Non-surgical deep uterine embryo transfer combined with electroporation-based genome editing enables scalable production of CD163-Knockout pigs.

Porcine reproductive and respiratory syndrome (PRRS) remains the most economically devastating viral disease in the global swine industry, causing annual losses exceeding $600 million in the United States alone. While CD163-knockout (KO) pigs have demonstrated complete resistance to the PRRS virus (PRRSV) infection, conventional production methods relying on microinjection-based genome editing and surgical embryo transfer present significant bottlenecks for industrial-scale application. Here, we demonstrate that the integration of electroporation-based CRISPR-Cas9 delivery with non-surgical deep intrauterine embryo transfer enables scalable production of CD163-KO pigs. Our approach achieved successful pregnancies and live births of genome-edited piglets without requiring surgical intervention or specialized microinjection expertise. Genotyping analysis revealed complete biallelic KOs in a subset of offspring, while others exhibited mosaic patterns reflecting the stochastic nature of electroporation-mediated editing. These findings establish a technically accessible and scalable pipeline for producing gene-edited pigs. By eliminating the need for complex surgical facilities and specialized microinjection equipment, this approach provides a practical and field-applicable strategy that can be readily implemented in standard commercial swine farms, representing a critical step toward the widespread deployment of genome-edited livestock in global agriculture.