Journal: Innate Immunity

Article Title: A genome-wide CRISPR screen identifies regulation factors of the TLR3 signalling pathway

doi: 10.1177/1753425920915507

Figure Lengend Snippet: Outline of the genome-wide CRISPR/Cas9 forward genetic screen to identify genes required for TLR3 signalling. (a) Transduction of KBM7 cells with TLR3 cDNA leads to the expression of both full-length (*) and cleaved/mature (**) TLR3. Immunoblot of cell lysates of KBM7 cells complemented by the indicated cDNA was revealed by an anti-TLR3 mAb. As a loading control, tubulin was revealed with a specific mAb. (b) Design of the forward genetic screen in human near-haploid KBM7 cells. Creation of the NF-κB reporter KBM7 cell line by transduction of three independent expression cassettes encoding for dscGFP, TLR3 and Cas9. DscGFP expression was controlled by a NF-κB dependent promoter. Cells were transduced with the lentiviral GeCKO v2 sgRNA library (122,411 sgRNAs). Cells that were successfully transduced were selected with puromycin. After selection, the population was split into two; half was not sorted to represent the entire library, while the remaining population was sorted by flow cytometry after poly(I:C) stimulation to enrich for dscGFP negative cells. After three rounds of poly(I:C) stimulation/enrichment, the DNA from the enriched populations (rounds 1, 2 and 3) was harvested, and enriched sgRNAs were identified by sequencing and compared to an unsorted library. FACS plots obtained after each sort on dscGFP-negative cells show progressive enrichment rates of dscGFP-negative cells. (c) and (d) Proportion of cells responding to poly(I:C) as judged by dscGFP expression before and after sorting during the sequential enrichment process. Polyclonal or clonal cell populations were stimulated with poly(I:C; twice at 35 µg/ml, 4 h apart) or TNF-α (10 ng/ml) for 16 h before sorting at each round. In both cases, the proportion of dscGFP negative cells increased at each round of enrichment. sgRNA: single-guide RNA; NF-κB TRE: NF-κB transcription responsive element; min CMV: minimal CMV; dscGFP: destabilized copepod GFP; SFFV: spleen focus-forming virus promoter; 2A: peptide bond skipping sequence; BFP: blue fluorescent protein: PGK: phosphoglycerate kinase promoter; Hygro: hygromycin resistance gene.

Article Snippet: The MOI of the GeCKO v2 sgRNA library A and B (generously deposited by Prof. Feng Zhang ; #1000000047; Addgene) was estimated on cells exposed to various volumes of viruses and cultured with or without puromycin before counting the remaining cells.

Techniques: Genome Wide, CRISPR, Transduction, Expressing, Western Blot, Control, Selection, Flow Cytometry, Sequencing, Virus

Journal: Innate Immunity

Article Title: A genome-wide CRISPR screen identifies regulation factors of the TLR3 signalling pathway

doi: 10.1177/1753425920915507

Figure Lengend Snippet: Genome-wide screen of the TLR3 pathway identifies expected and new genes. (a) –log P value of the results of sgRNA enrichments identified by sequencing, corresponding to 42 genes. After one, two or three rounds of enrichment, the DNA from the enriched cell population was harvested, and enriched sgRNAs were identified by sequencing and comparison to an unsorted library. This determination was carried out for the two independent screens performed in parallel, and sequencing was performed twice generating a total of 12 enriched cell populations. Genes are presented when –log( P value) > 4 for at least 2/12 comparisons. Each dot represents the gene enrichments calculated from one condition. The RSA algorithm was used to identify the significantly enriched genes targeted in the selected cells. Red: known key members of TLR3 pathway; blue: further validated genes. (b) Plot illustrating the hits from the genetic screen. Mean log( P values) determined with the clonal population as a function of those found with polyclonal population. Each dot is a gene. Red squares: known key members of the TLR3 pathway; light blue triangles: genes further validated; grey dots: genes further tested; density colours: remaining genes out of the 42 found in panel (a).

