Effect of CRISPR Knockout of or on Proliferation and Migration of Porcine Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive disease lacking effective treatment. Animal models of HCC are necessary for preclinical evaluation of the safety and efficacy of novel therapeutics. Large animal models of HCC allow testing image-guided locoregional therapies, which are widely used in the management of HCC. Models with precise tumor mutations mimicking human HCC provide valuable tools for testing precision medicine. and are two of the most frequently mutated genes in human HCC. Here, we investigated the effects of knockout of and/or on proliferation, migration, and chemotherapeutic susceptibility of porcine HCC cells and we developed subcutaneous tumors harboring these mutations in pigs. Gene knockout was achieved by CRISPR/Cas9 and was validated by Next Generation Sequencing. knockout increased the migration of porcine HCC cells but did not alter the cell proliferation. Knockout of increased both the proliferation and migration of porcine HCC cells. Simultaneous knockout of and increased the migration, but did not alter the proliferation of porcine HCC cells. The effect of gene knockout on the response of porcine HCC cells to two of the most commonly used systemic and locoregional HCC treatments was investigated; sorafenib and doxorubicin, respectively. Knockout of and/or did not alter the susceptibility of porcine HCC cells to sorafenib or doxorubicin. Autologous injection of CRISPR edited HCC cells resulted in development of subcutaneous tumors in pigs, which harbored the anticipated edits in and/or . This study elucidates the effects of CRISPR-mediated knockout of HCC-associated genes in porcine HCC cells, and lays the foundation for development and utilization of genetically-tailored porcine HCC models for testing of novel therapeutic approaches in a clinically-relevant large animal model.