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

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CRISPR Activation Screening Identifies VGLL3-TEAD1-RUNX1/3 as a Transcriptional Complex for PD-L1 Expression.

doi: 10.4049/jimmunol.2100917

Figure Lengend Snippet: FIGURE 1. CRISPR activation screen identifies novel regulators of PD-L1 expression. (A) Schematic setup of the screen. MelJuSo melanoma cells stably expressing MS2-p65-HSF1 were transduced with a pooled gRNA library containing dCAS9 and sorted by FACS for cells displaying high levels of PD-L1. (B) Genes for which at least two different gRNAs were significantly enriched (greater than fourfold) in the sorted population versus control population in both replicate sorts. Plotted are p val- ues based on RSA analysis. (C) MelJuSo MPH cells stably expressing the SAM vector with or without the indicated activation gRNAs were analyzed for cell surface expression of PD-L1 and MHC class I (HLA-ABC). Data represent three independent experiments (1SD), and statistical significance was determined by paired Student t test (*p < 0.05, **p < 0.01). (D) MelJuSo cells stably expressing FLAG (EV), GATA2-FLAG, or FLAG-VGLL3 were analyzed for cell surface expression of PD-L1 using flow cytometry. (E) MelJuSo cells as in D were either stimulated or not with IFN-g for 48 h, and cell surface expression of PD-L1 and PD-L2 was mea- sured using flow cytometry. (F) MelJuSo cells as in D were either stimulated or not with IFN-g for 24 h, and expression of the indicated proteins was determined by Western blot analysis. (G) MelJuSo cells as in D were treated with IFN-g for 24 h when indicated, and mRNA levels of the indicated genes were analyzed using quanti- tative real-time PCR and normalized to GAPDH. All data represent three independent experiments (1SD), and statistical significance was determined by ANOVA using Dunnett’s multiple comparison test (*p < 0.05, **p < 0.01).

Article Snippet: For knockout screening, we used the human CRISPR Brunello genomewide knockout library, a gift from David Root and John Doench (Addgene, 73178).

Techniques: CRISPR, Activation Assay, Expressing, Stable Transfection, Transduction, Control, Plasmid Preparation, Flow Cytometry, Western Blot, Real-time Polymerase Chain Reaction, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CRISPR Activation Screening Identifies VGLL3-TEAD1-RUNX1/3 as a Transcriptional Complex for PD-L1 Expression.

doi: 10.4049/jimmunol.2100917

Figure Lengend Snippet: FIGURE 4. VGLL3 cooperates with TEAD1 to drive PD-L1 expression. (A) Schematic setup of the screen. MelJuSo cells stably expressing FLAG- VGLL3 were transduced with the Brunello CRISPR knockout library and sorted by FACS twice for cells displaying low PD-L1 surface levels. (B) Results of the RSA analysis of the inserts from the biological duplicates, with three candidates indicated with gray dots. (C) Western blot validation of the knockout effi- ciency of the pooled MelJuSo VGLL3 knockout cells transduced with the indicated gRNAs. (D) MelJuSo FLAG-VGLL3 or FLAG-expressing cells were transduced with the indicated gRNAs, and pooled knockout lines were analyzed for surface PD-L1 expression using flow cytometry. (E) Left: Myc or Myc- TEAD1 were isolated from HEK293T cells using Myc-TRAP beads, and associated FLAG-VGLL3 or FLAG-VGLL3(vhfaaa) was detected by Western blot analysis. Right: MelJuSo cells transduced with the indicated expression constructs were analyzed for expression of PD-L1 using flow cytometry. (F) MelJuSo cells stably expressing FLAG or FLAG-VGLL3 were transfected with the indicated siRNAs and 3 d later were analyzed for PD-L1 expression using flow cytometry. (G) As in F, but 3 d after siRNA transfection. mRNA was isolated, and the expression of PD-L1 transcript was analyzed by qRT-PCR and normal- ized to GAPDH mRNA. All data represent three independent experiments (1SD); statistical significance was determined by ANOVA using Dunnett’s multi- ple comparison test (*p < 0.05, **p < 0.01).

Article Snippet: For knockout screening, we used the human CRISPR Brunello genomewide knockout library, a gift from David Root and John Doench (Addgene, 73178).

