mtko crispr library  (Addgene inc)

Journal: Science Advances

Article Title: Impaired mitochondrial metabolism is a critical cancer vulnerability for MYC inhibitors

doi: 10.1126/sciadv.adw5228

Figure Lengend Snippet: ( A ) Experimental design for genome-wide CRISPR screen in MycCaP cells. Cells were infected with the mouse Toronto KnockOut (mTKO) CRISPR library, selected, and treated with either vehicle or MYCi975 at LD20 (3.5 μM) for 9 days or 10 population doublings before next-generation sequencing. Created in BioRender. W. Yang (2025), https://BioRender.com/6x6pegu . ( B ) Norm Z scores for all screened genes plotted against gene rank. Genes that synergize with MYCi975 (red) exhibit strongly negative Norm Z values. Key metabolic hits are color coded by function as shown. ( C ) Gene Ontology (GO) analysis of synergistic hits reveals significant enrichment of mitochondrial pathways. Point size corresponds to gene number and color intensity reflects statistical significance [−log 10 ( P value)]. ( D ) Donut chart showing the proportion of MYC-regulated and synergistic components within each mitochondrial respiratory chain complex (I to V). Complex I exhibits the highest fraction of synthetic-lethal hits (43.9%), compared to ≤12.5% in other complexes.

Article Snippet: A genome-wide CRISPR KO screen was conducted in MycCaP cells using the mTKO CRISPR library (Addgene, no. 159393, RRID:Addgene_159393), which targets 19,463 mouse genes with 94,528 sgRNAs (five sgRNAs per gene) ( , ).

Techniques: Genome Wide, CRISPR, Infection, Knock-Out, Next-Generation Sequencing

Journal: Science Advances

Article Title: Impaired mitochondrial metabolism is a critical cancer vulnerability for MYC inhibitors

doi: 10.1126/sciadv.adw5228

Figure Lengend Snippet: ( A ) Western blot confirming small interfering RNA (siRNA)–mediated MYC knockdown and sgRNA-mediated Ndufa3 KO in MycCaP cells. Actin is a loading control. ( B ) Cell viability (manual counting) in MycCaP cells subjected to indicated siRNAs/sgRNAs for 3 days. Data are means ± SEM ( n = 3); unpaired two-tailed Student’s t test. ( C ) Western blot validation of Ndufa3 or Sod2 KO in MycCaP cells using two independent sgRNAs per gene. Actin is a loading control. ( D ) Cell viability of MycCaP cells expressing control or Ndufa3/Sod2 sgRNAs treated with dimethyl sulfoxide (DMSO) or 5 μM MYCi975. Data are means ± SEM ( n = 3); one-way analysis of variance (ANOVA), Tukey’s test. ( E and F ) Expression of yeast NADH [reduced form of nicotinamide adenine dinucleotide (oxidized form)] dehydrogenase (NDI1) rescues synthetic lethality between Ndufa3 loss and MYC inhibition but not Sod2 loss. Immunoblot validation (E) and quantification of cell viability (F) in cells treated with 4 μM MYCi975. Data are means ± SEM ( n = 3); one-way ANOVA, Tukey’s test. ( G ) Analysis of DepMap AVANA dataset showing differential gene dependencies between complex I–low and complex I–high cell lines. MYC exhibits enhanced dependency in complex I–low cells (red dot). Data visualized by authors; original CRISPR-dependency scores from DepMap AVANA .

Article Snippet: A genome-wide CRISPR KO screen was conducted in MycCaP cells using the mTKO CRISPR library (Addgene, no. 159393, RRID:Addgene_159393), which targets 19,463 mouse genes with 94,528 sgRNAs (five sgRNAs per gene) ( , ).

