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Ms Aruna Narula 2 Dr XuYu Yang Yanlan Li, 1, 2, * Xiangning Li, 1, 3, * Jiayao Qu, 1, 3 Dixian Luo, 1, 3 Zheng Hu 1, 3 1 Translational Medicine Institute, the First People’s Hospital of Chenzhou Affiliated to University of South China, Hunan 432000, People’s Republic of China; 2 Hunan Province Key Laboratory of Tumor Cellular and Molecular Pathology, Cancer Research Institute, University of South China, Hunan 421001, People’s Republic of China; 3 National & Local Joint Engineering Laboratory for High-Through Molecular Diagnosis Technology, The First People’s Hospital of Chenzhou, Hunan 432000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zheng Hu; Dixian LuoTranslational Medicine Institute, National & Local Joint Engineering Laboratory for High-Through Molecular Diagnosis Technology, The First People’s Hospital of Chenzhou, Hunan 432000, People’s Republic of ChinaTel/Fax +86 735 2343902Email hu48005@163.com; luodixian_2@163.com Purpose: Colorectal cancer (CRC) is one of the major contributors to cancer mortality and morbidity. Finding strategies to fight against CRC is urgently required. Mutations in driver genes of APC or β-catenin play an important role in the occurrence and progression of CRC. In the present study, we jointly apply CRISPR/Cas9-sgRNA system and Single-stranded oligodeoxynucleotide (ssODN) as templates to correct a heterozygous ΔTCT deletion mutation of β-catenin present in a colon cancer cell line HCT-116. This method provides a potential strategy in gene therapy for cancer. Methods: A Cas9/ β-catenin -sgRNA-eGFP co-expression vector was constructed and co-transfected with ssODN into HCT-116 cells. Mutation-corrected single-cell clones were sorted by FACS and judged by TA cloning and DNA sequencing. Effects of CRISPR/Cas9-mediated correction were tested by real-time quantitative PCR, Western blotting, CCK8, EDU dyeing and cell-plated clones. Moreover, the growth of cell clones derived tumors was analyzed at nude mice xenografts. Results: CRISPR/Cas9-mediated β-catenin mutation correction resulted in the presence of TCT sequence and the re-expression of phosphorylation β-catenin at Ser45, which restored the normal function of phosphorylation β-catenin including reduction of the transportation of nuclear β-catenin and the expression of downstream c-myc, survivin. Significantly reduced cell growth was observed in β-catenin mutation-corrected cells. Mice xenografted with mutation-corrected HCT-116 cells showed significantly smaller tumor size than uncorrected xenografts. Conclusion: The data of this study documented that correction of the driven mutation by the combination of CRISPR/Cas9 and ssODN could greatly remedy the biological behavior of the cancer cell line, suggesting a potential application of this strategy in gene therapy of cancer. Keywords: CRISPR/Cas9 , ssODN, targeted gene editing, β-catenin , colon cancer



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