Electroporation-mediated site-specific knock-in of a human coding sequence into the bovine ALB locus in embryos

Efficient delivery of genome-editing tools into embryos is essential for the production of gene-edited cattle. Somatic cell nuclear transfer and microinjection are commonly used techniques, but electroporation has recently gained attention owing to its technical simplicity, high-throughput capacity, and suitability for large-scale applications. However, its application has been largely restricted to gene knockout owing to the presence of the zona pellucida and size limitations of deliverable materials. To address these challenges, in this study, we optimized electroporation-mediated knock-in (KI) strategies in bovine embryos using single-stranded oligodeoxynucleotides (ssODNs) and adeno-associated virus (AAV) vectors as donors. Initial experiments using ssODN donors with asymmetric homology arms (10 bp left and 25 bp right) resulted in a KI blastocyst (BL) frequency of 13.9 ± 14.1%, although outcomes varied widely across replicates. By contrast, AAV donors carrying 61 bp cargo flanked by 500 bp homology arms enabled consistent KI events, after optimizing the concentration of AAV and length of homology arm. Using the optimized system, a 2.2 kb human albumin coding sequence was successfully inserted into the bovine albumin locus, yielding KI BL frequency of 55.6 ± 41.6%. Collectively, these findings demonstrate that electroporation combined with AAV-mediated donor delivery represents an efficient strategy for generating transgenic bovine embryos, which is a crucial first step toward producing transgenic cattle as bioreactors for high-value protein production.