Techniques: Expressing, Stable Transfection, Transduction, CRISPR, Knock-Out, Western Blot, Biomarker Discovery, Flow Cytometry, Isolation, Construct, Transfection, Quantitative RT-PCR, Comparison

Journal: Cell reports

Article Title: CRISPR screening reveals a dependency on ribosome recycling for efficient SARS-CoV-2 programmed ribosomal frameshifting and viral replication.

doi: 10.1016/j.celrep.2023.112076

Figure Lengend Snippet: Figure 1. Genome-wide CRISPR-Cas9 screen identifies host-encoded regulators of SARS-CoV-2 frameshifting (A) Schematic of the SARS-CoV-2 genome. Dotted box indicates close up of region shown in (B) harboring the coronavirus frameshifting element (FSE). (B) Secondary structure of the SARS-CoV-2 FSE containing the slippery sequence and three-stemmed pseudoknot. Based on structural data from Bhatt et al.13

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited data CRISPR screening data This paper GEO: GSE206101 Experimental models: Cell lines HCT116 ATCC CCL-247; RRID: CVCL_0291 HEK293T ATCC CRL-3216; RRID: CVCL_0063 VeroE6 ATCC CRL-1586; RRID: CVCL_0574 HCT116-SARS-CoV-2-PRF-1 reporter cell line 1 This paper N/A HCT116-SARS-CoV-2-PRF-1 reporter cell line 2 This paper N/A HCT116-SARS-CoV-2-PRF-0 reporter cell line 1 This paper N/A HCT116-SARS-CoV-2-PRF-0 reporter cell line 2 This paper N/A HCT116-ACE2-Blast cell line 1 This paper N/A HCT116-ACE2-Blast cell line 2 This paper N/A Oligonucleotides Sequences of oligonucleotides used in this study are provided in Table S2 This paper N/A Recombinant DNA Human CRISPR Knockout Pooled Library (Brunello) Addgene (David Root, John Doench) Cat# 73179; RRID: Addgene_73179 lentiCas9-Blast Addgene (Feng Zhang) Cat# 52962; RRID: Addgene_52962 lentiCRISPR v2 Addgene (Feng Zhang) Cat# 52961; RRID: Addgene_52961 pMD2.G Addgene (Didier Trono) Cat# 12259; RRID: Addgene_12259 psPAX2 Addgene (Didier Trono) Cat# 12260; RRID: Addgene_12260 pSCRBBL-ACE2-Blasticidin John Schoggins lab N/A lenti-mCh-HIV-PRF-1-P2A-eGFP This paper N/A lenti-mCh-HIV-PRF-0-P2A-eGFP This paper N/A lenti-mCh-SARS-CoV2-PRF-1-P2A-eGFP This paper N/A lenti-mCh-SARS-CoV2-PRF-0-P2A-eGFP This paper N/A lenti-nLuc-HKU1-PRF-1-ffLuc This paper N/A lenti-nLuc-HKU1-PRF-0-ffLuc This paper N/A lenti-nLuc-OC43-PRF-1-ffLuc This paper N/A lenti-nLuc-OC43-PRF-0-ffLuc This paper N/A lenti-nLuc-SARS-CoV1-PRF-1-ffLuc This paper N/A lenti-nLuc-SARS-CoV1-PRF-0-ffLuc This paper N/A lenti-nLuc-SARS-CoV2-PRF-1-ffLuc This paper N/A lenti-nLuc-SARS-CoV2-PRF-0-ffLuc This paper N/A lenti-nLuc-SARS-CoV2-UUA-PRF-1-ffLuc This paper N/A lenti-nLuc-SARS-CoV2-UUA-PRF-0-ffLuc This paper N/A lentiCRISPR-v2-sgRNA-hsa-ABCE1-1 This paper N/A lentiCRISPR-v2-sgRNA-hsa-ABCE1-2 This paper N/A lentiCRISPR-v2-sgRNA-hsa-DENR-1 This paper N/A lentiCRISPR-v2-sgRNA-hsa-DENR-2 This paper N/A lentiCRISPR-v2-gRNA-hsa-DOHH This paper N/A lentiCRISPR-v2-gRNA-hsa-DPH1 This paper N/A lentiCRISPR-v2-gRNA-hsa-DPH3 This paper N/A lentiCRISPR-v2-gRNA-hsa-EIF2D This paper N/A lentiCRISPR-v2-gRNA-hsa-EIF5A Manjunath et al.53 N/A lentiCRISPR-v2-gRNA-hsa-POLR3K This paper N/A lentiCRISPR-v2-sgRNA-hsa-ORAOV1 This paper N/A lentiCRISPR-v2-sgRNA-hsa-YAE1D1 This paper N/A (Continued on next page) Cell Reports 42, 112076, February 28, 2023 15

