human geckov2 crispr knockout pooled libraries  (Addgene inc)

Journal: Molecular Cancer

Article Title: Identification of HSPE1 as a new actionable cancer vulnerability leads to an innovative and effective combination therapy for pancreatic ductal adenocarcinoma

doi: 10.1186/s12943-026-02587-9

Figure Lengend Snippet: In vivo genome-wide CRISPR screening in PDAC. a Schematic representation of the loss-of-function genome-wide screen using the human lentiviral CRISPR/Cas9 library (GeCKOv2) library A in pancreatic ductal adenocarcinoma cell line (HPAF-II). b Average mass of extracted tumor from NSG mice subcutaneous transplanted with 30 million cells and grown for 28 days. Mean of three independent infection replicate experiments ( n = 5, 1 mouse per biological replicate was randomly selected for deep sequencing). Data are represented as mean ± SEM. c Normalized read count distribution from sequenced amplicons. d The unmapped percentage of sgRNAs in the library in cells before transplantation ( n = 3), and tumor samples ( n = 3) on day 28. e Statistical dispersion graphic (Gini index) of the sgRNA distribution within samples from cells and tumor replicates. f Cumulative distribution function (CDF) of library sgRNAs in the cell representation and tumor sample replicates. Shifts in tumor samples reflect altered read counts in subset of sgRNAs. g Pearson correlation of the sgRNA reads between all samples from in vitro and in vivo

Article Snippet: Human genome-wide CRISPR/cas9 knockout pooled library GeCKOv2 was a gift from Feng Zhang (Addgene#1,000,000,048).

Techniques: In Vivo, Genome Wide, CRISPR, Infection, Sequencing, Transplantation Assay, Dispersion, In Vitro

Journal: Frontiers in Pharmacology

Article Title: Genome-wide CRISPR screen identified NEK6 as a determinant of sensitivity to CDK4/6 inhibitor in endometrial cancer

doi: 10.3389/fphar.2025.1725886

Figure Lengend Snippet: Genome-scale CRISPR knockout screen and candidate identification. (A) Schematic representation of the CRISPR knockout screen in human endometrial cancer HEC-1A cells. (B) KEGG analysis the top 250 palbociclib negatively selected hits in the CRISPR-Cas9 screen. (C) The top 10 candidates that were most significantly depleted after palbociclib selection are ranked by a modified robust ranking aggregation (RRA score). (D) NEK6 expression in endometrial cancer was analyzed using RNA-Seq datasets from the TCGA database. (E) NEK6 gene expression level from GEPIA ( http://gepia.cancer-pku.cn ). It includes 174 TCGA-UCSC samples and 91 GTEx normal endometrial samples. (F) NEK6 was detected in 47 EC tissues and 19 normal endometrial tissues by qRT-PCR. (G) Representative images of NEK6 in paracancerous and EC tissues detected on a TMA. (H) Quantification of NEK6 staining intensities in human EC. The Y-axis shows the percentage of tumors with negative/weak, moderate and strong IHC staining.

Article Snippet: Human GeCKO v2 CRISPR knockout pooled library was a gift from Feng Zhang (Addgene # 1 000 000 048).

Techniques: CRISPR, Knock-Out, Selection, Modification, Expressing, RNA Sequencing, Gene Expression, Quantitative RT-PCR, Staining, Immunohistochemistry

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