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

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. 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. 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. 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.