Techniques: Genome Wide, CRISPR, Sequencing

Journal: Cell reports

Article Title: CRISPR screening reveals a dependency on ribosome recycling for efficient SARS-CoV-2 programmed ribosomal frameshifting and viral replication.

doi: 10.1016/j.celrep.2023.112076

Figure Lengend Snippet: Figure 6. Loss of ribosome recycling factors inhibits SARS-CoV-2 replication and reduces ribosomal frameshifting during infection (A) Experimental workflow for testing the effect of ribosome recycling on SARS-CoV-2 replication. (1) Lentiviral expression of ACE2 in HCT116 cells. (2) CRISPR- Cas9-mediated knockout of ABCE1 or DENR. (3) Infection with SARS-CoV-2. (4) Sample collection 7 h post-infection and qRT-PCR analysis of nucleocapsid (N) expression. (B) Immunoblotting of ABCE1 and DENR in HCT116-ACE2 CRISPR knockout pools. (C and D) qRT-PCR measurement of nucleocapsid mRNA expression 7 h after SARS-CoV-2 infection in cells transduced with non-target control sgRNA (sgNeg) or sgRNAs targeting ABCE1 (C) or DENR (D). Two distinct sgRNAs were used per gene in two independent ACE2-expressing HCT116 cell lines (ACE2-1 and ACE2-2). Nucleocapsid expression was normalized to host GAPDH expression. (E) Schematic of SARS-CoV-2 ORF1a and ORF1b non-structural proteins (NSPs). Antibody symbols indicate upstream (NSP1) and downstream (NSP16) NSPs that were detected by immunoblotting to assess relative frameshifting rate. (F) Representative western blot for NSP1, NSP16, and nucleocapsid from uninfected cells, infected control cells (sgNeg) and infected ABCE1 knockout pools generated with two independent sgRNAs (sgABCE1-1 and sgABCE1-2). (G) Quantification of NSP1, NSP16, and nucleocapsid protein levels, normalized to host GAPDH expression, from three independent experiments. Data are represented as the mean ± SD with individual replicates plotted. The p values for qRT-PCR experiments were calculated by two-way ANOVA with Dunnett’s multiple comparisons test. The p values for the immunoblotting results were calculated by two-way ANOVA with Tukey’s multiple comparisons test; **p % 0.01, ***p % 0.001; n = 3 biological replicates for all experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited data CRISPR screening data This paper GEO: GSE206101 Experimental models: Cell lines HCT116 ATCC CCL-247; RRID: CVCL_0291 HEK293T ATCC CRL-3216; RRID: CVCL_0063 VeroE6 ATCC CRL-1586; RRID: CVCL_0574 HCT116-SARS-CoV-2-PRF-1 reporter cell line 1 This paper N/A HCT116-SARS-CoV-2-PRF-1 reporter cell line 2 This paper N/A HCT116-SARS-CoV-2-PRF-0 reporter cell line 1 This paper N/A HCT116-SARS-CoV-2-PRF-0 reporter cell line 2 This paper N/A HCT116-ACE2-Blast cell line 1 This paper N/A HCT116-ACE2-Blast cell line 2 This paper N/A Oligonucleotides Sequences of oligonucleotides used in this study are provided in Table S2 This paper N/A Recombinant DNA Human CRISPR Knockout Pooled Library (Brunello) Addgene (David Root, John Doench) Cat# 73179; RRID: Addgene_73179 lentiCas9-Blast Addgene (Feng Zhang) Cat# 52962; RRID: Addgene_52962 lentiCRISPR v2 Addgene (Feng Zhang) Cat# 52961; RRID: Addgene_52961 pMD2.G Addgene (Didier Trono) Cat# 12259; RRID: Addgene_12259 psPAX2 Addgene (Didier Trono) Cat# 12260; RRID: Addgene_12260 pSCRBBL-ACE2-Blasticidin John Schoggins lab N/A lenti-mCh-HIV-PRF-1-P2A-eGFP This paper N/A lenti-mCh-HIV-PRF-0-P2A-eGFP This paper N/A lenti-mCh-SARS-CoV2-PRF-1-P2A-eGFP This paper N/A lenti-mCh-SARS-CoV2-PRF-0-P2A-eGFP This paper N/A lenti-nLuc-HKU1-PRF-1-ffLuc This paper N/A lenti-nLuc-HKU1-PRF-0-ffLuc This paper N/A lenti-nLuc-OC43-PRF-1-ffLuc This paper N/A lenti-nLuc-OC43-PRF-0-ffLuc This paper N/A lenti-nLuc-SARS-CoV1-PRF-1-ffLuc This paper N/A lenti-nLuc-SARS-CoV1-PRF-0-ffLuc This paper N/A lenti-nLuc-SARS-CoV2-PRF-1-ffLuc This paper N/A lenti-nLuc-SARS-CoV2-PRF-0-ffLuc This paper N/A lenti-nLuc-SARS-CoV2-UUA-PRF-1-ffLuc This paper N/A lenti-nLuc-SARS-CoV2-UUA-PRF-0-ffLuc This paper N/A lentiCRISPR-v2-sgRNA-hsa-ABCE1-1 This paper N/A lentiCRISPR-v2-sgRNA-hsa-ABCE1-2 This paper N/A lentiCRISPR-v2-sgRNA-hsa-DENR-1 This paper N/A lentiCRISPR-v2-sgRNA-hsa-DENR-2 This paper N/A lentiCRISPR-v2-gRNA-hsa-DOHH This paper N/A lentiCRISPR-v2-gRNA-hsa-DPH1 This paper N/A lentiCRISPR-v2-gRNA-hsa-DPH3 This paper N/A lentiCRISPR-v2-gRNA-hsa-EIF2D This paper N/A lentiCRISPR-v2-gRNA-hsa-EIF5A Manjunath et al.53 N/A lentiCRISPR-v2-gRNA-hsa-POLR3K This paper N/A lentiCRISPR-v2-sgRNA-hsa-ORAOV1 This paper N/A lentiCRISPR-v2-sgRNA-hsa-YAE1D1 This paper N/A (Continued on next page) Cell Reports 42, 112076, February 28, 2023 15

