mtKO: A dedicated guide RNA library for mitochondria research

Abstract

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.

Mitochondria are multifunctional organelles essential for energy metabolism, electron transport, ROS generation and detoxification, calcium regulation, and apoptosis and their functions are extensively reprogrammed in cancer to support rapid proliferation and Warburg-like metabolism (1). While mitochondrial involvement in oncogenesis, malignant transformation, and tumor progression is evident, its precise role—particularly in cancers driven by Krebs cycle deficiencies such as those involving IDH, SDH, or FH mutations—remains unclear (2). IDH1-mutant cancers, for example, exhibit vulnerability to oxidative stress and ferroptosis due to altered mitochondrial function shaped by distinct metabolic networks (3). Mitochondria are largely governed by nuclear-encoded proteins imported posttranslation, yet how nuclear signaling reprograms mitochondrial function during malignant progression—especially under metabolic constraints—remains poorly understood (4). To address this, we developed the mtKO guide RNA library targeting key mitochondria-associated genes across major pathways, enabling pooled screens to identify mitochondrial dependencies in normal and malignant cells. We identified altered mitochondrial dependencies in IDH1-mutated cancer cells for maintaining redox and metabolic homeostasis and contributing to tumor aggressiveness.

Results

To identify essential genes among mitochondria-associated genes, we queried the MitoCarta3.0 database (5–7) and built a Cas9 guide RNA (gRNA) library targeting nuclear-encoded 1,034 mitochondrial genes to establish the mtKO library, with essential, nonessential, and nontargeting guides as controls. With this robust tool, we performed a CRISPR knockout screen in RPE1 cells harboring the pathogenic IDH1^R132H mutation to explore the essentiality of mitochondrial pathways under oncogenic metabolic perturbation (Fig. 1A). High-throughput Illumina sequencing demonstrated excellent performance of the mtKO library (Fig. 1B). We identified consistent depletion of genes such as DNLZ, GPX4, and MTG2 (Fig. 1C). Conversely, enrichment was observed for SLC25A23 and REXO2. Notably, we found a differential dependency on superoxide dismutase 2 (SOD2), a key mitochondrial antioxidant enzyme. We observed that SOD2 exhibited mild depletion in IDH1^WT cells, whereas a strong and significant depletion was found in IDH1^R132H cells (Fig. 1 D and E). Besides, mtKO screens on patient-derived glioma stem cells (GSC) confirmed the connection between IDH1 mutation status and SOD2 dependency. The SOD2 guide RNA depletion was found significant in IDH1^R132H TS603 cells, whereas it was insignificant in IDH1^WT GS7-11 cells (Fig. 1F). This essential role of SOD2 is further evidenced in patient cohorts with IDH1-mutant glioma, where elevated SOD2 expression correlates with poorer outcomes and reduced overall survival (Fig. 1G).

Fig. 1.

mtKO screen to identify mitochondrial essential pathway in IDH1-mutant cells. (A) Schematic illustration shows mtKO screen in IDH1-mutant cells. (B) Bar graph shows changes in guide RNA abundancy for core essential genes (CEG) and nonessential genes (NEG). Each bar represents one biological replicate. (C) Gene-level guide RNA depletion in wild type (IDH1^WT) and mutant (IDH1^R132H) screens. Significantly depleted (blue) or enriched (orange) were highlighted. Nonsignificant gene was labeled in green. (D) Plot of gene depletion based on RPE1 IDH1^WT and IDH1^R132H screen. (E) Quantitative changes of SOD2 guide RNAs through the RPE1 cell mtKO screen. (F) Quantitative changes of SOD2 guide RNAs through the GSC mtKO screen. (G) Kaplan–Meier analysis reveals the predictive value of SOD2 expression in IDH1-mutant glioma datasets CGGA, TCGA LGG, and Gorovets (GSE35158).

To investigate the role of SOD2 in IDH1-mutated cancers, we generated SOD2 knockout (KO) derivatives of the IDH1-mutated cholangiocarcinoma cell line JJ012. Transcriptomic analysis revealed that SOD2 loss led to significant downregulation of pathways associated with cancer aggressiveness, including the replisome, electron transport chain (ETC), ribosome, and RNA metabolism (Fig. 2A). Additionally, SOD2 deficiency resulted in notable alterations in cellular proliferation and metabolic profiles, characterized by reduced gene set enrichment for oxidative phosphorylation (OXPHOS), ferroptosis pathway, E2F-mediated transcription, and mitochondria transportation pathway (MT TRANSPORT, Fig. 2B).

