Defining redox-dependent DNA damage response vulnerabilities after ionizing radiation using arrayed combination CRISPR screening and oxidative lesion imaging

Kurzfassung

Background Ionizing radiation threatens genomic integrity during long-duration space missions by inducing complex DNA damage through direct ionization and secondary reactive oxygen species. Internal biological countermeasures are needed, as physical shielding is limited by spacecraft mass constraints. Materials & Methods This study proposes the “Shield and Repair” model, where cellular redox pathways (“shield”) and DNA damage response pathways (“repair axis”) function redundantly to maintain genomic stability. Previously published gene screens were evaluated to establish predicted synthetic lethality pairs between redox and DNA repair pathways. High-throughput screening using machine learning further profiled genes via ionizing radiation and hydrogen peroxide sensitivity. A fluorescent reporter plasmid was engineered utilizing the SB100x transposon system to enable stable integration and real-time monitoring of Base Excision Repair dynamics. Functional validation employed an arrayed combinatorial CRISPR-Cas9 knockout matrix in hTERT-RPE1p53KO cells. Knockouts were processed, verified, and tested. Results The screening strategy for identifying synthetically lethal gene pairs was established and yielded numerous redox and DNA damage response targets. Initial investigation involved knockout generation, exposure to 2 Gy x-ray radiation, and ATP-based luminescence assays to assess survivability as a measure of possible synthetic lethal interactions. Thus far, pairs show decreased survivability but no definitive synthetic lethal interactions. However, immunofluorescence analysis of γ-H2AX foci suggests altered double-strand break signaling in double knockout backgrounds. Generation of the fluorescent reporter plasmid was successful and will allow improved visualization of altered repair dynamics. Further verification of CRISPR knockout screens with radiation is needed. Conclusion This work provides a framework for understanding synergy between metabolic detoxification and DNA repair, highlighting molecular vulnerabilities that could guide biological countermeasures for deep-space missions and precision strategies for cancer radiotherapy.