Kanser ilişkili hTERT mutasyonlarının 3d karaciğer organoidlerinde crıspr-Cas9 ile modellenmesi

Human TERT (hTERT) promoter mutations, C-124T and C-146T are the most common genetic alterations in hepatocellular carcinoma, has an early stage in hepatocarcinogenesis, and associate with the progression. Mechanism of TERT promotor mutations which collaborates with carcinogenesis is not well explained yet. In this study, we generated a hepatic organoid (eHEPO) culture system using human induced pluripotent stem cell (iPSC)-derived EpCAM-positive endodermal cells as an intermediate. eHEPOs can be produced within 2 weeks and expanded long term without any loss of differentiation capacity to mature hepatocytes. In this study, we aimed to introduce hTERT promoter mutations in hepatic organoids using CRISPR- Cas9 based genome editing. For the editing of TERT promoter, a homology-directed repair (HDR) based CRISPR Cas9 system called "pop in pop out" strategy was used. This system was optimized in HEK293T cells. Genome modified clones which included C-146T mutation in the promoter were obtained with small deletion mutations. We tried to utilize this system in hepatic organoids. Transfection of the hepatic organoids with CRISPR-Cas9 via chemical and physical-based transfection agents displayed different efficiencies. Low transfection efficiency hampers to edit promoter of hTERT in hepatic organoids. Therefore, to obtain higher transfection efficiency in organoids further, optimization is critical. Also, we tried to knockout the TP53 gene in hepatic organoids with a similar approach. Transfection and editing of hepatic organoids exhibited that the CRISPR-Cas9 system works in hepatic organoids and isogenic organoids clones can be obtained.