Techniques: Infection, Expressing, CRISPR, Knock-Out, Quantitative RT-PCR, Western Blot, Transduction, Control, Generated

Journal: Genome medicine

Article Title: Genome-scale CRISPR screens identify host factors that promote human coronavirus infection.

doi: 10.1186/s13073-022-01013-1

Figure Lengend Snippet: Fig. 1 Experimental design for genome-scale CRISPR screens performed in this study. Details of these screens are provided in the methods. A Vero E6 cells transduced with the newly generated Vervet sgRNA library were infected with SARS-CoV-2 or OC43 at MOI 0.01; resistant cells were expanded and reinfected at MOI 0.1. B Two screens were performed in HEK293T-hACE2 cells transduced with the Brunello sgRNA library. In the first screen, cells were infected with SARS-CoV-2 or OC43 at MOI 0.01 and resistant cells were reinfected with either MOI 0.01 or MOI 0.1 of the corresponding virus. In the second screen, cells were infected with SARS-CoV-2 at MOI 0.3 and reinfected at MOI 0.03. In all cases, genomic DNA was extracted from multiple replicates of control cells, the initial infections, and reinfections for the purpose of sgRNA sequencing

Article Snippet: Genome-wide CRISPR sgRNA screens The human CRISPR Brunello library (Addgene 73178) [19] was amplified following a previously published protocol [20].

