CRISPR/Cas9系統在肝癌研究之應用

Abstract

Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system (CRISPR/Cas9 system) is a highly efficient genome editing tool. It consists of two components: a strand of ribonucleic acid called small guide RNA (sgRNA) and an enzyme called CRISPR-associated endonuclease 9 (Cas9). In this system, sgRNA directs Cas9 to a targeted DNA sequence for site-specific cleavage. Recently, the mutagenic function of CRISPR/Cas9 system is widely used in genome and cancer researches. In this study, we exploited CRISPR/Cas9 system in two topics: CRISPR/Cas9 library screening in live cancer cell line and CRISPR/Cas9-mediated chromosomal translocation between Fibroblast growth factor receptor 2 (FGFR2) gene and BicC Family RNA Binding Protein 1 (BICC1) gene. In CRISPR/Cas9 library screen, we used HepG2 cell line to produce a mixture of cells with loss-of-function mutations of genes and then made tumor formation in NOD/SCID mice by subcutaneous injection of those cells. According to the consequences of next-generation sequencing, we found that some populations of CRISPR/Cas9 knockout cells dominated in quantity in late-phase tumors, so the possible explanation is that those genes knockout by the CRISPR/Cas9 system are suppressor of tumor progression. Further experiment revealed that one of the genes identified, DEDD2, can inhibit cell death caused by oxidative stress. In CRISPR/Cas9-mediated chromosomal translocation study, we used BICC1 and FGFR2-targeting CRISPR/Cas9 plasmids to produce the predicted FGFR2-BICC1 fusion DNA products. Although this method worked, the output was low. As a result, we developed the two-target CRISPR/Cas9 plasmids to improve the efficiency by increasing the possibility of cleavage of two target genes in one single cell. However, the experiment results showed that those plasmids were unable to produce the predicted chromosomal translocation products.