single guide rna sgrna library  (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

Journal: Nature Communications

Article Title: The O-glycosyltransferase C1GALT1 promotes EWSR1::FLI1 expression and is a therapeutic target for Ewing sarcoma

doi: 10.1038/s41467-025-56632-0

Figure Lengend Snippet: a Schematic of the reporter cell line construction (top) and genome-scale CRISPR/Cas9-based screening strategy (bottom). Created in BioRender.com . b False-discovery rate (FDR) plot showing the distribution of sgRNAs (shown as red lines) targeting candidate genes enriched in the tdTomato low EGFP high population. P -values were calculated using a two-tailed Fisher’s Exact test with Benjamini-Hochberg adjustment to control for FDR in the context of multiple comparisons. c Representative immunoblot showing EWSR1::FLI1 protein levels (monitored using an anti-FLI1 antibody) in A673 cells stably expressing an shRNA targeting each of the nine validated candidates, or as control a NS or EWSR1 shRNA. β-actin (ACTB) was monitored as a loading control. d qRT-PCR analysis monitoring relative EWSR1::FLI1 mRNA levels following knockdown of each of the nine candidates. EWSR1::FLI1 mRNA was detected using a primer pair spanning the fusion junction and is shown relative to that obtained with an NS shRNA, which was set to 1. Data are presented as mean ± SEM ( n = 3 biologically independent experiments). P -values were calculated using one-way ANOVA with post-hoc Dunnett’s multiple comparisons test. Source data are provided as a Source Data file.

Article Snippet: For the genome-wide CRISPR/Cas9 knockout screen, the A673/EWSR1::FLI1 tdTomato /EGFP/Cas9 reporter cell line was transduced with the Human CRISPR Knockout Pooled Library (Brunello) (Addgene, Cat# 73178) , consisting of ~76,441 sgRNAs targeting 19,114 genes at multiplicity-of-infection (MOI) of 0.3.

Techniques: CRISPR, Two Tailed Test, Control, Western Blot, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Knockdown

Journal: bioRxiv

Article Title: JNK pathway suppression drives resistance to combination endocrine therapy and CDK4/6 inhibition in ER+ breast cancer

doi: 10.1101/2025.01.08.631992

Figure Lengend Snippet: (A) A positive selection CRISPR/Cas9 screen was conducted in ER+ MCF-7-Cas9 expressing cells using the human Brunello library. Cells were treated with the combination of 500 nM tamoxifen and 250 nM palbociclib (early: 6 week and late: 10 week timepoint), or 500 nM monotherapy palbociclib (early: 2 week and late: 4 week timepoint) or vehicle (tetrahydrofuran) before genomic DNA was collected and sequenced. (B) Venn diagram showing single guide RNAs (sgRNAs) that increased following treatment with palbociclib and tamoxifen + palbociclib. sgRNAs selected with a false discovery rate <0.5, and occurrence in ≥3 screens, where at least one screen was with combination therapy. Selected sgRNAs indicated with dashed white line. (C) Gene set enrichment analysis of commonly downregulated genes with ≥2.5 aggregate β-score. (D) Top sgRNAs enriched following treatment with palbociclib and tamoxifen + palbociclib, and corresponding β-scores (sgRNAs with aggregate β-score ≥5 are shown). Orange * indicates sgRNAs in the JNK pathway. Black * indicates sgRNAs described in the literature to drive CDK4/6 inhibitor resistance. (E) Schematic of the JNK signalling pathway and enriched sgRNAs, as well as aggregate β- score scale. P indicates phosphorylation events.

Article Snippet: The human genome-wide pooled Brunello library (containing 77,441 sgRNAs) was provided by the VCFG (Addgene #73178).

Techniques: Selection, CRISPR, Expressing