Precise dual-gene knockout of MSTN and SOCS2 via cytidine base editing enhances muscling and growth in goats.

BACKGROUND: Myostatin (MSTN) and Suppressor of Cytokine Signaling 2 (SOCS2) are key negative regulators of muscle growth and development. Cytidine base editors (CBEs) offer a precise method for introducing stop codons to knock out these genes. This study aimed to apply the YE1-AncBE4max CBE system to edit MSTN and SOCS2 in Haimen goats, a local Chinese breed, to enhance growth traits. METHODS: Efficient single guide RNAs (sgRNAs) targeting MSTN and SOCS2 were screened in goat fetal fibroblasts via electroporation. Edited cells were analyzed by Sanger and TA-clone sequencing. Subsequently, a mixture of selected sgRNA and CBE mRNA was microinjected into 36 zygotes, which were then transferred to 9 recipient goats to generate gene-edited animals. Off-target effects were assessed by sequencing predicted sites. RESULTS: Cellular editing efficiencies for three MSTN sgRNAs were 25%, 37%, and 29%, and for one SOCS2 sgRNA was 31.57%. Microinjection and embryo transfer resulted in the birth of five kids, and one fetus (#S91) was aborted at 3 months of gestation; four with dual-gene (MSTN & SOCS2) edits (#241, #242, #S32, #S91) and two with single SOCS2 edits (#S31, #S11). Notably, two dual-edited goats (#241, #242) harbored biallelic MSTN mutations. No off-target mutations were detected. Phenotypic analysis showed that the edited goats exhibited significantly increased body weight and a "double-muscling" phenotype by 90 days of age compared to wild-type controls. This is the first study of generating any species with double gene knock out (MSTN, and SOCS2) to date to the best of our knowledge. CONCLUSION: This study successfully established a YE1-AncBE4max-based platform for efficient, precise dual-gene editing in goats. The generation of MSTN/SOCS2-edited Haimen goats with enhanced growth performance demonstrates the potential of base editing technology for rapid genetic improvement in livestock.