Abstract: The aim of this study was to edit the P53 gene, and analyze the expression of vital genes in its signaling pathway. Firstly, two designed sgRNAs were loaded into the PX459 V2.0 plasmid, and two single-stranded oligonucleotides (SSODN) carrying 241W, 242S, 266H mutations were ordered as donor DNA (one carrying 241W and 242S mutations, the other one carrying 266H mutation). The targeting vectors and SSODNs were co-transfected into PFF cells by electroporation, and monoclonal cells were collected. Then editing events in targeting region in isolated cells were detected, and expression of vital genes (MDM2, FAS, WIP1, BAX, P21 and DD1α) in P53 signaling pathway were detected and the proliferative capacity of the gene editing cells were identified. Among the isolated 107 monoclonal cells, each 4 clones were homozygous and heterozygous modification of R241 and R242, 1 clone was homozygous modification of R266, 63 clones contained homozygous deletions, 11 clones carried homozygous inversions, 11 clones contained homozygous insertions, 3 clones carried heterozygous deletions (or mixed clones) and 10 clones were wild-type. No cell with simultaneous modification of 3 sites was found. RT-PCR and Western blotting analysis showed that P53 expression was not detected in the cells with homozygous deletions. RT-PCR result indicated that the expression levels of MDM2, FAS, BAX and P21 were extremely significant decreased (P<0.01 or P<0.001) in cells carrying homozygous deletions and homozygous modification of R241 and R242. Western blotting result showed that P21 protein was undetectable, and MDM2 protein was obviously decreased in cells contained homozygous deletions. The CCK-8 test indicated that the proliferation capacity of the edited cells significantly (P<0.05) or extremely significantly (P<0.01 or P<0.001) increased compared with the wild-type cells. In conclusion, we successfully obtained PFF cells that simultaneously modified two sites in P53 gene related to human tumorigenesis. P53 gene editing obviously affected the expression levels of vital genes in its signaling pathway. The acquirement of cells with modification of two sites laid the solid foundation for further generating P53 point edited model in pigs.