MiniSdd7‐Derived Cytosine Base Editor Promotes Efficient Multiplex Gene Knockout in Porcine Primary Fibroblasts

ABSTRACT Efficient multiplex genome editing is essential for improving complex traits in livestock that are governed by multiple genes. However, achieving high‐efficiency simultaneous editing at multiple loci remains challenging for cytosine base editors (CBEs), largely due to variable editing efficiencies and limited deaminase activity. In this study, we developed and optimized a multiplex base editing framework targeting three economically important genes in pigs—APN, CD163, and MSTN. By integrating the high‐fidelity YE1‐BE4max‐SpG system with engineered sgRNA scaffolds, we first established a baseline editing platform capable of simultaneous multi‐gene modification. Despite successful editing, the efficiency of triple‐gene homozygous mutations remained limited. To overcome this bottleneck, we incorporated the engineered miniSdd7 deaminase into the BE4max‐SpG architecture and evaluated its performance in porcine multiplex genome editing. The resulting miniSdd7‐BE4max‐SpG system markedly enhanced editing efficiency across all three loci. Notably, the proportion of triple‐gene edited clones increased from 11.11% to 38.10%, representing more than a threefold improvement compared to the YE1‐based CBE system. Importantly, no detectable off‐target mutations were observed at the predicted candidate sites, indicating that the system maintains high specificity within the examined regions. Collectively, our study establishes an efficient multiplex base editing strategy in porcine cells and demonstrates that deaminase engineering is a key determinant of editing performance. The miniSdd7‐BE4max‐SpG system provides a powerful tool for precise multi‐gene modification and holds great potential for accelerating the genetic improvement of livestock.