human pooled genome wide sgrna library brunello sequences addgene addgene  (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: Cells

Article Title: Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9

doi: 10.3390/cells14151165

Figure Lengend Snippet: Overview of CRISPR-Cas9-based strategies for generating fluorescent reporter cell lines via in-frame knock-in. ( A ) Schematic illustration of the general strategy for generating fluorescent knock-in reporter cell lines. Cas9-expressing cells are transduced with a gene-specific sgRNA to introduce a double-stranded break at the target locus. A donor DNA template containing a fluorescent reporter is provided for homology-directed repair (HDR), enabling precise insertion of the reporter sequence into the genomic locus of interest. Created with BioRender.com. ( B ) Schematic representation of the dual-plasmid and single-plasmid systems. In the dual-plasmid system, the sgRNA and the donor DNA template are delivered by two separate vectors. The sgRNA plasmid features a doxycycline-inducible sgRNA expression cassette and a miRFP670 fluorescent reporter as a marker for transient expression of the plasmid. The donor DNA vector includes a 5′ homology arm, a fluorescent reporter in frame with a T2A peptide and lacking a start codon, and a 3′ homology arm. In the single-plasmid system, a multiple cloning site (MCS) was engineered into the sgRNA plasmid to enable subcloning of the donor DNA template. Other than expressing sgRNA induced by doxycycline, this plasmid also acts as the donor DNA for HDR within a limited time window after infection. Moreover, cells expressing the miRFP670 reporter from randomly integrated donor DNA can be eliminated by FACS sorting from a few days post-infection onwards. Created with BioRender.com.

Article Snippet: The Human Two Plasmid Activity-Optimized CRISPR Knockout sgRNA Library, developed by David Sabatini, Eric Lander and colleagues, was obtained from Addgene (Cat# 1000000095, Watertown, MA, USA) [ ].

Techniques: CRISPR, Knock-In, Expressing, Transduction, Introduce, Sequencing, Plasmid Preparation, Marker, Cloning, Subcloning, Infection

Journal: Cells

Article Title: Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9

doi: 10.3390/cells14151165

Figure Lengend Snippet: Generation of biallelic knockout cells with fluorescent knock-in reporter as a surrogate of gene disruption. ( A ) Workflow for generating TSPAN8 knock-out cell lines with a GFP knock-in reporter. The donor DNA template consists of an 800 bp 5′ homology arm, T2A-GFP sequence, SV40 polyA signal, and an 800 bp 3′ homology arm flanking exon 2 of the TSPAN8 gene. The TSPAN8 sgRNA target site was abolished in this donor template to prevent further DNA breaks after integration. Cas9-expressing cells were transfected with dual plasmids (sgRNA and donor template) by electroporation. Immediately following transfection, cells were treated with doxycycline (Dox) to induce sgRNA expression for 48 h. After 7–10 days of culture, cells were enriched based on GFP expression levels to isolate targeted populations. ( B ) Representative FACS plot showing the initial sorting of cells. Only cells transfected with TSPAN8 sgRNA exhibit a GFP + population, confirming successful targeting and reporter integration. The GFP + population was gated and sorted for subsequent analyses and enrichment. Both MEC and JHH5 cells were used in this experiment, with 20 µg of total plasmids (1:1 ratio of sgRNA and donor template plasmids) transfected per million cells. ( C ) FACS plot showing TSPAN8 protein expression in relation to GFP expression in the initially sorted cell population (GFP + cells gated in ( B )). Notably, the majority of GFP high cells exhibited an absence of TSPAN8 expression, confirming the efficacy of the reporter enrichment strategy. GFP high cells were sorted to further enrich the homozygous reporter cell population and establish the final cell line. ( D ) FACS analysis demonstrates that most cells in the final homozygous cell line derived from the sorted GFP high population were TSPAN8-negative. ( E ) Western blotting validates the absence of TSPAN8 protein in the homozygous reporter cell line. An established TSPAN8 knockout (KO) and non-targeting (NT) cells generated in our previous study were used as negative and positive controls, respectively, for TSPAN8 expression.

Article Snippet: The Human Two Plasmid Activity-Optimized CRISPR Knockout sgRNA Library, developed by David Sabatini, Eric Lander and colleagues, was obtained from Addgene (Cat# 1000000095, Watertown, MA, USA) [ ].

Techniques: Knock-Out, Knock-In, Disruption, Sequencing, Expressing, Transfection, Electroporation, Derivative Assay, Western Blot, Generated

Journal: Cells

Article Title: Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9

doi: 10.3390/cells14151165

Figure Lengend Snippet: Establishment of biallelic knock-in reporter cells retaining endogenous protein expression. ( A ) Schematic illustration of the targeting strategy for the TSPAN8 gene locus by CRISPR-Cas9. In this strategy, the stop codon of the human TSPAN8 gene is replaced with a T2A-eGFP sequence. The resulting mRNA from the modified allele encodes two separate proteins: TSPAN8 and GFP. ( B ) Representative FACS analysis of electroporated cells. Different dosages of plasmids were tested on MEC cells. Notably, in the absence of TSPAN8 sgRNA (i.e., non-target sgRNA), no GFP-positive cells were observed. ( C ) Representative FACS plots of the reporter cell line generated after multiple rounds of sorting of the GFP high population. The established reporter cell line exhibits high GFP and intact TSPAN8 expression. ( D ) PCR genotyping analysis of the established reporter cell line. The total GFP + cells exhibited two bands corresponding to knock-in (KI) and wild-type (WT) alleles, respectively. The WT allele is absent in the reporter cell line established from the GFP high population.

