crispr-ko screening library  (Twist Bioscience)

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