Synthetic Lethality: Whole Exome CRISPR to Identify Therapeutic Targets for SCCOHT
SCCOHT (Small Cell Carcinoma of the Ovary Hypercalcemic Type) is a rare and aggressive ovarian cancer. The survival rate is diminutive because there are no current treatments specific for this ovarian cancer, therefore a new approach is needed. SCCOHT is characterized by a mutation of BRG1 which encodes SMARCA4, a sub-unit in the SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complex. This single inactivating mutation can be exploited by using synthetic lethality, where the simultaneous alteration of two genes leads to cell death but the alteration of one gene does not affect the cell viability. The goal is to use synthetic lethality to selectively take down SCCOHT cells while avoiding any damage to healthy cells. Whole Exome CRISPR has the ability to knockdown specific genes using Cas9 and synthetic guide RNAs (sgRNAs). Using this cutting edge technology, gene libraries from Synthego can be used to screen for gene knockouts which kill SCCOHT cells. Using isogenic SCCOHT cell lines that re-express SMARCA4 as a control, and testing synthetic lethality when SMARCA4 is absent, potential targets can be identified by analyzing the survival rate of the cells. Genes identified as synthetically lethal can then be tested to determine how cells are affected by their loss. Gene knockouts that only kill SCCOHT cells can lead to potential therapeutic targets. Screening with the DNA damage and repair library from Synthego has revealed multiple potential targets. Targets of interest are being validated by testing the cell growth rates with CellTiterGlo, gene expression with RT-PCR, and protein expression by western blots. The results have confirmed the use of synthetic lethality as an approach to identify vulnerabilities in SCCOHT. Future experiments include performing CRISPR screens on the remaining Synthego libraries which consist of 19,000 genes
