CRISPR-Cas9 mediated PIK3CA modification as a novel treatment modality in cancer

CRISPR-Cas9 is the leading technology among all genome modification methods. It has been utilized in cancer etiology, tumor pathogenesis, tumor microenvironment, drug efficiency studies, and developing new strategies via novel biomarker discoveries. PI3K/Akt/mTOR signaling pathway have great impact on cancer cell growth and proliferation as well as cancer cells' escape from immune system and apoptosis. Thus, it is especially promising for targeted anti-cancer therapies. In this study, CRISPR-Cas9 induced knockdown was performed on PIK3CA gene which is located on this important cellular pathway, and the impacts of this gene modification on both the behavior of cancer cells and whole PI3K pathway were investigated using Cukurova University AGENTEM and Cukurova Technopolis InfoGenom infrastructure. Experiments were carried on MCF-7 cell line, and electroporation was used for all transfection steps. Electroporation optimization for this cell line was performed by tracking cell viability and transfection efficiency under florescence microscope together with flow cytometry assays. CRISPR-Cas9 mediated modification of PIK3CA gene was made by the most efficient electroporation protocols and showed using Sanger and next-generation sequencing methods. Also, impacts of gene modification were shown via invert microscope, flow cytometry and real-time PCR expression assay. The results of this study indicates knockdown of PIK3CA gene via CRISPR-Cas9 induces cell death and has potential for the new therapeutic approaches. Finally, with this thesis, we aimed to contribute to the literature for novel treatments associated to CRISPR-Cas9-based modification of PIK3CA gene.