mtKO: A dedicated guide RNA library for mitochondria research.

Mitochondria are multifunctional organelles central to both physiological and pathological processes. In malignant cancer cells, mitochondrial reprogramming establishes the metabolic foundation to meet cellular demands, which is particularly important in tumor cells with existing metabolic perturbations. To identify key mitochondrial pathways supporting cancer development, we developed mitochondria Knockout (mtKO), a robust and unbiased CRISPR screening platform to pinpoint critical mitochondria-associated pathways. The mtKO screen revealed that the mitochondrial antioxidant enzyme SOD2 is essential for cells harboring IDH1 mutations. Mechanistically, SOD2 activity determines the disease manifestation of IDH1-mutated cancers, through maintaining redox homeostasis and mitochondrial fitness. This study introduces a powerful functional genomic tool to identify mitochondrial-centered pathways and reveals the selective mitochondrial vulnerability in Krebs cycle-deficient cancers for future therapeutic intervention.