mtKO: A dedicated guide RNA library for mitochondria redox biology research

Mitochondria are multifunctional organelles essential for both physiological regulation and pathological progression. In malignant cancer cells, mitochondrial reprogramming establishes a metabolic foundation that supports tumor growth, which is essential for cancer cells to overcome intrinsic metabolic abnormalities and stress. To uncover key mitochondrial pathways involved in cancer development, we developed mitochondrial Knockout (mtKO) - a robust, flexible, and unbiased CRISPR/Cas9 guide RNA screening platform designed to systematically identify critical mitochondria-associated functions. The mtKO library target genes involved in diverse mitochondrial-guided processes, including biosynthesis, transmembrane transport, oxidative phosphorylation, and redox regulation. Through a mtKO dropout screen, we identified the mitochondrial antioxidant enzyme superoxide dismutase 2 (SOD2) as indispensable for the fitness and survival of cancer cells harboring oncogenic mutations in isocitrate dehydrogenase 1 (IDH1). Mechanistically, SOD2 mitigates mitochondrial reactive oxygen species (ROS) generated by dysfunctional Krebs cycle activity in IDH1-mutant glioma and chondrosarcoma cells. Functionally, SOD2 maintains redox homeostasis and preserves mitochondrial integrity thereby controlling disease manifestation both in vitro and in vivo . Overall, our study introduces a powerful functional genomics approach to interrogate mitochondrial biology and uncovers a selective mitochondrial redox vulnerability in Krebs cycle-deficient cancers, highlighting mitochondrial redox imbalance as a potential therapeutic target.