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Background  Genetic engineering technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides a powerful tool for developing disease models and determining gene functions. Recent interests in canine cancer models have highlighted the necessity of developing genetic engineering tools for dogs. In this study, we attempted to generate optimized CRISPR/Cas9 system to target canine tumor protein 53 ( TP53 ), one of the most crucial tumor suppressor genes, to establish TP53 knockout canine cells for canine cancer research.    Results  We constructed CRISPR/Cas9 vectors using each of three  TP53  gene-targeting guide RNAs (gRNAs) with minimal off-target potential. After transfection, we obtained several clones of  TP53  knockout cells containing “indel” mutations in the targeted locus which had infinite cellular life span, resistance to genotoxicity, and unstable genomic status in contrast to normal cells. Of the established TP53 knockout cells, TP53KO#30 cells targeted by TP53 gRNA #30 showed non-cancerous phenotypes without oncogenic activation both in vitro and in vivo. More importantly, no off-target alteration was detected in TP53KO#30 cells. We also tested the developmental capacity of TP53 knockout cells after application of the somatic cell nuclear transfer technique.    Conclusions  Our results indicated that  TP53  in canine cells was effectively and specifically targeted by our CRISPR/Cas9 system. Thus, we suggest our CRISPR/Cas9-derived canine  TP53  knockout cells as a useful platform to reveal novel oncogenic functions and effects of developing anti-cancer therapeutics.    Electronic supplementary material  The online version of this article (10.1186/s12896-018-0491-5) contains supplementary material, which is available to authorized users.

Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research