Techniques: CRISPR, Transduction, Generated, Infection, Virus, Control, Sequencing

Journal: Genome medicine

Article Title: Genome-scale CRISPR screens identify host factors that promote human coronavirus infection.

doi: 10.1186/s13073-022-01013-1

Figure Lengend Snippet: Fig. 4 Identification of host factors that promote OC43 infection of HEK293T-hACE2 cells. HEK293T-hACE2 cells transduced with the Brunello sgRNA library were infected with OC43 at MOI 0.01 and sgRNAs in resistant clones sequenced. Resistant clones were reinfected with OC43 at MOI 0.01 or MOI 0.1 and sgRNAs in resistant clones sequenced. For all three infections, MAGeCK analysis of multiple replicates compared to uninfected control library replicates yielded log2fold changes (log2FC) that were plotted on the x-axis. Negative log10-transformed FDR were plotted on the y-axis. Data are presented for the initial infection (A), MOI 0.01 reinfection (B), and MOI 0.1 reinfection (C). D The heat map displays the log2FC for top-scoring genes (FDR < 0.25) across the three infections

Article Snippet: Genome-wide CRISPR sgRNA screens The human CRISPR Brunello library (Addgene 73178) [19] was amplified following a previously published protocol [20].

Techniques: Infection, Transduction, Clone Assay, Control, Transformation Assay

Journal: Genome medicine

Article Title: Genome-scale CRISPR screens identify host factors that promote human coronavirus infection.

doi: 10.1186/s13073-022-01013-1

Figure Lengend Snippet: Fig. 5 Comparison of multiple CRISPR screens identifying host factors promoting SARS-CoV-2 infection of human cell lines. A Data from four recently published CRISPR screens for SARS-CoV-2 in various human cell lines were reanalyzed and compared to our data to identify common top-scoring genes (FDR < 0.25). Using this criterion, there were 74 genes identified in our study, 53 in Daniloski et al., 707 in Schneider et al., 13 in Wang et al., and 1 in Baggen et al. No common genes were identified in all studies, 1 gene was identified in four studies, 6 genes were identified in three studies, and 25 genes were identified in two studies. Fifty-three genes were uniquely identified in our study as significant. B The heat map displays the log2FC for the 32 genes found in common across two or more of the published studies with FDR < 0.25. C A heat map displaying the log2FC for the 53 genes uniquely identified as significant in our studies compared to their observed log2FC across the other published studies

Article Snippet: Genome-wide CRISPR sgRNA screens The human CRISPR Brunello library (Addgene 73178) [19] was amplified following a previously published protocol [20].

Techniques: Comparison, CRISPR, Infection

Journal: Genome medicine

Article Title: Genome-scale CRISPR screens identify host factors that promote human coronavirus infection.

doi: 10.1186/s13073-022-01013-1

Figure Lengend Snippet: Fig. 6 Confirmation of host factor involvement by targeted shRNA knockdown and CRISPR knockout. Lentivirus-packaged shRNA clones directed to CTSL, CCZ1, and EDC4 were transduced into HEK293T-hACE2 cells and selected with puromycin. Lentivirus-packaged sgRNA directed to EDC4 and XRN1 were transduced into SAEC-hACE2 cells and selected with puromycin. A Gene knockdown was assessed using western blotting with antibodies directed to CTSL, CCZ1, and EDC4 in cells transduced with a gene-specific shRNA or empty vector control (EV). Actin expression served as a loading control. B Triplicate wells of knockdown cells were infected with SARS-CoV-2 or OC43 at MOI 0.01. At 2 dpi, viral genome copy numbers were determined by RT-qPCR and normalized to GAPDH levels as a housekeeping control. The data are reported as the relative normalized viral genome copy number in shRNA-expressing cells compared to the EV control (n = 3 experiments). Error bars denote standard errors of mean and P values were determined using one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001). C EDC4 and XRN1 knockout in SAEC-hACE2 was assessed using western blotting with antibodies directed to EDC4 and XRN1 in cells transduced with a gene-specific sgRNA or in wild-type cells (WT). Actin expression served as a loading control. D Triplicate wells of SAEChACE2 WT, EDC4ko, and XRN1ko cells were infected with SARS-CoV-2 or OC43 and MOI 0.01. At 0hpi, 1dpi, 2dpi, and 3dpi, cell supernatants were harvested and infectious viral particles were measured by TCID50 (n = 2 experiments). Error bars denote standard errors of mean and P values were determined using one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

Article Snippet: Genome-wide CRISPR sgRNA screens The human CRISPR Brunello library (Addgene 73178) [19] was amplified following a previously published protocol [20].

