genome wide crispr cas9 screen  (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: Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy

Article Title: A quiescence-like/TGF-β1-specific CRISPRi screen reveals drug uptake transporters as secondary targets of kinase inhibitors in AML.

doi: 10.1016/j.drup.2025.101242

Figure Lengend Snippet: Fig. 5. CRISPRi screening reveals a potential link between vactosertib-induced resistance to Ara-C and alterations in drug uptake mechanisms in AML cells. (A) Schematic of the CRISPRi library screen workflow. THP1-dCas9-KRAB cells were transduced with CRISPRi library at an MOI of approximately 0.3 and selected with puromycin. Cells were treated with either Ara-C alone or in combination with vactosertib for 48 hours and were maintained for 21 days in culture. sgRNA sequences were amplified from extracted DNA and analyzed by NGS. MAGeCK was used for data processing. (B) and (C) Volcano plots indicating sgRNA distribution in vactosertib + Ara-C and Ara-C alone conditions, respectively. Data represent three independent biological replicates (n = 3). For every gene, the x-axis displays its enrichment (positive hits) or depletion (negative hits) after treatment, while the y-axis shows the score as calculated by MAGeCK. Relevant hits were annotated. (D) Validation of CRISPRi library screen results by cell growth competition assay. THP1-dCas9-KRAB cells were transduced with sgENT1 (two independent sgRNAs: sgENT1#1 and sgENT1#2), sgDCK, sgNT and sgMYBL2. The effect of each sgRNA on cell proliferation under different treatment conditions with vactosertib and Ara- C was determined by tracking the percentage of BFP-positive cells over time using flow cytometry. Results are shown as the percentage of BFP-positive cells at the indicated times relative to day 0 (n = 3).

Article Snippet: A drug-focused CRISPR interference (CRISPRi) library screen reveals TGFBR1 as the primary mediator of the quiescence-like phenotype induced by TGF-β1 To explore potential therapeutic interventions targeting TGF-β1induced dormancy, we employed a CRISPR interference (CRISPRi) screening approach using a sgRNA library targeting kinases, phosphatases and drug targets (Addgene #83971) (Horlbeck et al., 2016).

Techniques: Transduction, Amplification, Biomarker Discovery, Competitive Binding Assay, Flow Cytometry

Journal: Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy

Article Title: A quiescence-like/TGF-β1-specific CRISPRi screen reveals drug uptake transporters as secondary targets of kinase inhibitors in AML.

doi: 10.1016/j.drup.2025.101242

Figure Lengend Snippet: Fig. 4. Vactosertib-induced Ara-C resistance phenotype is not mediated through the TGF-β signaling pathway. (A-D) XTT cytotoxicity assays demonstrating the effects of TGF-β1 signaling pathway component knockdowns on Ara-C response: (A) TGFBR1, (B) ACVR1B, (C) double knockdown of TGFBR1 and ACVR1B, and (D) SMAD4. Knockdown efficiency for each target gene was validated by qPCR, with results shown in the inset graphs (n = 3, two-tailed t-test). (E) Schematic repre sentation of the RNA sequencing experimental design. (F) Volcano plot depicting significantly deregulated genes (red: upregulated, blue: downregulated) in THP1 cells upon treatment with TGF-β1 (10 ng/mL), vactosertib (10 μM), or a combination of TGF-β1 + vactosertib (10 ng/mL and 10 μM, respectively) (p-value < 0.05, | log fold change| > 1.5). (G) Heatmap illustrating the expression of negative feedback regulators of TGF-β signaling including SMAD6, SMAD7, TGFBR1, SKI, SMURF, and SPP1 in THP1, HL60, and MV4-11 cells following treatment with TGF-β1 (10 ng/mL), vactosertib (10 μM), or a combination of TGF-β1 + vactosertib (10 ng/mL and 10 μM, respectively). All p-values were less than 0.05 (two-tailed t-test). Color intensity values correspond to fold change. p-value significance is represented * ** , < 0.001.

Article Snippet: A drug-focused CRISPR interference (CRISPRi) library screen reveals TGFBR1 as the primary mediator of the quiescence-like phenotype induced by TGF-β1 To explore potential therapeutic interventions targeting TGF-β1induced dormancy, we employed a CRISPR interference (CRISPRi) screening approach using a sgRNA library targeting kinases, phosphatases and drug targets (Addgene #83971) (Horlbeck et al., 2016).

Techniques: Knockdown, Two Tailed Test, RNA Sequencing, Expressing

Journal: Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy

Article Title: A quiescence-like/TGF-β1-specific CRISPRi screen reveals drug uptake transporters as secondary targets of kinase inhibitors in AML.

doi: 10.1016/j.drup.2025.101242

Figure Lengend Snippet: Fig. 5. CRISPRi screening reveals a potential link between vactosertib-induced resistance to Ara-C and alterations in drug uptake mechanisms in AML cells. (A) Schematic of the CRISPRi library screen workflow. THP1-dCas9-KRAB cells were transduced with CRISPRi library at an MOI of approximately 0.3 and selected with puromycin. Cells were treated with either Ara-C alone or in combination with vactosertib for 48 hours and were maintained for 21 days in culture. sgRNA sequences were amplified from extracted DNA and analyzed by NGS. MAGeCK was used for data processing. (B) and (C) Volcano plots indicating sgRNA distribution in vactosertib + Ara-C and Ara-C alone conditions, respectively. Data represent three independent biological replicates (n = 3). For every gene, the x-axis displays its enrichment (positive hits) or depletion (negative hits) after treatment, while the y-axis shows the score as calculated by MAGeCK. Relevant hits were annotated. (D) Validation of CRISPRi library screen results by cell growth competition assay. THP1-dCas9-KRAB cells were transduced with sgENT1 (two independent sgRNAs: sgENT1#1 and sgENT1#2), sgDCK, sgNT and sgMYBL2. The effect of each sgRNA on cell proliferation under different treatment conditions with vactosertib and Ara- C was determined by tracking the percentage of BFP-positive cells over time using flow cytometry. Results are shown as the percentage of BFP-positive cells at the indicated times relative to day 0 (n = 3).

Article Snippet: A drug-focused CRISPR interference (CRISPRi) library screen reveals TGFBR1 as the primary mediator of the quiescence-like phenotype induced by TGF-β1 To explore potential therapeutic interventions targeting TGF-β1induced dormancy, we employed a CRISPR interference (CRISPRi) screening approach using a sgRNA library targeting kinases, phosphatases and drug targets (Addgene #83971) (Horlbeck et al., 2016).

Techniques: Transduction, Amplification, Biomarker Discovery, Competitive Binding Assay, Flow Cytometry