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: 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

Journal: Journal of Virology

Article Title: TAF Family Proteins and MEF2C Are Essential for Epstein-Barr Virus Super-Enhancer Activity

doi: 10.1128/JVI.00513-19

Figure Lengend Snippet: CRISPR screen for cell factors essential for ESE. (A) Schematic diagram of the genome wide CRISPR screen. (B) Differentially enriched genes essential for 525ESE1 function.

Article Snippet: The human CRISPR knockout pooled plasmid library (Brunello) used for the CRISPR screen was purchased from Addgene (catalog number 73178).

Techniques: CRISPR, Genome Wide

Journal: Journal of Virology

Article Title: TAF Family Proteins and MEF2C Are Essential for Epstein-Barr Virus Super-Enhancer Activity

doi: 10.1128/JVI.00513-19

Figure Lengend Snippet: MEF2C is essential for 5252ESE1 function. (A) LCLs stably expressing CAS9 and 525ESE1-driven GFP-luciferase reporter were transduced with lentiviruses expressing control sgRNA or sgRNA targeting MEF2C. After puromycin selection, GFP levels were determined by FACS. (B) LCLs stably expressing CAS9 and SV40 enhancer-driven GFP-luciferase reporter were transduced with lentiviruses expressing control sgRNA or sgRNA targeting MEF2C. GFP levels were determined by FACS. (C) MEF2C expression in LCLs expressing 525ESE1- or SV40 enhancer-driven reporters. (D) MYC expression levels determined by qRT-PCR following MEF2C knockout. The level of control sgRNA was set to 1. **, P < 0.01; *, P < 0.05. (E) MEF2C expression was first knocked out by CRISPR in MUTU III cells. 525ESE1-driven reporter or control reporter was then electroporated into these cells. Luciferase activities were normalized by Renilla luciferase. Control reporter levels were set to 1. ***, P < 0.001; **, P < 0.01. (F) V5-tagged MEF2C cDNA with the 525ESE1 sgRNA2 PAM site mutated was expressed in LCLs. MEF2C sgRNA2 efficiently knocked out endogenous MEF2C but not MEF2C from rescue cDNA. (G) 525ESE1-driven GFP levels in cells with MEF2C knockout or cells expressing rescue cDNA. MEF2C knockout efficiently repressed MYC expression. (H) In cells expressing CRISPR-resistant MEF2C, knockout did not decrease MYC expression. MYC expression levels in control sgRNA treated cells were set to 1. **, P < 0.01; NS, not significant.

Article Snippet: The human CRISPR knockout pooled plasmid library (Brunello) used for the CRISPR screen was purchased from Addgene (catalog number 73178).

Techniques: Stable Transfection, Expressing, Luciferase, Transduction, Control, Selection, Quantitative RT-PCR, Knock-Out, CRISPR

Journal: Journal of Virology

Article Title: TAF Family Proteins and MEF2C Are Essential for Epstein-Barr Virus Super-Enhancer Activity

doi: 10.1128/JVI.00513-19

Figure Lengend Snippet: MEF2C knockout reduces IRF4, EBNA2, and SPI1 SE binding and LCL growth. (A) MEF2C, IRF4, EBNA2, SPI1, and H3K27ac ChIP-seq tracks at 525ESE1. (B) MEF2C CRISPR knockout was first done in LCLs. ChIP-qPCR was used to measure IRF4, EBNA2, and SPI1 binding to 525ESE1. **, P < 0.01. (C) Expression of MEF2C, IRF4, EBNA2, and SPI1 followed by MEF2C knockout. (D) IRF4 was knocked out in LCLs. MEF2C DNA binding was determined by ChIP-qPCR. **, P < 0.01. (E) LCL growth followed by MEF2C knockout. ***, P < 0.001. (F) LCL cell cycle distribution followed by MEF2C knockout.

Article Snippet: The human CRISPR knockout pooled plasmid library (Brunello) used for the CRISPR screen was purchased from Addgene (catalog number 73178).

Techniques: Knock-Out, Binding Assay, ChIP-sequencing, CRISPR, ChIP-qPCR, Expressing

Journal: Cell death discovery

Article Title: Kinome-wide CRISPR-Cas9 knockout screens revealed PLK1 as a therapeutic target for osteosarcoma.

doi: 10.1038/s41420-023-01526-7

Figure Lengend Snippet: Fig. 1 Kinome-wide CRISPR-Cas9 knockout screens reveal a cohort of kinases essential for human osteosarcoma cells. A Workflow of CRISPR-Cas9 knockout screen using human kinome CRISPR knockout library (Brunello) in U2OS, Saos-2 and OS-732 osteosarcoma cancer cell lines. Two infection replicates for each cell line was carried out. B Heatmap of Pearson correlation coefficients of gene beta scores across cell lines as well as replicates. Beta score was calculated using MAGeCK MLE algorithm. Top essential genes in knockout screen of U2OS (C), Saos-2 (D), and OS-732 cells (E). F Top essential genes of osteosarcoma, which used U2OS, Saos-2 and OS-732 cells as biological replicates. G Pathway enrichment of the essential genes using Metascape database. H Frequency histograms of sgRNA beta scores showing essentiality of PLK1 in three osteosarcoma cells as biological replicates. Lines representing the beta scores of individual sgRNAs targeting PLK1 are marked red.

Article Snippet: Cell Death Discovery (2023) 9:231 transfection reagent (Neofect, TF201201) at the following ratio, Human kinome CRISPR knockout library (Brunello, Addgene Cat# 1000000083): psPax2 (Addgene Cat# 12260, RRID: Addgene_12260): pMD2.G (Addgene Cat# 12259, RRID: Addgene_12259)= 7: 5: 2.

Techniques: CRISPR, Knock-Out, Infection