Abstract
BackgroundGenetic 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.ResultsWe 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.ConclusionsOur 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.
Highlights
Canine cancer is among the most common causes of death of pet dogs
Construction of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems for canine tumor protein 53 (TP53) gene knockout To target the canine TP53 locus via the CRISPR/Cas9 system, we selected three guide RNAs (gRNAs) with the lowest off-target potentials (Fig. 1a, b). These gRNAs were applied to our CRISPR/Cas9 expression vector and transiently transfected into canine fetal fibroblast cells (K9 Fetus 1), in which cellular senescence phenotypes appeared at passages 6–8 (Fig. 1c)
After culturing the control cells until they were senescent, consecutively proliferating cell colonies were obtained from cells targeted by TP53 gRNA #30 and #39 (Additional file 1: Figure S1)
Summary
Canine cancer is among the most common causes of death of pet dogs. According to a report from the Swiss Canine Cancer Registry, 51.83% (67,943 of 126,693) of dogs were diagnosed with various cancers from 1955 to 2008 [1]. Canine TP53 modulating tools and canine experimental model of TP53 deficiency are the most fundamental requirement to study canine cancers. Genetic engineering technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas 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. We attempted to generate optimized CRISPR/Cas 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
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