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: Cell reports

Article Title: CRISPR-Cas9 screening identifies an IRF1-SOCS1-mediated negative feedback loop that limits CXCL9 expression and antitumor immunity.

doi: 10.1016/j.celrep.2023.113014

Figure Lengend Snippet: Figure 5. Targeting SOCS1 in macrophages enhances CXCL9 expression CRISPR-Cas9 editing was used to generate control or Irf1- or Socs1-deficient bone marrow-derived macrophages as per Figure 3E. 1 3 105 macrophages were stimulated for 17 h with 1 ng/mL IFNg. (A and B) Expression of Cxcl9 determined (A) by flow cytometry and (B) by cytometric bead array. *p < 0.05, ****p < 0.0001, two-way ANOVA. (C) MA plot of RNA-seq analysis performed on control and Socs1 KO bone marrow-derived macrophages stimulated with 1 ng/mL IFNg for 17 h.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited data Raw data This paper GSE237974 Murine scRNA sequencing data Qu et al.6 GSE150970 Human scRNA sequencing data Bassez et al.29 EGAS00001004809 STAT1 CHIPseq Hogg et al.28 GSE94134 Experimental models: Cell lines Mouse: AT-3 Trina Stewart N/A Mouse: E0771 Robin Anderson N/A Mouse: B16F10 ATCC CRL-6475 Human: OVCAR-3 ATCC HTB-161 Human: MCF7 ATCC HTB-22 Human: HEK293T ATCC CRL-3216 Experimental models: Organisms/strains Mouse: Human-Her2 (hHer2) Bred in house N/A Mouse: OTI Bred in house N/A Mouse: C57BL/6 WEHI N/A Mouse: C57BL/6 Rag1 / WEHI, Australian Bioresources N/A Mouse: Ly5.1 WEHI, Australian Bioresources N/A Oligonucleotides See Table S3 for sgRNA Sequences Synthego N/A See Table S4 for Primer Sequences Integrated DNA Technologies N/A Recombinant DNA Plasmid: FUCas9Cherry Aubrey et al.45 Addgene Plasmid:70182 Pooled library: Mouse CRISPR Knockout Pooled Library (Brie) Doench et al.46 Addgene Cat:73633 Pooled library: Mouse CRISPR Knockout Pooled Library (Gecko) Sanjana et al.47 Plasmid: pMDLg/pRRE Dull et al.48 Addgene Plasmid:12251 Plasmid: pRSV-Rev Dull et al.48 Addgene Plasmid:12253 Plasmid: pMD2.G Dider Trono: Trono Laboratory Packaging and Envelope Plasmids (unpublished) Addgene Plasmid:12259 Homology repair template: Cxcl9-GFP Knock In (See Table S5 for sequence) This Paper N/A Homology repair template: BFP into Irf1 Knock In (See Table S5 for sequence) This Paper N/A Homology repair template: Irf1 + BFP into Irf1 Knock In (See Table S5 for sequence) This Paper N/A Software and algorithms CutAdapt Martin.49 https://cutadapt.readthedocs.io/en/stable/ MAGeCK v0.5.7 Li et al.50 https://sourceforge.net/projects/mageck/ RNASeQC DeLuca et al.51 www.broadinstitute.org/rna-seqc/ HISAT2 Kim et al.52 https://github.com/DaehwanKimLab/ hisat2 EdgeR v3.8.5 Robinson, McCarthy, and Smyth53 McCarthy, Chen, and Smyth54 https://bioconductor.org/packages/ release/bioc/html/edgeR.html Seurat v4.0.4 Stuart et al.55 https://github.com/satijalab/seurat/ releases/tag/v4.0.4 GENIE3 v1.16.0 Huynh-Thu et al.56 https://github.com/vahuynh/GENIE3 Bowtie2 v2.3.3 Langmead et al.57 https://bowtie-bio.sourceforge.net/index. shtml (Continued on next page) Cell Reports 42, 113014, August 29, 2023 19

Techniques: Expressing, CRISPR, Control, Derivative Assay, Cytometry, RNA Sequencing

Journal: Molecular Therapy Oncolytics

Article Title: Advancements in CRISPR screens for the development of cancer immunotherapy strategies

doi: 10.1016/j.omto.2023.100733

Figure Lengend Snippet: CRISPR screens in T cells to develop advanced T-cell-based products for cancer immunotherapy

Article Snippet: Mouse , CD4 + T cells , loss of function , library pMSCV-U6gRNA(lib)-PGKpuroT2ABFP (Addgene: #104861) , retrovirus transduction , expression of IRF4, XBP1, or GATA3 , Pparg and Bhlhe40 , PPARG (peroxisome proliferator activated receptor gamma) and BHLHE40 (basic-helix-loop-helix protein 40) , PPARG and BHLHE40 are crucial to TH2 gene regulation and differentiation. Genes regulating TH2 activation and genes regulating TH2 differentiation are highly overlapped. , Henriksson et al. (2019) .

Techniques: CRISPR, Transduction, Selection, Expressing, Activation Assay, Retroviral, Genome Wide, Knock-Out, In Vitro, Activity Assay, Electroporation, Membrane, Adoptive Transfer Assay, Transgenic Assay, Infection, In Vivo, Migration, Biomarker Discovery, Immunofluorescence, Staining, Flow Cytometry