained through a sequential strategy of first creating X3 and then performing additional editing. Whether the mutant protein fully retains trans-dimer formation and its ability to support adherens junctions has not yet been confirmed. Under the test conditions, however, viral internalization, intracellular replication, and progeny virus release were at levels similar to those in conventional nectin-1 KO cell lines, with no statistically significant difference observed between the two groups.

Standardizing programmable smart-breeding infrastructure and establishing agricultural and livestock biosecurity standards This integrated white paper on agricultural genetics and synthetic biology data comprehensively reset livestock disease-control governance from approaches based on post hoc feed additives and hygienic isolation to “programmable infection-inhibition infrastructure based on scanning host-genome code.” In the off-target analysis, the top three predicted candidate sites for each sgRNA were amplified from the genomic DNA of edited cells using two-step nested PCR and examined by high-throughput sequencing. The established nectin-1 prime-editing correction factors and structural dynamics values will serve as a backbone for precomputing CMC (chemistry, manufacturing, and controls) safety metrics in future IND regulatory pathways for next-generation transgenic livestock. They will function as a master reference that dramatically compresses global regulatory approval timelines for sustainable, environmentally friendly antiviral breeding businesses.