Article Snippet: The MOI of the GeCKO v2 sgRNA library A and B (generously deposited by Prof. Feng Zhang ; #1000000047; Addgene) was estimated on cells exposed to various volumes of viruses and cultured with or without puromycin before counting the remaining cells.

Techniques: Genome Wide, Sequencing, Comparison

Journal: Innate Immunity

Article Title: A genome-wide CRISPR screen identifies regulation factors of the TLR3 signalling pathway

doi: 10.1177/1753425920915507

Figure Lengend Snippet: DLX1, SOD1 and AhR are required for a proper TLR3 response. (a) Heat map of the GFP expression results obtained with KBM7Rep cells transduced with the various sgRNAs ( n = 3 independent experiments). KBM7Rep cells transduced with the indicated sgRNAs were exposed to increasing doses of poly(I:C) or TNF-α (10 ng/ml) for 16 h before measurement of the GFP expression by FACS. The cluster of genes containing two sgRNAs targeting UNC93B1 and one targeting TLR3 is magnified. red: positive controls; blue: genes further studied; grey: negative controls. (b) NF-κB activity or (c) IL-8 and (d) IP-10 were measured by FACS or cytometric bead assay (CBA), respectively, in response to poly(I:C) exposure of the indicated cells (two-way ANOVA between one sgRNA condition and ctrl Cas9 ctrl). (e), (f) and (g) The indicated parameters were measured in the absence or presence of TNF-α ( n = 3 independent experiments). *, **, *** or ****: significantly different from ctrl Cas9 (Friedman test, with Dunn’s multiple comparison test between sgRNA conditions and Cas9 ctrl). UNC: UNC93B1. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: The MOI of the GeCKO v2 sgRNA library A and B (generously deposited by Prof. Feng Zhang ; #1000000047; Addgene) was estimated on cells exposed to various volumes of viruses and cultured with or without puromycin before counting the remaining cells.

Techniques: Expressing, Transduction, Activity Assay, Comparison

Journal: eLife

Article Title: DBT is a metabolic switch for maintenance of proteostasis under proteasomal impairment

doi: 10.7554/eLife.91002

Figure Lengend Snippet: ( A ) Workflow of the CRISPR screen in retinal pigment epithelium (RPE1) cells, which were transduced with a lentiviral Genome-Scale CRISPR Knock-out (GeCKO) single guide RNA (sgRNA) library and selected for the sgRNA expression and then survival after treatments with the proteasome inhibitor MG132. Individual surviving cell colonies were collected for sequencing and subsequent analysis. ( B ) Left: The cytotoxicity analysis of wild-type (WT) and DBT knockout (KO) RPE1 cells treated with MG132 at different doses for 96 hr (n=3). Right: The time course analysis of MG132-induced cytotoxicity in the WT and DBT KO cells (n=3). ( C ) Immunoblot analysis of WT RPE1, DBT KO, and DBT’ cells. The DBT’ cells expressed an engineered DBT cDNA that resisted DBT-targeted Cas9 cleavage and rescued the DBT expression in the KO cells. ( D ) Cell viability was measured by Calcein-AM staining in WT RPE1, DBT KO, and DBT’ cells treated with MG132 (2 μM, 96 hr). Scale bar, 100 μm. ( E ) Quantification of the cell viability measured by Calcein-AM staining in ( D ) (n=9). ( F ) Left: Immunoblot analysis of RPE1 cells transfected with DBT shRNAs and non-targeting control shRNAs. Right: Quantification of the cell viability under treatment with MG132 (2 μM, 48 hr), as measured by Calcein-AM staining (n=4). ( G ) Immunoblotting and quantification of cleaved PARP as an MG132-induced cell death marker (n=4). ( H ) Immunoblotting and quantification of cleaved Caspase 3 as an MG132-induced cell death marker (n=3). Error bars represent means ± SEM. *p≤0.05; **p≤0.01; ****p≤0.0001. Figure 1—source data 1. Original and uncropped blots for . Figure 1—source data 2. Original and uncropped blots for . Figure 1—source data 3. Original and uncropped blots for . Figure 1—source data 4. Original and uncropped blots for .

