Journal: Research Square

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

doi: 10.21203/rs.3.rs-555275/v1

Figure Lengend Snippet: a. Schematic of pooled screen to identify SARS-CoV-2 regulators in Vero E6 cells. b. Scatter plot showing the gene-level mean z-scores of genes when knocked out in Vero E6 cells. The top genes conferring resistance to SARS-CoV-2 are annotated and shown in blue. c. Comparison between this Vero E6 screen to the Vero E6 screen conducted by the Wilen lab 27. Genes that scored among the top 20 resistance hits and sensitization hits in both screens are labeled. d. Venn diagram comparing hits across screens conducted in Vero E6, A549, and Huh7 (or derivatives) cells (ectopically expressing ACE2 and TMPRSS2 or not) 23–28. The top 20 genes from each cell line are included, with genes considered a hit in another cell line if the average z-score was > 3.

Article Snippet: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described .

Techniques: Comparison, Labeling, Expressing

Journal: Research Square

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

doi: 10.21203/rs.3.rs-555275/v1

Figure Lengend Snippet: Properties of SARS-CoV-2 host factor screens assayed by cell viability. For each library, the number of unique guides per gene is indicated in parentheses. The essential gene QC serves as a metric for screen quality (see Methods ) in the untreated arm, when applicable; the number of days post-library introduction until the end of the experiment is written after the semicolon.

Article Snippet: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described .

Techniques:

Journal: Research Square

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

doi: 10.21203/rs.3.rs-555275/v1

Figure Lengend Snippet: Calu-3-Cas9 cells were stably transduced to express 2 different sgRNAs (g1, g2) per indicated gene and selected for 10–15 days. a. Cells were infected with SARS-CoV-2 bearing the mNG reporter and the infection efficiency was scored 48h later by flow cytometry. b. The expression levels of ACE2 were analyzed by immunoblot, Actin served as a loading control. c. Relative surface ACE2 expression was measured using a Spike-RBD-Fc fusion and a fluorescent secondary antibody followed by flow cytometry analysis. d. Cells were incubated with SARS-CoV-2 at MOI 5 for 2h at 37°C and then treated with Subtilisin A followed by RNA extraction and RdRp RT-qPCR analysis as a measure of viral internalization. e. Cells were infected with Spike del19 and VSV-G pseudotyped, GFP expressing VSV and infection efficiency was analyzed 24h later by flow cytometry. f. Cells were infected with SARS-CoV-2 at MOI 0.05 and, 24h later, lysed for RNA extraction and RdRp RT-qPCR analysis. g. Aliquots of the supernatants from F were harvested and plaque assays were performed to evaluate the production of infectious viruses in the different conditions. h. Cells were infected with MERS-CoV and 16h later, infectious particle production in the supernatant was measured by TCID 50 . The mean and SEM of at least 5 ( a ), 3 ( c , d , e , f , h ) independent experiments or representative experiments ( b and g ) are shown. The red dashed line represents 50% inhibition ( a , c-f ).

Article Snippet: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described .

Techniques: Stable Transfection, Infection, Flow Cytometry, Expressing, Western Blot, Control, Incubation, RNA Extraction, Quantitative RT-PCR, Inhibition

Journal: Research Square

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

doi: 10.21203/rs.3.rs-555275/v1

Figure Lengend Snippet: sgRNA sequences used for CRISPR KO perturbations

Article Snippet: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described .

Techniques: CRISPR, Sequencing

Journal: Research Square

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

doi: 10.21203/rs.3.rs-555275/v1

Figure Lengend Snippet: sgRNA sequences used for CRISPRa perturbations

Article Snippet: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described .

Techniques: Sequencing

Journal: Cells

Article Title: NSP4 and ORF9b of SARS-CoV-2 Induce Pro-Inflammatory Mitochondrial DNA Release in Inner Membrane-Derived Vesicles

