Obtaining human glioblastoma NRF2 knockout via CRISPR / Cas9

Abstract

Glioblastoma is an extremely aggressive type of cancer and patients with this type of tumor, even undergoing surgery, radiotherapy and chemotherapy, have an average survival of 12 to 15 months. Standard chemotherapy treatment is performed by administering temozolomide (TMZ) which is an alkylating agent that induces cell death by the formation of various DNA damage. Despite aggressive and multimodal treatment, the prognosis of patients with glioblastoma remains very poor. This is a strong indication that mechanisms other than those already described and established may be contributing to the determination of resistance to TMZ. Therefore, we believe it is vitally important to unravel the mechanisms behind TMZ resistance and, from this, to determine intervention strategies so that the tumor responds to treatment and results in patient healing. In this sense, our group demonstrated that the transcription factor NRF2 can mediate resistance to TMZ through induction of synthesis and utilization of glutathione. However, these results were obtained using gene silencing by shRNA technique rather than gene knockout, which can minimize the real role of NRF2 in TMZ resistance. In this research project we will evaluate the role of NRF2 in TMZ resistance in a knockout cell model for NRF2. For this we will use the CRISPR/Cas9 gene editing tool. We will then evaluate the cellular responses in these strains against TMZ treatment, always compared to the wild strain.