Techniques: shRNA, Knockdown, CRISPR, Knock-Out, Clone Assay, Western Blot, Transduction, Plasmid Preparation, Control, Expressing, Infection, Quantitative RT-PCR

Journal: Genome medicine

Article Title: Genome-scale CRISPR screens identify host factors that promote human coronavirus infection.

doi: 10.1186/s13073-022-01013-1

Figure Lengend Snippet: Fig. 9 Summary of genes found in this and other studies and their potential roles in the SARS-CoV-2 life cycle. The host factors identified in CRISPR screens are presented adjacent to the putative stage of viral replication where they function. The genes are color-coded based on their identification in our and other published studies, as indicated in the legend. Candidate pan-HCoV host factors are indicated with red asterisks. The virus replicates through a series of well-defined molecular steps. 1–2 After virion binding to ACE2, SARS-CoV-2 can fuse at the plasma membrane or following endocytosis. Heparan sulfate proteoglycans enhance viral attachment to cells so host factors involved in heparan sulfate biosynthesis (B3GAT3, EXT1, EXTL3, SLC35B2) and glycosylation (A4GALT, ALG5, ALG9) may play a role in viral entry. The IFITM proteins are proposed to promote fusion at the cell surface but inhibit fusion in endosomes. Host factors involved in endocytosis (C18orf8, CCZ1, CCZ1B, CLTC, EPN1, WDR81, WDR91), vesicular transport (DNM2, PIK3C3, RAB7A, TMEM106B, SNX27, VAC14, VPS35), and amphisome maturation/lysosome fusion (ATP6VIE1, ATPCV1G1, ATP6V1A, CTSL, GDI2, TMEM41B) likely facilitate virion uncoating. 3 The positive-sense RNA genome is then translated to produce the nonstructural polyproteins which are co-translationally cleaved to form the mature nsps. Certain host factors like RNH1 and DAZ3 may serve to protect the viral genome from degradation by host enzymes. 4 The nsps form the viral replicase which assembles on organellar membranes to form the replication and transcription complexes (RTCs) where progeny genomes and structural/accessory protein transcripts are produced, respectively. P-body components EDC4 and XRN1, identified in this study, may play a role in maintaining viral RNA stability or assembly of the RTC. 5 Structural and accessory proteins are translated, and structural proteins insert into the ER membrane. ER- localized SLC39A1 may play a role in this process. 6 Nucleocapsids bud into the ERGIC, potentially aided by host factors ERGIC3, SEC63, SLC33A1, and SCAP. 7 Progeny virions form as they traverse through the Golgi and structural proteins are glycosylated. 8 Virions exit the cell through either typical exocytosis (DNM2, EXOC2, EXT1, EXTL3, MYH13, SNX27, VPS35) or nonclassical lysosomal egress (GNPTAB, GNPTG, NAGPA, NPC1, TMEM106B, PIP4P1). Numerous host factors with less obvious direct roles in promoting steps in the viral life cycle have also been identified in CRISPR screens. For example, numerous factors regulating the cell cycle (BAX, CDK4, CDKN1A, DYRK1A, HRK, MPLKIP, PTCH1, STRADA, TP53) were identified in our screens in AGM and human cells. Furthermore, multiple nuclear-localized host factors including diverse transcriptional regulators and two components of the integrator complex (INTS6, INTS12) were identified. Overall, the large number of diverse host factors that promote SARS-CoV-2 replication illustrates the large-scale exploitation of cellular processes required for successful viral propagation. Adapted from BioRender template titled Life Cycle of Coronavirus generated by the Britt Glaunsinger laboratory. Created with BioRender.com

Article Snippet: Genome-wide CRISPR sgRNA screens The human CRISPR Brunello library (Addgene 73178) [19] was amplified following a previously published protocol [20].

Techniques: CRISPR, Virus, Binding Assay, Clinical Proteomics, Membrane, Glycoproteomics, Produced, Generated