Microinjection
When performing microinjection, Cas9 mRNA (Sigma–Aldrich, Cat. no. CAS9MRNA) and sgRNA were divided into 4 groups to find the most appropriate concentration (CB; only TE microinjection, RNA1X; Cas9 mRNA: 100 ng/μl, sgRNA: 50 ng/μl, RNA2X; Cas9 mRNA: 200 ng/μl, sgRNA: 100 ng/μl, RNA 4X; Cas9 mRNA: 400 ng/μl, sgRNA: 200 ng/μl). After 18 h of IVF, presumptive zygotes were injected with Cas9 mRNA and sgRNA synthesized by GeneArt™ Precision gRNA Synthesis Kit (Thermofisher, Cat. no. A29377) for MSTN using a microinjector machine (Eppendorf, Femtojet®). The amount of injected Cas9 mRNA and sgRNA for MSTNwere different each group (Fig 1 A-b). After 7 days of microinjection, pre-implantation stage embryos were collected and the mutation ofMSTN was observed in vitro or transferred in vivo into a surrogate cow.