gecko v2 human crispr pooled libraries (a and b)  (GenScript corporation)

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Identification of host factors essential for PEDV infection via genome-wide CRISPR/Cas9 screening. ( A ) Overview of the genome-wide CRISPR/Cas9 screening process conducted in Huh7 cells. Cas9-expressing Huh7 cells were transduced with a genome-wide sgRNA lentiviral library and then infected with PEDV at an MOI of 0.01 or 0.1. Surviving cells were harvested, and sgRNAs were amplified via PCR, followed by quantification of their abundance via next-generation sequencing. The sgRNA abundance from the genome-wide CRISPR/Cas9 screen is shown for infections with 0.01 MOI ( B ) or 0.1 MOI ( C ) of PEDV. The x -axis indicates the number of sgRNAs, whereas the y -axis represents the log 10 value of the normalized sgRNA reads. The 10 most enriched sgRNAs are highlighted in blue and red for clarity. ( D ) IFITM3-wild-type (WT) and IFITM3-KO Huh7 cells were inoculated with PEDV SD at an MOI of 0.05 or 0.1. At 12 h post-infection, the cells were fixed and visualized via immunofluorescence staining with a mouse monoclonal antibody targeting the PEDV nucleocapsid ( N ) protein (red). The cell nuclei were stained with DAPI (blue). Scale bar: 200 µm. ( E ) The viral RNA in the supernatants was quantified by quantitative RT-PCR and is presented as the viral RNA copy number per milliliter. The error bars indicate the standard deviations (s.d.) of three biological replicates ( n = 3). ( F ) Infectious PEDV particles in the supernatants of IFITM3-WT and -KO Huh7 cells were assessed via the TCID 50 assay. The error bars indicate the s.d. from three independent experiments. ***, P < 0.001.

Article Snippet: The human GeCKO v2 CRISPR knockout pooled library module B (Addgene #1000000049) containing 58,028 sgRNAs targeting 19,050 genes was a gift from Feng Zhang ( ).

Techniques: Infection, Genome Wide, CRISPR, Expressing, Transduction, Amplification, Next-Generation Sequencing, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: eLife

Article Title: Ribosome subunit attrition and activation of the p53–MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition

doi: 10.7554/eLife.90683

Figure Lengend Snippet: ( A ) Tier 1 screen: daily cell counts of MV4;11 Cas9 and MV4;11 Cas9+GeCKOv2 (Library) populations treated with either DMSO or 2 µM C6. The two replicates of this screen are shown separately. ( B ) Normalized counts of each sgRNA (x-axis) in the GeCKOv.2 library targeting TP53 in the initial transduced cells (red; not visible on this scale) and the C6-treated population (blue). Data represents means of replicates; * indicates false discovery rate (FDR) < 0.05. ( C ) As in ( B ) but for sgRNAs targeting CDKN2A . ( D ) Schematic of the CDKN2A gene locus with indicated sites complementary to tier 1 and tier 2 screen sgRNAs. Red sgRNAs increase in representation in CRISPR screens. ( E ) miRNet 2.0 analysis of the 27 miRNAs enriched in the tier 1 screen produced a single significant hit corresponding to the KEGG p53 signaling pathway. The miRNAs are represented as blue boxes and target genes as red circles; the connections between them are indicated. ( F ) As in ( B ) but for sgRNAs targeting RPL22 . ( G ) Volcano plots, showing gene-level changes from the tier 2 screen in sgRNA representation in C6- (left) and C16- (right) treated populations compared to DMSO control cultures (orange indicates FDR < 0.05). ( H ) Graph depicting gene-level Log2 FC and FDR values for genes that were flagged as C6- (squares) or C16- (circles) specific in the tier 2 screen. ( I ) GO enrichment analysis of the 57 C6/C16 common genes emerging from tier 2 of the screen. Italics represent the number of genes in each category.

Article Snippet: Briefly, the Human GeCKOv2 CRISPR Knockout Pooled Library (A+B) in the lentiGuide-Puro vector backbone (gift from Feng Zhang; Addgene plasmid # 1000000048) was amplified and purified as directed by Addgene.