doi: 10.3390/cells11192969

Figure Lengend Snippet: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins NSP4 and ORF9b synergistically induce the extracellular release of mitochondrial DNA (mtDNA). ( a ) Heatmap showing the integrated density of mtDNA associated with the mitochondria in A549 cells transfected with 29 SARS-CoV-2 proteins. A549 cells were individually transfected with the constructs encoding the proteins and probed with the anti-TFAM antibodies. Images were acquired at 24 h post-transfection and subjected to densitometric analysis. The combination of actinomycin D, ABT-737, and zVD (ActD + ABT + zVD) (treatment for 2 h) was used as a positive control for the mtDNA release experiments. ( b ) Quantitative analysis of images of the indicated groups probed with the anti-TFAM (representing mtDNA inside mitochondria) antibodies (n = 9). A549 cells were transduced (48 h) with lentiviral-encoded shortlisted SARS-CoV-2 protein plasmids (as shown in ( a )) and probed with the anti-TFAM antibodies. Images were subjected to densitometric analysis. Corresponding representative images are shown in . Data are plotted as integrated density. ( c ) Quantification of Cyt c levels in the mitochondria. A549 cells transduced with the selected plasmids were subsequently probed with the anti-Cyt c antibodies and imaged after 48 h. Images were subjected to densitometric analysis (n = 9). Data are plotted as integrated density. ( d , e ) Representative flow cytometry histograms of cells stained with tetramethylrhodamine, ethyl ester (TMRE) (representing ΔΨm), and mitoSOX (representing mitochondrial reactive oxygen species (mtROS)) under the conditions mentioned in ( b , c ). ( f ) Heatmap of mtDNA and Cyt c release in A549 cells transduced with the selected protein-encoding constructs, which upregulated mtROS and downregulated ΔΨm. The values were calculated from the analysis data shown in ( b – e ). ( g ) Quantification of mtDNA release (n = 9) from cells co-transduced with various plasmids in the images. The combination of NSP4 and ORF9b robustly promoted the release of mtDNA. Data are plotted as integrated density. ( h ) Representative confocal images showing the localization of mtDNA (probed with anti-TFAM antibodies; magenta) with the mitochondria (probed with the anti-TOM20 antibodies; green) in cells transduced with the combination of NSP4 and ORF9b (N4 + 9b). Insets show the association of mtDNA with the mitochondria in vector (VEC)-transduced cells ( blue arrowheads ). In infected cells, mtDNA is mostly localized outside the mitochondria ( yellow arrowheads ) or the mitochondria are devoid of mtDNA ( red arrowheads ). ( i ) Representative immunofluorescence images showing Cyt c (red) localization with the mitochondria (probed with the anti-TOM20 antibodies; green) in cells transduced with VEC or N4 + 9b. ( j ) Quantitative analysis of images shown in ( i ) (n = 10). Data are plotted as integrated density. ( k ) Immunoblot of Cyto c in the cytosolic extract prepared from cells after transduction with N4+9b or VEC. GAPDH was used as a loading control. ( l ) Representative immunoblot showing the expression of TFAM in the cytosol. GAPDH served as the reference control. The cytosolic extract was prepared from cells transduced with VEC or N4 + 9b (48 h). The combination of ActD + ABT + zVD (treatment for 2 h) was used as the positive control for the experiments evaluating mtDNA release. ( m ) Representative immunoblots of concentrated cell supernatant from the cells treated as indicated in ( k ). E-Cadherin (E-Cad) was used as the reference control. ( n ) Densitometry analysis of proteins in the immunoblots shown in ( k , l ) (n = 4). Data are plotted as the ratio of TFAM and the reference control. ( o ) Representative immunoblot showing TFAM (pulled down using the anti-TFAM antibodies; mtDNA marker) expression in the immunoprecipitated samples. Chromatin immunoprecipitation (ChIP) was performed using the concentrated cell supernatant (Sup) with three different volumes as the starting materials (2, 4, and 6 mL). IgG was used as the isotype control and tested only at a higher concentration (6 mL). ( p ) Quantification of mtDNA copy number (calculated using polymerase chain reaction and represented as copies/mL). mtDNA was extracted from various supernatant volumes and subjected to ChIP with the anti-TFAM antibodies (n = 6). ( q ) Quantitative analysis of IL1B expression in normal human bronchial epithelial (NHBE) cells incubated with various mtDNA concentrations (24 h). Total RNA was extracted from the cells and subjected to quantitative real-time PCR (n = 8). ( r ) Quantitative analysis of IL-1β levels in the total protein samples extracted from NHBE cells, which were treated as described in ( p ). ( s , t ) Representative flow cytometry histogram plots showing NHBE cell death (determined using Sytox dye) induced by different mtDNA concentrations (n = 6). Data are plotted as percentage of cell death. All data are represented as mean ± standard error of mean from three independent experiments. Statistical analyses (one-way analysis of variance ( b , c , h , n , p ) and unpaired t -tests ( j , q , r , t )) were performed using Graphpad Prism software. * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant. Scale bar: 10 μm.

Article Snippet: A549 cells were transduced with lentiviral particles derived from the ACE2/TMPRSS2-expressing vector (Addgene #154987), and cells stably expressing ACE2/TMPRSS2 were selected using puromycin selection.

Techniques: Transfection, Construct, Positive Control, Transduction, Flow Cytometry, Staining, Plasmid Preparation, Infection, Immunofluorescence, Western Blot, Control, Expressing, Marker, Immunoprecipitation, Chromatin Immunoprecipitation, Concentration Assay, Polymerase Chain Reaction, Incubation, Real-time Polymerase Chain Reaction, Software