Advancing inducible gene-inactivation systems to explore synthetic lethality

Abstract Synthetic lethality offers opportunities to identify therapeutic targets for cancer research, facilitating the development of targeted tumour therapy protocols. However, current gene knockout approaches may cause compensatory changes in cellular function and, often, RNAi does not align with the ideal time window for studies and can be inconsistent, limiting the study of molecular interactions, especially when the disruption of two genes causes cell death. To circumvent this problem, we developed the IGIS (Inducible Gene-Inactivation Systems) platform, which uses transient or targeted integration of tetracycline-regulated gene-silencing constructs into human cell lines and fluorescent markers, permitting precise timing of gene inactivation, avoiding transfection variability, and enabling follow-up assays. The applicability of the IGIS systems was validated by investigating the functional interplay between the BRCA1 and RAD18 genes. Combining IGIS with cell survival, DNA fibre, BrdU alkaline comet assays, and pRPA immunostaining, we show that BRCA1 and RAD18 work in different pathways in replication-fork restart and post-replicative gap filling, and thus combined loss of these factors leads to accumulation of ssDNA gaps and replication catastrophe. This synthetic-lethal interaction study with our newly developed IGIS method highlights RAD18-dependent tolerance mechanisms as potential therapeutic vulnerabilities in BRCA-deficient tumours.