Many genome-edited animals have been produced using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology to edit specific genes.However, there are few guidelines for the application of this technique to cattle. The goal of this study was to produce trait-improved cattle using the genome-editing technology CRISPR-Cas9. Myostatin (MSTN) was selected as a target locus, and synthetic mRNA of sgRNA and Cas9 were microinjected into fertilized bovine embryos in vitro. As a result, 17 healthy calves were born, and three of them showed MSTN mutation rates of 10.5%, 45.4%, and 99.9%, respectively. Importantly, the offspring with the 99.9% MSTN mutation rate had a biallelic mutation (-12 bps) and a doublemuscling phenotype. In conclusion, we demonstrate that the genome-editing technology CRISPR-Cas9 can produce genetically modified calves with improved traits.

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