Abstract: Objective: To knock out p53 gene in skeletal muscle satellite cells in tree shrews and detect its proliferative activity through utilization of the CRISPR/Cas9 system. Methods: The homology analysis of amino acid sequences of the p53 protein in tree shrews and 15 mammals was conducted using a bioinformatics approach. Based on the genomic information of tree shrews, a specific sgRNA targeting the p53 mutation site was designed. Subsequently, a lenti CRISPR v2-sgp53 gene editing recombinant plasmid was constructed and lentiviral packaging was performed with 293T cells to infect skeletal muscle satellite cells in tree shrews. Polyclonal cells were then screened and amplified using puromycin. The effectiveness of gene editing was determined by sequencing analysis. Cell proliferation activity was assessed through cell counting kit-8 (CCK8) assay, and the expression level of p53 protein was verified by western blotting. Results: Compared to the amino acid sequence homology of human p53 and mouse p53 (96.92%), the homology between human p53 and tree shrew p53 (98.21%) was higher. Evolutionary tree analysis revealed a closer relationship between humans and tree shrews than mice. Three recombinant vectors targeting p53 editing in tree shrews were successfully constructed, namely lenti CRISPR v2-sgp53-g1, lenti CRISPR v2-sgp53-g2, and lenti CRISPR v2-sgp53-g3, and they all successfully achieved p53 gene editing in skeletal muscle satellite cells in tree shrews to produce mutations, resulting in loss of p53 function. The mutation of p53 improved the proliferation of skeletal muscle satellite cells in tree shrews.The p53 gene was knocked out successfully in skeletal muscle satellite cells in tree shrews, and the expression level of p53 protein was significantly decreased (P< 0.01). Conclusion: In this study, the CRISPR/Cas9 system is used to successfully mutate and disable p53 gene in skeletal muscle satellite cells in tree shrews, which provides an important molecular biological basis for the subsequent establishment of p53 gene knockout model in tree shrews.