crispr brunello lentiviral pooled library plasmid #73138  (Addgene inc)

Journal: bioRxiv

Article Title: Glycosylphosphatidylinositol biosynthesis restricts coronavirus infection via the regulation of LY6E

doi: 10.1101/2025.02.08.637211

Figure Lengend Snippet: A. Workflow of the genome-wide CRISPR screening. A549-ACE2 or HeLa cells expressing the Cas9 were transduced with a CRISPR knockout sgRNA library, followed by infection with coronaviruses expressing fluorescent protein reporter. Infected reporter-positive cells were sorted for genomic extraction and sgRNA sequence analysis. B-D. Genes identified from the CRISPR screens in A549-ACE cells using SARS-CoV-2 trVLP-GFP (B) , HCoV-OC43-mGreen (C) , or PEDV-GFP (D) at an MOI of 0.1 for 24 h. The genes were analyzed by MAGeCK software and sorted based on the -log 10 (MAGeCK score). E-G. KEGG pathway analysis of 100 top-ranked genes from the screens in B-D .

Article Snippet: The human Brunello CRISPR knockout pooled library encompassing 76,441 different sgRNAs targeting 19,114 genes was a gift from David Root and John Doench (Addgene #73178) and was packaged in HEK293T cells after co-transfection with psPAX2 and pMD2.G at a ratio of 2:2:1 using Fugene ® HD (Promega).

Techniques: Genome Wide, CRISPR, Expressing, Transduction, Knock-Out, Infection, Extraction, Sequencing, Software

Journal: bioRxiv

Article Title: Glycosylphosphatidylinositol biosynthesis restricts coronavirus infection via the regulation of LY6E

doi: 10.1101/2025.02.08.637211

Figure Lengend Snippet: A. Genes identified from the CRISPR knockout screen in HeLa cells using PEDV-GFP. The genes were analyzed by MAGeCK software and sorted based on the -log 10 (MAGeCK score). B. Validation of the 50 top-ranked genes from the screen in HeLa cells. Two independent sgRNAs per gene were used, and cells were infected with PEDV (MOI 0.5, 24 h) and infection was assessed by flow cytometry. Data shown are pooled from three independent experiments, and each performed in duplicate.

Article Snippet: The human Brunello CRISPR knockout pooled library encompassing 76,441 different sgRNAs targeting 19,114 genes was a gift from David Root and John Doench (Addgene #73178) and was packaged in HEK293T cells after co-transfection with psPAX2 and pMD2.G at a ratio of 2:2:1 using Fugene ® HD (Promega).

Techniques: CRISPR, Knock-Out, Software, Biomarker Discovery, Infection, Flow Cytometry

Journal: bioRxiv

Article Title: Glycosylphosphatidylinositol biosynthesis restricts coronavirus infection via the regulation of LY6E

doi: 10.1101/2025.02.08.637211

Figure Lengend Snippet: A. The sequence traces of the gene locus of WT (upper) and PIGA-, PIGV -, or GPAA1 -knockout (bottom) HeLa cells. The sgRNA target site is indicated, and knockout efficiency was determined using Inference of CRISPR Edits (ICE) analysis.

Article Snippet: The human Brunello CRISPR knockout pooled library encompassing 76,441 different sgRNAs targeting 19,114 genes was a gift from David Root and John Doench (Addgene #73178) and was packaged in HEK293T cells after co-transfection with psPAX2 and pMD2.G at a ratio of 2:2:1 using Fugene ® HD (Promega).

Techniques: Sequencing, Knock-Out, CRISPR

Journal: bioRxiv

Article Title: Glycosylphosphatidylinositol biosynthesis restricts coronavirus infection via the regulation of LY6E

doi: 10.1101/2025.02.08.637211

Figure Lengend Snippet: A. Schematic of focused CRISPR knockout screen of known or predicted GPI-AP genes. The sub-library of 772 sgRNAs targeting 193 known or predicted GPI-AP genes was generated, and the screens were conducted in A549-ACE2 cells infected with SARS-CoV-2 trVLP-GFP, HCoV-OC43-mGreen, HCoV-229E-mGreen, or PEDV-GFP at an MOI 0.5 for 24 h. Infected GFP-positive cells were sorted for genomic extraction, sequencing, and sgRNA analysis with MAGeCK software. B. The results of focused CRISPR knockout screening with four coronaviruses. The genes were ranked based on the -log 10 (MAGeCK score). C. Venn diagram analysis of the 10 top-ranked genes from each screen. D-G. Validation of the 11 genes combined from the 5 top-ranked genes from each infection screen in A549-ACE2 cells. Cells were infected with SARS-CoV-2 trVLP-Nluc (MOI 1, 15 h), HCoV-OC43 (MOI 0.5, 48 h), HCoV-229E (MOI 0.75, 48 h), or PEDV (MOI 1, 48 h), followed by flow cytometry analysis. Data shown are from four independent experiments. D-G, two-way ANOVA with Dunnett’s test; the mean of two sgRNAs was compared with the control sgRNA; mean ± s.d.; *P < 0.05; ****P < 0.0001; ns, not significant.

Article Snippet: The human Brunello CRISPR knockout pooled library encompassing 76,441 different sgRNAs targeting 19,114 genes was a gift from David Root and John Doench (Addgene #73178) and was packaged in HEK293T cells after co-transfection with psPAX2 and pMD2.G at a ratio of 2:2:1 using Fugene ® HD (Promega).

Techniques: CRISPR, Knock-Out, Generated, Infection, Extraction, Sequencing, Software, Biomarker Discovery, Flow Cytometry, Control