Article Snippet: Briefly, 6×10 7 RPE1 cells were infected with the human sgRNA library (Human GeCKO v2 Library Cat#1000000048, Addgene) at an MOI of ~0.3.

Techniques: CRISPR, Transduction, Knock-Out, Expressing, Sequencing, Western Blot, Staining, Transfection, Control, Marker

Journal: Advances in Virus Research

Article Title: Functional Genomic Strategies for Elucidating Human–Virus Interactions

doi: 10.1016/bs.aivir.2015.11.001

Figure Lengend Snippet: Functional Genomic Screens for Elucidating Host–Viral Interactions

Article Snippet: Next, we stably transduced the H1-HeLa-Cas9 cells at a moi of 0.2 with a complex lentiviral pool expressing the human GeCKO v.2 sgRNA library (Addgene #1000000049), which targets 19,052 genes in the human genome with six sgRNAs per gene across two half-libraries (library A and B) ( ).

Techniques: Functional Assay, Virus, Knockdown, Selection, Biomarker Discovery, Suspension, Mutagenesis, Immunofluorescence, Microscopy, TALENs, Flow Cytometry, Comparison, CRISPR, Recombinant, Expressing, Luciferase, Disruption, Infection, Activity Assay, Inhibition, Western Blot, Genome Wide, In Vivo, Construct, Over Expression, Standard Deviation, Membrane, Staining, Viability Assay, In-Cell ELISA, Transfection, Immunoprecipitation, Control, Northern Blot, Fluorescence, Gene Expression, Microarray, Dominant Negative Mutation, Transmission Assay, Electron Microscopy, Transduction, Amplification, Clone Assay, Enzyme-linked Immunosorbent Assay, Modification, Two Hybrid Assay, Plaque Assay, Single Vesicle Fusion Assay, shRNA, Binding Assay, Sequencing, esiRNA, Glycoproteomics, Blocking Assay, Cell Cycle Assay, Ubiquitin Proteomics, Conjugation Assay, Plasmid Preparation, Mass Spectrometry, Reverse Transcription, Polymerase Chain Reaction

Journal: Advances in Virus Research

Article Title: Functional Genomic Strategies for Elucidating Human–Virus Interactions

doi: 10.1016/bs.aivir.2015.11.001

Figure Lengend Snippet: CRISPR/Cas9 screen for HRV host factors. (A) The HRV-HF CRISPR/Cas9 screen workflow showing the generation of the Cas9 expressing H1-HeLa cells containing the sgRNA libraries followed by their subsequent challenge with HRV14 and the assessment of the enriched sgRNAs using next-gen sequencing. (B) HeLa-H1-Cas9 cells were transduced with Moloney Leukemia virus (MLV)-GFP, then supra-transduced with either an empty vector control (parent population) or one expressing a sgRNA against GFP. The cells were selected for puromycin resistance and cultured for 11 days then fixed and imaged for GFP expression. Differential interference contrast (DIC) images are provided below. 4 × magnification. (C) DIC images of cells transduced with either library A or B that survived the HRV14 challenge were expanded and tested for their susceptibility to HRV14’s cytopathic effect over 2 days (bottom row) compared to the unselected parent cell population and the respective uninfected cell populations (top row). (D) Cells from (C) were fixed and immunostained for ICAM1 surface expression by flow cytometry. (E) A chart showing the relative proportion of total sequencing reads for the recovered sgRNAs from the HRV14 CRISPR/Cas9 pooled screen based upon the analysis of genomic DNA from the surviving cells from library A or B. Gene names are provided for each sgRNA with the associated numbers designating their unique identifying library number.

Article Snippet: Next, we stably transduced the H1-HeLa-Cas9 cells at a moi of 0.2 with a complex lentiviral pool expressing the human GeCKO v.2 sgRNA library (Addgene #1000000049), which targets 19,052 genes in the human genome with six sgRNAs per gene across two half-libraries (library A and B) ( ).

Techniques: CRISPR, Expressing, Sequencing, Transduction, Virus, Plasmid Preparation, Control, Cell Culture, Flow Cytometry