gecko v2 human sgrna library  (Addgene inc)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting E2F8 sensitizes gemcitabine-resistant gallbladder cancer to PARP inhibitors by disrupting RRM2-driven DNA repair

doi: 10.1186/s13046-025-03586-2

Figure Lengend Snippet: Generation of gemcitabine-resistant GBC cell lines and genome-wide CRISPR screen for therapeutic targets ( A ) Dose-response curves and IC 50 values of gemcitabine in parental (NOZ and GBC-SD) and gemcitabine-resistant (NOZ-R and GBC-SD-R) cells, assessed by CellTiter-Glo viability assays after 5 days treatment. Data are presented as mean ± SD from three independent experiments ( B ) Volcano plots of differentially expressed genes (DEGs): NOZ-R versus NOZ (left) and GBC-SD-R versus GBC-SD (right). Significantly upregulated (red) and downregulated (blue) genes are defined by log 2 fold-change > 1 or < −1 and adjusted p < 0.05 ( C ) Venn diagrams illustrating the overlap of significantly upregulated (top) and downregulated (bottom) DEGs in NOZ-R and GBC-SD-R cells ( D ) Genome-wide CRISPR-Cas9 loss-of-function screen in NOZ-R cells under Olaparib versus DMSO treatment, highlighting top candidate genes with significant depletion (negative selection, left) or enrichment (positive selection, right) based on fold-change and p -value ( E ) Normalized sgRNA counts for the top 10 candidate genes identified from the CRISPR screen, with six sgRNAs per gene ( F ) Validation of selected screen hits (E2F8, PHF12, PTCH1 and C2orf73) by CRISPR-mediated knockout in NOZ-R and GBC-SD-R cells, followed by treatment with Olaparib (1 µM) or DMSO for 5 days. Cell viability was measured using CellTiter-Glo. Data represent mean ± SD from three independent experiments. Statistical significance was determined using two-tailed Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001; ns: not significant)

Article Snippet: A genome-wide CRISPR-Cas9 loss-of-function screen was performed using the human GeCKO v2 pooled sgRNA library (Addgene, #52961), which encodes both sgRNAs and Cas9 in a single lentiviral construct.

Techniques: Genome Wide, CRISPR, Biomarker Discovery, Selection, Knock-Out, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting E2F8 sensitizes gemcitabine-resistant gallbladder cancer to PARP inhibitors by disrupting RRM2-driven DNA repair

doi: 10.1186/s13046-025-03586-2

Figure Lengend Snippet: E2F8 deficiency sensitizes gemcitabine-resistant gallbladder cancer cells to PARP inhibition. ( A ) Knockdown of E2F8 enhances sensitivity to Olaparib and gemcitabine in NOZ-R cells. Cells were transduced with control sgRNA (sgNC) or two independent sgRNAs targeting E2F8 (sgE2F8 #1 and sgE2F8 #2), followed by treatment with DMSO, Olaparib (1 µM), or gemcitabine (0.1 µM). Cell viability was measured at the indicated time points using the CellTiter-Glo assay. Data are shown as mean ± SD from three independent experiments (two-tailed t-test; * p < 0.05, ** p < 0.01, *** p < 0.001) ( B ) Apoptosis analysis of NOZ-R cells transduced with sgNC or sgE2F8 (#1 and #2) and treated with DMSO, Olaparib (1 µM), or gemcitabine (0.1 µM) for three days. Apoptotic cells were detected by Annexin V/PI staining followed by flow cytometry. Data are presented as mean ± SD from three independent experiments (t-test; *** p < 0.001) ( C ) Quantification of apoptotic cells in GBC-SD-R cells transduced with sgNC or sgE2F8 (#1 and #2) and treated with DMSO, Olaparib (1 µM), or gemcitabine (0.1 µM) for three days, as determined by Annexin V/PI staining and flow cytometry ( D - E ) Apoptosis assays in NOZ-R ( D ) and GBC-SD-R ( E ) cells stably expressing control (shNC) or E2F8-targeting shRNA (shE2F8), with or without E2F8 overexpression, followed by treatment with DMSO or Olaparib (1 µM). Apoptotic cells were quantified by flow cytometry after Annexin V/PI staining. E2F8 expression levels were confirmed by western blotting ( F ) Western blot analysis of γ-H2AX levels in NOZ, NOZ-R, GBC-SD, and GBC-SD-R cells pretreated with gemcitabine (0.5 µM) for 30 min and harvested 4 h later. Quantitative of γ-H2AX levels was performed using ImageJ software and normalized to total H2AX ( G ) Immunofluorescence detection and quantification of γ-H2AX foci per nucleus in the same panel of cell lines treated as in (F). Foci were quantified using Image-Pro Plus software. Scatter dot plots represent mean ± SD ( n = 3 independent experiments, unpaired t-test; *** p < 0.001)

Article Snippet: A genome-wide CRISPR-Cas9 loss-of-function screen was performed using the human GeCKO v2 pooled sgRNA library (Addgene, #52961), which encodes both sgRNAs and Cas9 in a single lentiviral construct.