Techniques: CRISPR, Produced, Control

Journal: eLife

Article Title: Ribosome subunit attrition and activation of the p53–MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition

doi: 10.7554/eLife.90683

Figure Lengend Snippet:

Article Snippet: Briefly, the Human GeCKOv2 CRISPR Knockout Pooled Library (A+B) in the lentiGuide-Puro vector backbone (gift from Feng Zhang; Addgene plasmid # 1000000048) was amplified and purified as directed by Addgene.

Techniques: Recombinant, Plasmid Preparation, CRISPR, Knock-Out, Protease Inhibitor, Staining, Reverse Transcription, Mass Spectrometry, Transfection, Gene Knockout, Negative Control, Cell Culture, Plex Assay, Bicinchoninic Acid Protein Assay, Western Blot, Sensitive Assay, Cell Viability Assay, Software, Luminex, Real-time Polymerase Chain Reaction, Imaging

Journal: Journal of Virology

Article Title: A Genome-Wide CRISPR-Cas9 Screen Identifies the Dolichol-Phosphate Mannose Synthase Complex as a Host Dependency Factor for Dengue Virus Infection

doi: 10.1128/JVI.01751-19

Figure Lengend Snippet: Haploid CRISPR-Cas9 screen identifies DPM1 and -3 as host factors for DENV infection. (A) Schematic representation of the CRISPR-Cas9 screen to identify host factors for DENV2 JAM in HAP1 cells. (B) Results of the DENV2 JAM screen analyzed by MAGeCK. Each circle represents individual gene. Genes of interest were colored according to their biological pathways. The y axis represents the significance of sgRNA enrichment of genes in the selected population compared to the unselected control population. The x axis represents a random distribution of the genes. (C) Sanger sequencing of DPM1 and DPM3 in control and DPM1 and DPM3 KO cells. PAM, protospacer adjacent motif. (D) Immunoblot of DPM1 in control, DPM1KO, and DPM3KO cells. (E) Cell surface CD59 staining on control and DPM1KO or DPM3KO HAP1 cells. (F) Control, DPM1KO, and DPM3KO HAP1 cells were plated and cell viability was assessed over a 72-h period using the CellTiter-Glo assay. (G and H) Control and DPM1 or -3 KO HAP1 cells were challenged with DENV2 JAM (MOIs of 2 and 20 in panel G and MOI of 5 in panel H). Levels of infection were quantified 48 hpi by flow cytometry using MAb 2H2 (G) or by immunofluorescence using MAb 2H2 or antibodies against NS3 (H). (I) Quantification of the viral particles released in the supernatant of inoculated HAP1 cells collected at 48 hpi. Virus titer was determined on Vero E6 cells by flow cytometry. FIU, flow cytometry infectious units. (C, D, E, F, and H) All data are representative of results from at least two independent experiments. (G and I) Data are means ± SD from three independent experiments performed in duplicate. Significance was calculated using a two-way ANOVA with Dunnett’s multiple-comparison test. n.s, nonsignificant. ***, P < 0.001; ****, P < 0.001.

Article Snippet: The GeCKO v2 human CRISPR pooled libraries (A and B) encompassing 123,411 different sgRNAs targeting 19,050 genes were purchased from GenScript.

Techniques: CRISPR, Infection, Control, Sequencing, Western Blot, Staining, Glo Assay, Flow Cytometry, Immunofluorescence, Virus, Comparison

Journal: Journal of Virology

Article Title: A Genome-Wide CRISPR-Cas9 Screen Identifies the Dolichol-Phosphate Mannose Synthase Complex as a Host Dependency Factor for Dengue Virus Infection

doi: 10.1128/JVI.01751-19

Figure Lengend Snippet: Key resources a

Article Snippet: The GeCKO v2 human CRISPR pooled libraries (A and B) encompassing 123,411 different sgRNAs targeting 19,050 genes were purchased from GenScript.

Techniques: Sequencing, Virus, Control, CRISPR, Mutagenesis