human crispr knockout pooled library brunello  (Addgene inc)

Journal: mBio

Article Title: Soluble MFGE8 mediates cell entry of Crimean-Congo hemorrhagic fever virus

doi: 10.1128/mbio.01617-25

Figure Lengend Snippet: CRISPR knockout screen identifies common host factors required for rVSV-CCHFV pseudovirus infection. ( A ). Bubble plot of genes significantly enriched in a genome-wide CRISPR knockout screen in wild-type A549 (A549-WT) cells challenged with rVSV-CCHFV pseudovirus. The virus-resistant A549-WT cells were collected for analysis, and genes were ranked according to the MAGeCK score. ( B ) KEGG (Kyoto Encyclopedia of Genes and Genomes) and Go (Gene Ontology) analysis of top 100 enriched genes. ( C and D ) Flow cytometry ( C ) and fluorescence imaging ( D ) analysis of A549-WT and A549-BAT (B3GAT3, AXL, and TIM-1 triple-knockout cells) infected with rVSV-CCHFV (MOI 3). The percentage of GFP-positive cells was analyzed at indicated time points using flow cytometer, and images were taken using fluorescence microscope at 24 h post-infection (hpi). Scale bar, 400 µm. Two-way ANOVA with Sidak’s multiple-comparison test. **** P < 0.0001.

Article Snippet: The human Brunello CRISPR knockout pooled library targeting 19,114 genes (Addgene #73178) or Calabrese activation pooled library targeting 18,885 genes (Addgene #92379) was a gift from David Root and John Doench ( ) and packaged in 293 FT cells after co-transfection with psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) using FugeneHD (Promega).

Techniques: CRISPR, Knock-Out, Infection, Genome Wide, Virus, Flow Cytometry, Fluorescence, Imaging, Triple Knockout, Microscopy, Comparison

Journal: mBio

Article Title: Soluble MFGE8 mediates cell entry of Crimean-Congo hemorrhagic fever virus

doi: 10.1128/mbio.01617-25

Figure Lengend Snippet: CRISPR activation screen identifies MFGE8 as a proviral host factor for rVSV-CCHFV infection. ( A ) Identification of genes from CRISPR screen in A549-BAT cells. Cells transduced with the CRISPR activation library were infected with rVSV-CCHFV for 24 h. GFP-positive cells were sorted for sgRNA abundance analysis and ranked based on the MAGeCK score and P value. ( B and C ) Validation of MFGE8 gene. Gene expression was activated using two or representative sgRNAs in A549-BAT cells, followed by infection with rVSV-CCHFV (MOI 3, 18 h) ( B ) and rVSV (MOI 0.01, 15 h) ( C ). The percentage of GFP-positive cells were analyzed by flow cytometry. ( D ) Representative fluorescence images of A549-BAT cell infected with respective virus from ( B ) and ( C ) were taken before harvesting the cells. Scale bar, 400 µm. ( E ) Overexpression of MFGE8 enhances rVSV-CCHFV infection in A549-BAT cells. ( F ) Growth kinetics of rVSV-CCHFV in vector control and MFGE8-overexpressing cells. Cells were infected with rVSV-CCHFV at an MOI of 0.3, and viral titers in the supernatants at indicated time points were determined by plaque-forming assay. ( G–I ) Overexpression of MFGE8 enhances rVSV-CCHFV infection in A549-WT ( G ), Hela ( H ), and SW-13 ( I ) cells. The percentage of GFP-positive cells were analyzed by flow cytometry at 16 hpi. (J) Knockout of MFGE8 decreases the rVSV-CCHFV infection. A549-WT cells edited with two different nontargeting control or MFGE8 -specific sgRNAs were infected with rVSV-CCHFV, followed by flow cytometry analysis of GFP-positive cells at 16 hpi. Two-way ANOVA with Sidak’s multiple-comparison test. ns, not significant; *** P < 0.001; **** P < 0.0001.

Article Snippet: The human Brunello CRISPR knockout pooled library targeting 19,114 genes (Addgene #73178) or Calabrese activation pooled library targeting 18,885 genes (Addgene #92379) was a gift from David Root and John Doench ( ) and packaged in 293 FT cells after co-transfection with psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) using FugeneHD (Promega).

Techniques: CRISPR, Activation Assay, Infection, Transduction, Biomarker Discovery, Gene Expression, Flow Cytometry, Fluorescence, Virus, Over Expression, Plasmid Preparation, Control, Knock-Out, Comparison