Article Snippet: The Human Two Plasmid Activity-Optimized CRISPR Knockout sgRNA Library, developed by David Sabatini, Eric Lander and colleagues, was obtained from Addgene (Cat# 1000000095, Watertown, MA, USA) [ ].

Techniques: Knock-In, Expressing, CRISPR, Sequencing, Modification, Generated

Journal: Cells

Article Title: Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9

doi: 10.3390/cells14151165

Figure Lengend Snippet: Generation of knock-in reporter cells using a single-plasmid system integrating both sgRNA and donor DNA. ( A ) Workflow for generating GFP knock-in cell lines using a single plasmid. Cas9-expressing MEC cells were transfected with the single plasmid by electroporation and cultured for 7–10 days. GFP + /miRFP670 − populations were enriched by FACS. ( B ) Representative FACS plots for cells electroporated with different amounts of single plasmid and analysed 10 days post-transduction. Transduced cells with the single plasmid lacking TSPAN8 sgRNA (i.e., non-target sgRNA) were also tested as a negative control. ( C ) Representative FACS plots of the knock-in reporter cell line generated after multiple rounds of GFP high sorting. ( D ) PCR genotyping of the established reporter cell line. The total GFP + cells exhibited bands corresponding to both knock-in (KI) and wild-type (WT) alleles. The WT allele is absent in the reporter cell line derived from the GFP high population.

Article Snippet: The Human Two Plasmid Activity-Optimized CRISPR Knockout sgRNA Library, developed by David Sabatini, Eric Lander and colleagues, was obtained from Addgene (Cat# 1000000095, Watertown, MA, USA) [ ].

Techniques: Knock-In, Plasmid Preparation, Expressing, Transfection, Electroporation, Cell Culture, Transduction, Negative Control, Generated, Derivative Assay

Journal: Cells

Article Title: Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9

doi: 10.3390/cells14151165

Figure Lengend Snippet: Generation of knock-in reporter cells by the IDLV system. ( A ) Workflow for generating GFP knock-in cell lines using the IDLV system. Cas9-expressing MEC cells were infected with lentivirus packaged by the IDLV system. Following infection, cells were treated immediately with doxycycline to induce sgRNA expression. GFP + /miRFP670 − populations were enriched by FACS. ( B ) Representative FACS plots of cells at 10 days post-infection with the indicated amount of lentivirus generated by the IDLV system. Lentivirus lacking sgRNA was used as a negative control. ( C ) Representative FACS plots showing a uniform GFP expression in the established knock-in reporter cells, established by multiple rounds of sorting of the GFP high population. ( D ) Validation of correct integration of GFP reporter into the TSPAN8 locus. Two distinct sgRNAs targeting early exons of TSPAN8 were used for validation. ( E ) PCR analysis of random integration. PCR analysis was conducted using indicated primers targeting the sequences spanning the plasmid backbone and homology arms. Unsorted cells after transduction or the established reporter cell lines by single-plasmid electroporation or IDLV were analysed. ( F ) Droplet digital PCR (ddPCR) analysis of GFP and TSPAN8 was performed to determine their genomic copy numbers. Primers targeting the GFP insert and the endogenous TSPAN8 locus were used to assess the relative abundance of each sequence. Each sample was analysed in triplicate.

Article Snippet: The Human Two Plasmid Activity-Optimized CRISPR Knockout sgRNA Library, developed by David Sabatini, Eric Lander and colleagues, was obtained from Addgene (Cat# 1000000095, Watertown, MA, USA) [ ].

Techniques: Knock-In, Expressing, Infection, Generated, Negative Control, Biomarker Discovery, Plasmid Preparation, Transduction, Electroporation, Digital PCR, Sequencing

Journal: Cells

Article Title: Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9

doi: 10.3390/cells14151165

Figure Lengend Snippet: Genome-wide CRISPR-Cas9 screen for transcriptional regulators of TSPAN8. ( A ) Schematic illustrating the workflow of the genome-scale CRISPR/Cas9 loss-of-function screen to enrich the GFP low cells. JHH5 reporter cells stably expressing Cas9 were transduced with a lentiviral sgRNA library. The GFP low cell population was enriched through repeated rounds of sorting and culture. The enriched sgRNAs were identified via next-generation sequencing (NGS). ( B ) FACS plots showing sequential enrichment of the cells with reduced GFP expression. The GFP low cells were sorted over multiple rounds, with one week of expansion between rounds. ( C ) Candidate genes were identified by comparing the sgRNA read counts between the initial pool and the final GFP low population. ( D ) Scatterplot displaying the sgRNA enrichment for selected candidate genes. ( E – G ) Representative FACS plots showing the reduction in both cell-surface TSPAN8 ( E ) and GFP reporter expression ( G ) following knockout of NF2, DYRK1A, and SOX9. ( H , I ) FACS and qPCR analysis showing that knockout of SOX9 in JHH5 parental cells significantly downregulates TSPAN8 expression. ( J ) Representative FACS plots showing knockout of NF2, DYRK1A, and SOX9 reduced cell-surface TSPAN8 expression in the human HCC cell line SNU878.

Article Snippet: The Human Two Plasmid Activity-Optimized CRISPR Knockout sgRNA Library, developed by David Sabatini, Eric Lander and colleagues, was obtained from Addgene (Cat# 1000000095, Watertown, MA, USA) [ ].

Techniques: Genome Wide, CRISPR, Stable Transfection, Expressing, Transduction, Next-Generation Sequencing, Knock-Out