Objective To obtain gene-targeted mouse embryos expressing Cas9 protein specifically in muscleusing CRISPR/ Cas9, to lay the foundation for the construction of animal model expressing Cas9 protein specifically inmuscle. Methods The sgRNA targeted Rosa26 locus was designed and synthesized according to the sequence of theRosa26 gene in mice. The editing efficiency was tested by in vitro cleavage assay. Rosa26 sgRNA and Cas9 protein weresubsequently co-injected into mouse pronuclear-stage embryos to examine the editing efficiency in vivo. A mouse Rosa26locus homologous targeting vector for the muscle-specific expression of Cas9 protein was designed and constructed. Finally,gene-targeting embryos were obtained through co-injecting the homologous targeting vector and Rosa26 sgRNA with Cas9protein into mouse embryos. Results The sgRNA designed and synthesized according to the sequence of mouse Rosa26gene was effective at gene editing in the in vitro digestion experiments. The homologous targeting vector for the musclespecific expression of Cas9 protein, Donor-SP-px459, was successfully constructed by PCR and homologous recombinationligation. Mouse embryos injected with Rosa26 sgRNA and Cas9 protein showed normal cleavage and supported blastocystdevelopment. PCR and sequencing result demonstrated that gene editing occurred in the obtained embryos, after which theembryo grew into a Rosa26 gene edited mouse. Mouse embryos for the muscle-specific expression of Cas9 protein weresuccessfully obtained after co-injection of a homologous targeting vector and Rosa26 sgRNA with Cas9 protein. Conclusions Rosa26 gene-edited embryos and mice are obtained using the CRISPR/ Cas9 system, and mouse embryos with musclespecificexpression of Cas9 protein are successfully obtained, laying the foundation for the construction of animal models expressing Cas9 protein specifically in muscle.