sam pooled sgrna library  (Addgene inc)

Journal: bioRxiv

Article Title: A YIPF5-GOT1A/B complex directs a transcription-independent function of ATF6 in ER export

doi: 10.1101/2023.12.12.569033

Figure Lengend Snippet: a) Schematic of pharmacogenetic screens to identify factors that modulate the effect of secretion inhibitors. b) General procedure of CRISPR activation and CRISPR knock-out (KO) screening approaches. MCF10A cells were infected with the genome-wide lentiviral SAM (synergistic activation mediator) or GeCKO v2 sgRNA library. After selection with Brefeldin A (BFA) or FLI-06, the sgRNA distribution was determined by NGS (next generation sequencing) and analyzed using the RSA (redundant siRNA activity) algorithm in comparison with unselected control cells. c) CRISPR activation screen for Brefeldin A (BFA, 100 nM) resistance in clonal MCF10A cells using the SAM system. Displayed is the RSA analysis for sgRNA enrichment from two MCF10A-SAM clones (#9 and #28). Each dot represents one gene, gene names of selected hits are shown. d-e ) Validation of GBF1 as BFA target. qPCR expression analysis ( d ) of GBF1 and IncuCyte growth curve analysis upon BFA treatment ( e ) in MCF10A-SAM cells stably expressing sgGBF1 or non-targeting (NT) sgRNAs. Displayed is the confluency over time. f ) Principle to identify genetic perturbations that confer resistance or sensitivity to secretion inhibitors by sgRNA enrichment or depletion, respectively. g ) CRISPR a screen for FLI-06 (10 µM) resistance in clonal MCF10A cells (#9 and #28) using the SAM system. RSA analysis for both clones was performed for sgRNA enrichment (left) or sgRNA depletion (right). Each dot represents one gene. h ) CRISPR-KO screen for FLI-06 (10 µM) resistance in human BJ-Tert cells using the GeCKO sgRNA library. RSA analysis was performed for sgRNA enrichment. Each dot represents one gene. i-n ) Validation of GOLT1A , GOLT1B and YIPF5 as mediators of resistance/sensitivity to FLI-06. qPCR expression analysis ( i ) of GOLT1A and IncuCyte growth curve analysis upon FLI-06 (10 µM) treatment ( j ) in MCF10A-SAM clone #9 stably expressing sgGOLT1A or non-targeting sgRNAs. qPCR expression analysis ( k ) of GOLT1B and IncuCyte growth curve analysis upon FLI-06 treatment ( l ) in MCF10A cells stably expressing shGOLT1B or shRenilla as control. m ) Immunoblotting analysis with total protein as loading control and n ) IncuCyte growth curve analysis upon FLI-06 treatment in YIPF5 KO and YIPF5 or empty vector re-expressing MCF10A cells. For qPCR, B2M was used as housekeeping gene. Data are shown as mean of three biological replicates and error bars represent SEM ( d, e, i, j, k, l, n ). Statistical testing: one-way-ANOVA test with Tukey HSD post-hoc test ( d, e, i, j, l, n ) and unpaired two-sided Welch t-test ( k ).

Article Snippet: The genome-wide human SAM sgRNA library (kind gift from Feng Zhang, Addgene #1000000057) was amplified as previously described ( ).

Techniques: CRISPR, Activation Assay, Knock-Out, Infection, Genome Wide, Selection, Next-Generation Sequencing, Activity Assay, Comparison, Control, Clone Assay, Biomarker Discovery, Expressing, Stable Transfection, Western Blot, Plasmid Preparation

Journal: bioRxiv

Article Title: Pathogen-driven CRISPR screens identify TREX1 as a regulator of DNA self-sensing during influenza virus infection

doi: 10.1101/2023.02.07.527556

Figure Lengend Snippet: A, Cartoon of re-engineered TRPPC NS genome segment and TRPPCa-mediated gene expression. The sgRNA directs VP64-dCas9 to specific genome targets while two MS2 hairpins inserted in the sgRNA recruit MCP-p65-HSF1. B, TRPPCa of a luciferase reporter in 293T cells. Cell were transfected with vectors expressing viral genomic RNA for NS, Split NS that lacks an sgRNA, or TRPCC NS targeting the reporter promoter. Activation was measured in the presence (+RNP, right) or absence (left) of the viral replication machinery. C, Multicycle replication of IAV harboring a TRPPC- NS segment in A549 cells. D, Virally delivered sgRNA activates reporter gene expression in a multicycle infection. A549-CRISPRa cells were inoculated with virus encoding the indicated NS segment (MOI = 0.05), or mock treated, and luciferase reporter was measured over the course of infection. E, Virally delivered sgRNAs activate expression of host genes from the endogenous locus. A549-CRISPRa cells were inoculated with TRPPC viruses (MOI = 5) targeting the indicated gene, a non-targeting control (C) or mock. Host gene expression was measured at 8 hpi via RT-qPCR. F, A pool of 34 TRPPC viruses targeting a collection of 10 potential pro- or antiviral host genes were subject to 4 rounds of selection in A549-CRISPRa cells cells. Viruses present at each stage of selection were quantified by deep-sequencing and normalized sgRNA composition is depicted. Viruses activating proviral genes enriched at least 3-fold are colored green, while viruses activating antiviral genes that are depleted at least 3-fold are colored red. Graph is representative of mean values for 2 replicate screens. G, TRPPCa screens are highly reproducible. Comparison of two biological replications shows nearly identical relative enrichment of TRPPC viruses targeting the indicated host genes after 4 rounds of selection. H, TRPPC results reflect changes in viral replication. Multicycle replication in A549-CRISPRa cells of individual TRPPC viruses targeting specific host genes (MOI = 0.01). Data are shown as grand mean of 3 replicates ± SEM (B, D) or mean ± s.d. (C, E, F, H). T tests (B), two-way ANOVA with Dunnett’s multiple comparisons test against WT (C, D, H), and one-way ANOVA with Dunnett’s multiple comparisons tests (E) were performed (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: The human SAM CRISPRa sgRNA library (Addgene #1000000078) was cloned into the pHW-TRPPC-NS rescue plasmid backbone for PR8 ( , ). sgRNAs were PCR-amplified from the SAM library in 24 low-cycle reactions using primers that append restriction sites compatible with the pHW-TRPPC-NS cloning vector.

