knockout crispr library version 2  (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: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet: Receptor-ligand CRISPR-Cas9 activation screen reveals that CD55 interacts with HLA-C∗07:01-VRIG tetramers (A) Schematic of the receptor ligand CRISPR-Cas9 activation screen. K562 cells transduced with a genome-wide activation library were stained with a pool of three HLA tetramers (HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, and HLA-C∗07:01-VRIG), and enriched gRNAs in stained cells were identified using NGS. (B) SigmaFC scores of genes from two replicate screens. SigmaFC scores were calculated using PinAplPy, and top hits are annotated. (C) K562 cells stably expressing dCas9 and transduced with a gRNA upregulating CD55 or a control guide were stained with the HLA-A, -B, -C, or tetramers as in (A) or with HLA-E∗01:01-VMAP tetramers and analyzed by flow cytometry. (D) In vitro co-immunoprecipitation of recombinant CD55-Fc with HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, HLA-C∗07:01-VRIG, or HLA-E∗01:01-VMAP tetramers. (E) Three different cell lines (HeLa, PC-3M, or SiHa) that express CD55 endogenously were stained for CD55 (top) or with HLA-C∗07:01-VRIG tetramers (bottom) and analyzed by flow cytometry. (F) HeLa wild-type or HeLa CD55 KO cells were stained with αCD55 or HLA-C∗07:01-VRIG tetramers and analyzed by flow cytometry. All data except (B) represent at least three independent experiments. CRISPRa, CRISPR activation screen; TMs, tetramers; WT, wild-type; KO, knockout. Related to Figure S1 and Table S1 .

Article Snippet: Genome-wide mouse CRISPR Brunello knockout library , Dr. D Root and Dr. J Doench , Addgene cat: 73178.

Techniques: CRISPR, Activation Assay, Transduction, Genome Wide, Staining, Stable Transfection, Expressing, Control, Flow Cytometry, In Vitro, Immunoprecipitation, Recombinant, Knock-Out

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet: CRISPR-Cas9 activation and KO screens identify an interaction between HLA-C∗07:02-YRFR and heparan sulfate chains (A) HeLa cells were stained with a variety of HLA-C∗07:02 tetramers loaded with different peptides and analyzed by flow cytometry. (B) The indicated cell lines were stained with HLA-C∗07:02-YRFR tetramers and analyzed by flow cytometry. Staining is normalized to unstained levels of that cell line. (C) Schematic of CRISPR-Cas9 KO screen in MelJuSo cells. Cells were stained with HLA-C∗07:02-YRFR, and enriched gRNAs in non-binding cells were identified using NGS. (D) SigmaFC scores of genes from two replicate screens. SigmaFC scores were calculated using PinAplPy, and genes involved in the heparan sulfate biosynthesis pathway are depicted in purple. Hits used for further validation are annotated. (E) Schematic of the CRISPR-Cas9 activation screen. K562 cells stably expressing dCas9 transduced with a genome-wide activation library were stained with HLA-C YRFR∗07:02 tetramers, and enriched gRNAs in positive cells were identified. (F) SigmaFC scores of genes from two replicate screens. SigmaFC scores were calculated using PinAplPy, and top hits are annotated. Proteoglycans of interest are depicted in purple. CRISPRa, Crispr activation screen. Related to Figure S3 , Table S1 and .

Article Snippet: Genome-wide mouse CRISPR Brunello knockout library , Dr. D Root and Dr. J Doench , Addgene cat: 73178.

Techniques: CRISPR, Activation Assay, Staining, Flow Cytometry, Binding Assay, Biomarker Discovery, Stable Transfection, Expressing, Transduction, Genome Wide

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet:

Article Snippet: Genome-wide mouse CRISPR Brunello knockout library , Dr. D Root and Dr. J Doench , Addgene cat: 73178.

Techniques: Virus, Recombinant, Blocking Assay, Genome Wide, Activation Assay, CRISPR, Knock-Out, Mutagenesis, Plasmid Preparation, Software, Imaging