Optimized prime editing in pigs through La protein-enhanced pegRNA stabilization

Prime editor (PE) enables precise genome modification without introducing double-strand breaks or requiring donor DNA templates, yet its efficiency in porcine cells remains limited, constraining its utility in pig breeding. Here, we systematically evaluated and optimized a series of PE variants in porcine cells. Among them, PE6c and PE6d demonstrated markedly improved editing efficiency and product purity compared with PE2 and uPEn. To further enhance RNA stability and PE performance, we introduced a ZIKV-derived xrRNA structural motif into pegRNAs and fused a small RNA-binding protein La to the PE complex. The combined use of La protein and xrpegRNA significantly boosted editing efficiency, achieving up to a 6.5-fold average increase in porcine cells. Notably, the optimized PE6c-La system exhibited robust activity in porcine embryonic fibroblasts and enabled efficient generation of precisely edited, disease-resistant cell lines. Using a twinPE strategy, we successfully introduced two amino acid substitutions (N736A and T738V) into the ANPEP gene, conferring complete resistance to transmissible gastroenteritis virus (TGEV) in porcine cells. Collectively, these results establish PE6c-La as a highly efficient prime editing platform for the porcine genome, providing a valuable tool for precision breeding, biomedical model development, and genome engineering in other agricultural species.