Abstract

Introduction The rapid development of genome editing technologies has enabled precise manipulation of livestock genomes for the improvement of production traits such as meat yield and milk quality. Myostatin ( MSTN ) and β-lactoglobulin ( BLG ) are key gene targets for enhancing muscle growth and reducing lactose intolerance, respectively. Methods In this study, we employed an optimized CRISPR/EOCas12i system to simultaneously target MSTN and BLG in bovine fetal fibroblasts (BFFs) using a single plasmid. Results T7E1 and Sanger sequencing confirmed efficient editing at multiple target sites, with EOCas12i producing deletions ranging from tens to over 100 bp. Furthermore, no off-target (OT) effects were detected at predicted loci, supporting the high specificity of this system in large animals. Gene-edited single-cell clones (SCCs) were expanded in conditioned medium, and selected double-knockout (DKO) clones served as nuclear donors for somatic cell nuclear transfer (SCNT) to produce MSTN / BLG double gene-edited cattle embryos. Discussion Collectively, this study demonstrates the feasibility of generating MSTN / BLG double gene-edited cattle embryos using a single CRISPR/EOCas12i plasmid and SCNT, providing a robust platform for multiplex genome editing aimed at improving meat production and milk traits, with potential applications in both agricultural and biomedical research.