A simple, rapid, and transgene-free strategy for the generation of transgenic pigs via precise editing of monoclonal porcine fetal fibroblasts

• The IRE-DSRNP method enables transgene-free, precise editing of porcine fetal fibroblasts with monoclonal selection, reducing donor cell generation time from 3-4 weeks to 1 week. • This improved method overcomes the limitations of plasmid-based editing, such as antibiotic resistance genes and prolonged culture, by using ribonucleoprotein complexes and fluorescent RNA reporters. • The strategy enhances editing efficiency and reduces off-target effects, making it a reliable approach for generating genetically edited pigs for biomedical and agricultural applications. • The method's simplicity and speed facilitate the production of gene-edited pigs for disease models, xenotransplantation, and breed improvement.