Techniques: Inhibition, Knockdown, Transduction, Control, Glo Assay, Two Tailed Test, Staining, Flow Cytometry, Stable Transfection, Expressing, shRNA, Over Expression, Western Blot, Software, Immunofluorescence

Journal: bioRxiv

Article Title: A YIPF5-GOT1A/B complex directs a transcription-independent function of ATF6 in ER export

doi: 10.1101/2023.12.12.569033

Figure Lengend Snippet: a) Schematic of pharmacogenetic screens to identify factors that modulate the effect of secretion inhibitors. b) General procedure of CRISPR activation and CRISPR knock-out (KO) screening approaches. MCF10A cells were infected with the genome-wide lentiviral SAM (synergistic activation mediator) or GeCKO v2 sgRNA library. After selection with Brefeldin A (BFA) or FLI-06, the sgRNA distribution was determined by NGS (next generation sequencing) and analyzed using the RSA (redundant siRNA activity) algorithm in comparison with unselected control cells. c) CRISPR activation screen for Brefeldin A (BFA, 100 nM) resistance in clonal MCF10A cells using the SAM system. Displayed is the RSA analysis for sgRNA enrichment from two MCF10A-SAM clones (#9 and #28). Each dot represents one gene, gene names of selected hits are shown. d-e ) Validation of GBF1 as BFA target. qPCR expression analysis ( d ) of GBF1 and IncuCyte growth curve analysis upon BFA treatment ( e ) in MCF10A-SAM cells stably expressing sgGBF1 or non-targeting (NT) sgRNAs. Displayed is the confluency over time. f ) Principle to identify genetic perturbations that confer resistance or sensitivity to secretion inhibitors by sgRNA enrichment or depletion, respectively. g ) CRISPR a screen for FLI-06 (10 µM) resistance in clonal MCF10A cells (#9 and #28) using the SAM system. RSA analysis for both clones was performed for sgRNA enrichment (left) or sgRNA depletion (right). Each dot represents one gene. h ) CRISPR-KO screen for FLI-06 (10 µM) resistance in human BJ-Tert cells using the GeCKO sgRNA library. RSA analysis was performed for sgRNA enrichment. Each dot represents one gene. i-n ) Validation of GOLT1A , GOLT1B and YIPF5 as mediators of resistance/sensitivity to FLI-06. qPCR expression analysis ( i ) of GOLT1A and IncuCyte growth curve analysis upon FLI-06 (10 µM) treatment ( j ) in MCF10A-SAM clone #9 stably expressing sgGOLT1A or non-targeting sgRNAs. qPCR expression analysis ( k ) of GOLT1B and IncuCyte growth curve analysis upon FLI-06 treatment ( l ) in MCF10A cells stably expressing shGOLT1B or shRenilla as control. m ) Immunoblotting analysis with total protein as loading control and n ) IncuCyte growth curve analysis upon FLI-06 treatment in YIPF5 KO and YIPF5 or empty vector re-expressing MCF10A cells. For qPCR, B2M was used as housekeeping gene. Data are shown as mean of three biological replicates and error bars represent SEM ( d, e, i, j, k, l, n ). Statistical testing: one-way-ANOVA test with Tukey HSD post-hoc test ( d, e, i, j, l, n ) and unpaired two-sided Welch t-test ( k ).

Article Snippet: The genome-wide human GeCKO lentiviral sgRNA library v2 (kind gift from Feng Zhang, Addgene #1000000048), consisting of the 2 pools A and B, was amplified as previously described ( ) and the balanced library distribution was verified by NGS for both of them.

Techniques: CRISPR, Activation Assay, Knock-Out, Infection, Genome Wide, Selection, Next-Generation Sequencing, Activity Assay, Comparison, Control, Clone Assay, Biomarker Discovery, Expressing, Stable Transfection, Western Blot, Plasmid Preparation