Techniques: Western Blot, Small Interfering RNA, Knockdown, Control, Two Tailed Test, Biomarker Discovery, Expressing, Inhibition, CRISPR

Journal: Acta Neuropathologica

Article Title: Functional profiling of murine glioma models highlights targetable immune evasion phenotypes

doi: 10.1007/s00401-024-02831-w

Figure Lengend Snippet: Mechanisms of CT2A-intrinsic immune evasion. a Workflow for mTKO genome-scale pooled CRISPR screens to identify immune-evasion genes. CRISPR-mutagenized CT2A cells were propagated in the present or absence of various immune cell lines (microglia; BV-2, macrophages; Raw 264.7 and J774.1, phagocytes; J774.1 treated with anti-CD29, cytotoxic T-lymphocytes, or natural killer cells) to apply selective pressure and CT2A cells were subjected to deep sequencing to identify sgRNA that were enriched (i.e., resister genes) or depleted (i.e., sensitizer genes) relative to untreated cells. b Rank-ordered z-score of sgRNA enriched/depleted in mutagenized CT2A cells after exposure to immune cells. Hits at FDR < 5% are highlighted in yellow (resistor genes) and blue (sensitizer genes). Point size is inversely scaled by FDR. c – e STRING network analysis of myeloid c and lymphoid d sensitizer genes, and resister genes ( e ). Clusters determined by Markov clustering. Nodes represents genes, and solid and broken edges represented intra- and inter-cluster connectivity, respectively. f Precision-recall ( top ) and ROC analysis ( bottom ) illustrating recovery of core CTL sensitizers and resisters identified by Lawson et al. g Enrichment maps comparing CTL resisters ( yellow ) and resisters ( blue ) between CT2A and core sets. Nodes represent gene sets, and edges represent Jaccard similarities between gene sets. h GSEA for select pathways in in vivo ΔAtg12 CT2A tumors, compared to parental tumors, using snRNA-seq data. i Survival of C57BL/6 mice orthotopically engrafted with parental and ΔAtg12 CT2A cells. AUPRC, area under precision-recall curve; AUROC, area under receiver operating characteristic curve; CTL, cytotoxic T-lymphocytes; GSEA, gene set enrichment analysis; NK, natural killer cells; STRING, Search Tool for the Retrieval of Interacting Genes/Proteins

Article Snippet: In brief, 1.5 × 10 8 cells were infected with the mTKO lentiviral library (Addgene #159393) at an MOI of around 0.3.

Techniques: CRISPR, Sequencing, In Vivo

Journal: Acta Neuropathologica

Article Title: Functional profiling of murine glioma models highlights targetable immune evasion phenotypes

doi: 10.1007/s00401-024-02831-w

Figure Lengend Snippet: Genetic dependencies in murine and human glioblastoma. a Workflow for mTKO genome-scale pooled CRISPR screens to identify fitness genes in CT2A and GL261 cells. b Distribution of gene-level differential logFC of sgRNAs in CT2A and GL261, stratified by essentiality. Gene fitness was scored using BAGEL. c Comparison of CT2A and GL261 gene-level fitness. Scatter plot shows CT2A and GL261 scaled BFs. Scaled BF was calculated as BF – 5 such that scaled BF > 0 represents essential genes. d Ranked differential fitness between GL261 and CT2A. Y-axis for differential fitness is signed log 10 (FDR) derived from difference between scaled BF scores. e Enrichment map illustrating CT2A and GL261-specific dependencies. Nodes represent gene sets, and edges represent Jaccard similarities between gene sets. f Scatter plot of scaled BF scores for human GBM cells and non-CNS cells. Scores were retrieved from Project Score Database ( see methods ). g Ranked differential fitness between human GBM and non-CNS cell lines. Genes were ranked by signed log 10 (FDR) derived from difference between scaled BF scores. h Venn diagram of human (GBM and non-CNS) and murine (CT2A and GL261) essential genes (scaled BF > 0). i Boxplot of scaled BFs from CT2A and GL261 screens grouped by human essentiality gene sets ( as defined in f ). j Dot plot of GBM-specific fitness genes that are common to human GBM and murine gliomas. BAGEL, Bayesian analysis of gene essentiality; BF, Bayes factor; CNS, central nervous system; ETC, electron transport chain; logFC, log fold-change

Article Snippet: In brief, 1.5 × 10 8 cells were infected with the mTKO lentiviral library (Addgene #159393) at an MOI of around 0.3.

Techniques: CRISPR, Comparison, Derivative Assay