Highly Efficient Production of MSTN-Edited Hu Sheep Mediated by the CBE System

The cytosine base editor (CBE) enables precise C-to-T substitution without DNA double-strand breaks, which offering a promising tool for editing livestock genomes to enhance economically valuable traits. In this study, using Hu sheep, characterized by high reproductive performance but suboptimal meat production as the research subject, two CBE-editing sgRNAs (sgM1 and sgM2) targeting the negative regulator MSTN (Myostatin) gene were designed. The results revealed a 75% editing efficiency of sgM2 at the parthenogenetically activated embryonic level and no detectable off-target effects. Thirty-four Hu sheep zygotes microinjected with sgM2 and CBE mRNA mixtures were transferred into four recipient ewes, yielding four lambs with confirmed MSTN editing and no off-target activity. Growth performance data revealed that MSTN -edited Hu sheep exhibited higher body weights at 120-180 days, and significantly enlarged muscle fiber cross-sectional areas compared to wild-type controls. Edited Hu sheep displayed reduced MSTN protein expression, elevated p-AKT levels, and diminished p-ERK and p-p38 signaling. In conclusion, MSTN -edited Hu sheep were highly efficient generated using CBE, and further analysis demonstrate that MSTN editing activates the AKT pathway while suppressing MAPK signaling, leading to muscle fiber hypertrophy and accelerated growth, which provides technical methodologies and breeding materials for developing fast-growing, meat-type Hu sheep strains.