SpCas9-mediated gene editing in bovine embryo via single adeno-associated virus infection using a novel micro-sized promoter.

Genome editing in livestock offers practical solutions to address challenges related to land use, climate change, and food production. However, conventional delivery methods such as electroporation and microinjection impose physical stress on embryos, limiting scalability. This study aimed to develop a simplified, non-invasive, and scalable genome editing system for bovine embryos by designing an all-in-one adeno-associated virus (AAV) vector. A novel micro-sized promoter (50 bp), derived from the core regulatory region upstream of the bovine MSTN gene, was constructed to enable expression of Streptococcus pyogenes Cas9 (spCas9) within the AAV packaging limit (∼4.7 kb). This promoter was incorporated into an AAV cassette containing spCas9, a polyadenylation signal, a U6 promoter, and a single-guide RNA (sgRNA) targeting the bovine ALB gene. After confirming editing activity in bovine fibroblasts, the AAV6 vector was added directly to in vitro fertilization (IVF) cultures without physical manipulation. Genome editing was successfully induced, with insertion/deletion (indel) mutations detected in 33.8 ± 23.2 % of the blastocysts. Although blastocyst development was moderately reduced, gene editing was achieved without invasive techniques. These results demonstrate that a micro-promoter-based AAV system can support spCas9-mediated genome editing in bovine embryos through a single-vector infection strategy. The system presents a promising platform for producing gene-edited livestock and may contribute to more efficient and less labor-intensive applications in animal biotechnology.