Techniques: Gene Expression, Luciferase, Transfection, Expressing, Activation Assay, Infection, Virus, Control, Quantitative RT-PCR, Selection, Sequencing, Comparison

Journal: bioRxiv

Article Title: Pathogen-driven CRISPR screens identify TREX1 as a regulator of DNA self-sensing during influenza virus infection

doi: 10.1101/2023.02.07.527556

Figure Lengend Snippet: A, Experimental design of a genome-wide TRPPC screen in CRISPRa cells. B, Viral titers (left axis, solid line) and population richness (right axis, dashed line) were measured of 5 sequential rounds of TRPPCa selection. Data for independent screens A, B and C are shown. C, Stack plot of the abundance of individual TRPPC viruses in three independent genome-wide screen. Viruses enriched >4-fold at passage 5 are plotted for each replicate, with number of enriched viruses indicated for each screen. Colors are used to distinguish each member, but are not unique to any specific sgRNA. D, Final abundance of individual TRPPC viruses at passage 5 as a function of their abundance at passage 0 for all replicates. Colors represent viruses >4-fold enriched (green) or >4-fold depleted (red) or unchanged (grey). E, Robust ranking aggregation for top hits. MAGeCK gene scores for the top 30 genes in the TRPPC screens. F, Venn diagram of genes enriched >4-fold in the 3 screen replicates. G, Bubble plot of positive selection values for all genes in the screen. Bubble size indicates the number of replicate screens in which that gene was detected. Colored dots represent genes >10-fold enriched, with labelled dots representing genes >20-fold enriched. Genes are randomly positioned along the x-axis.

Article Snippet: The human SAM CRISPRa sgRNA library (Addgene #1000000078) was cloned into the pHW-TRPPC-NS rescue plasmid backbone for PR8 ( , ). sgRNAs were PCR-amplified from the SAM library in 24 low-cycle reactions using primers that append restriction sites compatible with the pHW-TRPPC-NS cloning vector.

Techniques: Genome Wide, Selection

Journal: bioRxiv

Article Title: Pathogen-driven CRISPR screens identify TREX1 as a regulator of DNA self-sensing during influenza virus infection

doi: 10.1101/2023.02.07.527556

Figure Lengend Snippet: A, Multiple TRPPC viruses with distinct targeting sequences activate TREX1 expression. A549-CRISPRa cells were inoculated (MOI = 1) with viruses targeting different sites in the TREX1 promoter or a non-targeting control. TREX1 expression was measured relative to mock by RT-qPCR at 10 hpi and western blotting at 12 hpi. B, Multicycle replication of TREX1 - or non-targeting TRPPC viruses in A549-CRISPRa cells (MOI = 0.01). Titers determined by plaque assay. C, A pool of TRPPC viruses were competed for 48 h during replication in A549-CRISPRa cells (pooled MOI = 0.05). Relative abundances at the start (input) and end (output) of the infection for each virus was determined by sequencing and shown for 2 independent replicates. D, Multicycle replication of a WSN influenza A reporter virus in WT A549 cells or lines stably expressing TREX1 or the catalytic mutant TREX1 D18N . Replication was normalized to viral titers in WT cells at 24 hpi. E, Viral replication was measure at 48 hpi (MOI = 0.05) in 3 distinct TREX1-KO clones inoculated with a WSN influenza A reporter virus. Clones were complemented with TREX1 or TREX1 D18N , where indicated. Values are relative to replication in parental WT A549 cells. For statistical analyses, KO clones were compared to WT, whereas complemented clones were compared to the matched KO. F, Multicycle replication of a WSN influenza A reporter virus (MOI = 0.05) in WT A549 cells, TREX1-KO cells, or complemented cell lines. Values are compared to replication in parental WT A549 cells. G. Viral titers at 48 hpi (above) and NP protein levels at 24 hpi (below) in cells inoculated with PR8 (MOI = 0.01) H, Replication of reporter influenza viruses based on the primary viral isolates CA04 (MOI = 0.5), S009 (MOI = 0.05), B/Bris (MOI = 0.2) at 48 hpi or VSV (MOI = 0.001) at 24 hpi. Values are compared to replication in parental WT A549 cells. Data are shown as grand mean for 3 replicates ± SEM (D-F, H) or mean ± s.d. (A-B, D, G). One-way ANOVA with post-hoc Dunnett’s tests were performed except for (E), which used a one-way ANOVA with post-hoc Tukey’s test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant).

Article Snippet: The human SAM CRISPRa sgRNA library (Addgene #1000000078) was cloned into the pHW-TRPPC-NS rescue plasmid backbone for PR8 ( , ). sgRNAs were PCR-amplified from the SAM library in 24 low-cycle reactions using primers that append restriction sites compatible with the pHW-TRPPC-NS cloning vector.

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Plaque Assay, Infection, Virus, Sequencing, Stable Transfection, Mutagenesis, Clone Assay