Fig. 2.

Validation of gene essentiality of SOD2. (A) RNA sequencing-based network analysis shows depleted genes and pathways in SOD2 KO vs. parental JJ012 cells. (B) GSEA on the significantly depleted pathways. (C) Mitochondrial ROS measurement in JJ012 parental (Par) and SOD2 KO cells using MitoSox flow cytometry. (D) Statistical analysis on MitoSox signal measured in C (**P < 0.01). (E) Statistical analysis on MitoSox signal ROS measurement in GSC (**P < 0.01). (F) Mitochondrial membrane potential measurement in JJ012 cells using JC-1 flow cytometry (**P < 0.01). (G) Dose response curve for JJ012 cells exposed to RSL3. (H) Phase contrast microscopy shows the morphology of JJ012 cells exposed to RSL3 and/or Ferr-1. (I) Cell proliferation measurement in JJ012 cells. (J) Measurement of JJ012 xenograft. (**P < 0.01). (K) Quantification of EdU positive labeled GSC with SOD2 RNA interference (**P < 0.01).

To investigate the role of SOD2 in the aggressive phenotype of IDH1-mutant cancers, we assessed mitochondrial function in SOD2-deficient (SOD2^KO) cells. SOD2 loss significantly increased mitochondrial reactive oxygen species (ROS) levels (Fig. 2 C and D). Similarly, SOD2 depletion led to markedly elevated mitochondrial ROS in IDH1^R132H GSC403 cells, but not in IDH1^WT GS7-11 cells (Fig. 2E). Assessment of mitochondrial membrane potential (ΔΨm) using JC-1 dye revealed a significantly impaired mitochondrial function in SOD2^KO cells (Fig. 2F). Furthermore, SOD2^KO cells showed increased sensitivity to RSL3-induced cell death, which was rescued by the ferroptosis inhibitor ferrostatin-1 (Fer-1, Fig. 2H). Further, SOD2 loss significantly impaired the proliferation of IDH1^R132H JJ012 cells in vitro and reduced the oncogenic potential of JJ012 subcutaneous xenografts (Fig. 2 I and J). Similarly, SOD2 depletion reduced proliferation in IDH1^R132H GSC403 and TS603 cells, but not in IDH1^WT GSC268 or GS7-11 cells (Fig. 2K). Collectively, these findings indicate that SOD2 supports the survival and growth of IDH1-mutant cancer cells by limiting mitochondrial ROS and protecting against ferroptosis, thereby promoting tumor progression.

Discussion

In this study, we developed a gRNA library, mtKO, to investigate essential mitochondrial pathways cancer cells with Krebs cycle deficiency as an alternative approach from existing platform (8). Further, we identified SOD2 as a novel metabolic “Achilles heel” in IDH1-mutated cells. SOD2 was found to be indispensable in the IDH1-mutated genetic background but not in their wild-type counterparts (Fig. 1 C and D), which predicts unfavorable disease outcomes (Fig. 1E).

Our study emphasizes the critical role of antioxidant pathways in cancers with Krebs cycle deficiencies. Evolutionarily conserved mechanisms, notably the Nrf2-guided glutathione metabolism, are vital for these malignancies, influencing disease progression and therapeutic responses (9, 10). Our findings highlight the mitochondrial antioxidant enzyme SOD2 as central to maintaining organelle function (Fig. 2 A and B), through detoxifying mitochondrial reactive oxygen species, preventing ferroptosis, and contributing to disease manifestation (Fig. 2 C–K). Targeting SOD2-guided antioxidant pathway may be valuable to impact the metabolic foundation of these malignancies with improved outcome.

Materials and Methods

Development of mtKO Library.

The list of human mitochondria-associated genes was obtained from MitoCarta 3.0 database (7). A total of 6,204 guides targeting 1,034 of these genes were obtained from the Brunello Human CRISPR Knockout Pooled Library (Addgene #73178) or designed using Guidescan (11). To establish the complete mtKO library, the following controls were included: guides targeting core essential genes (CEG) (1,409 genes, 8,454 guides), nonessential genes (NEG) (405 genes, 2,430 guides), and intergenic nontargeting regions (435 guides) (12, 13). See SI Applendix for details.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

All study data are included in the article and/or SI Appendix.