Journal: Nature biotechnology

Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks

doi: 10.1038/nbt.4062

Figure Lengend Snippet: a , Schematic of genome-scale CRISPRa screening approach (see text for details). b , Overview of CRISPRa screen results. Negative τ values indicate depletion and positive values enrichment of cells following imatinib selection. Significant candidate genes (FDR<0.05, p<0.001) are in colour (blue = depleted, red = enriched). Validated candidate genes are labelled in black. Mann-Whitney U test was used to calculate p-values as described previously . To correct for multiple hypothesis testing, we first performed random sampling with replacement among the set of τ values for non-targeting control sgRNAs and calculated p-values for each sampling. Then, we calculated the false discover rate (FDR) based on the distribution of p-values for all genes in the library and for non-targeting controls generated above. c, Candidate gene validation. Enrichment of candidate sgRNA expressing cells was measured over time. Values represent the mean of three different sgRNAs targeting each gene with s.e.m. Grey shading = two standard deviations of sgNTCs at day 15. All values from separate sgRNAs on days 7, 11 and 15 normalised to baseline or untreated cells are shown in .

Article Snippet: For the CRISPRa library, the designed 20 nt target specific sgRNA sequences were synthesised as a pool, on microarray surfaces (CustomArray, Inc.), flanked by overhangs compatible with Gibson Assembly into the pSico based sgLenti sgRNA library vector (see for vector map).

Techniques: Selection, MANN-WHITNEY, Sampling, Generated, Expressing

Journal: Nature biotechnology

Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks

doi: 10.1038/nbt.4062

Figure Lengend Snippet: a, Concept of the application of the orthogonal system for directional gene interaction studies. In the same cell, one gene is activated (CRISPRa) while another gene in knocked out (SaCas9 nuclease). b–d, Correlation of τ values from two clonal cell line replicates is shown for b, gene activation, c, gene knockout and d, all possible combinations thereof. Correlation values (r) are Pearson product-moment correlation coefficients. e , Schematic of perturbation data set from each gene pair (blue = depleted, red = enriched, NTC = non-target control sgRNA) f, Formula for calculating Ψ scores. Negative Ψ scores define interactions in which directionality could be inferred. g , To determine which of both interaction partners acts up- or downstream, τ activation values were multiplied with genetic interaction scores. Positive values indicate a downstream function, negative values an upstream function of the activated gene. h , Based on GI and Ψ scores determined by the full orthogonal interaction screen, a genetic interaction model was constructed. For positive regulators of cell fitness, nodes are shown in red and negative regulators in blue. Arrow-shaped edges indicate inferred directional interactions between nodes. Line-shaped edges symbolise genetic interactions where directionality could not be inferred. Node sizes are proportional to the degree of connectivity. In total, 2258 gene:gene combinations that passed the filter criteria were considered for the construction of the directional genetic interaction network.

Article Snippet: For the CRISPRa library, the designed 20 nt target specific sgRNA sequences were synthesised as a pool, on microarray surfaces (CustomArray, Inc.), flanked by overhangs compatible with Gibson Assembly into the pSico based sgLenti sgRNA library vector (see for vector map).

Techniques: Activation Assay, Gene Knockout, Construct

Journal: bioRxiv

Article Title: Ras-dependent activation of BMAL2 regulates hypoxic metabolism in pancreatic cancer

doi: 10.1101/2023.03.19.533333

Figure Lengend Snippet: ( A ) Oxygen tension (y-axis) as determined by an OxyLite probe in tumors from KPC mice breathing ambient air or pure oxygen (x-axis). Dark blue circles represent averages per tumor and boxplots illustrate their distribution. Light blue circles represent repeat measurments per tumor. ( B ) HIF target genes (orange) controlled directly or indirectly by BMAL2 (yellow). Indirect control involves both BMAL2’s negative influence on first (dark blue) or second tier (light blue) RP repressing HIF target genes, and positive influence on first (dark red) and second (light red) tier RP activating HIF target genes. ( C ) Fold change in cell growth relative to cells expressing non-targeting sgRNA in normoxic (21% O2) and hypoxic (1% O2) conditions ( D ) Number of migrated cells for cells expressing non-targeting or BMAL2-directed sgRNA in the indicated oxygen environment. P-values are derived from testing the indicated coefficients from a linear regression model. Significant interaction term suggests synergistic effects of BMAL2 pertubation and hypoxia on cell migration. ( E ) Media pH levels after 72 hours incubation. P-values derived from Welch’s t-test ( F ) Fold change in lactate levels in the indicated oxygen environment expressing either non-targeting or BMAL2-directed sgRNA ( G ) Western Blot for HIF1a, HIF2a,

Article Snippet: The sgRNA (small-guide RNA) to knocking-out BMAL2 as well as non-targeting (NT) sequence were purchased from GenScript (Piscataway, NJ) using pLentiGuide-Puro vector as a backbone.

Techniques: Control, Expressing, Derivative Assay, Migration, Incubation, Western Blot

Journal: Frontiers in Immunology

Article Title: Establishment of a CRISPR/Cas9 knockout library for screening type I interferon-inducible antiviral effectors in pig cells

doi: 10.3389/fimmu.2022.1016545

Figure Lengend Snippet: VSV-eGFP possessed a sensitivity to type I IFN-triggered antiviral response. (A) Treatment with exogenous type I IFNs induces the expression of hundreds of ISGs, allowing the establishment of a so-called antiviral state against pathogen invasion in host cells. (B) Representative Flow cytometry analysis of EGFP expression rate in VSV-eGFP-infected IBRS-2 cells mock-treated (top), treated with RUX (500 nM) (middle, upper) and IFN (10 ng/mL) (middle, lower) alone or in combination (bottom). Cells without infection were tested as a background fluorescence intensity control (not shown in the histogram). VSV-eGFP replication was effectively inhibited by type I IFN treatment but subsequently restored by supplementation of RUX. (C) Graphs showing Flow cytometry data analysis indicating the reversion of IFN inhibition by addition of RUX. Data are expressed as mean ± SEM. ***P < 0.001.

Article Snippet: Oligos encoding the sgRNA library with ~1908 specific sgRNA sequences targeting 359 ISGs were synthesized by a programmable microarray using the Synthesizer (GenScript, Wuhan) and cloned as a pool into lentiCRISPRv2 vector.

Techniques: Expressing, Flow Cytometry, Infection, Fluorescence, Control, Inhibition

Journal: Frontiers in Immunology

Article Title: Establishment of a CRISPR/Cas9 knockout library for screening type I interferon-inducible antiviral effectors in pig cells

doi: 10.3389/fimmu.2022.1016545

Figure Lengend Snippet: Identification of ISGs induced by exogenous type I IFNs using RNA-Seq. (A) Differentially expressed genes ( p value < 0.05, twofold or more change and FPKM value greater than 1 in at least one sample) for each IFN-treated sample are depicted numerically. (B) Correlation matrix of all 5 IFN-treated samples (based on Pearson correlation coefficients). (C) Upset plot of up-regulated DEGs in IFN-treated samples. Of note, all the up-regulated DEGs in the blue bars are clustered into ISG family.

Article Snippet: Oligos encoding the sgRNA library with ~1908 specific sgRNA sequences targeting 359 ISGs were synthesized by a programmable microarray using the Synthesizer (GenScript, Wuhan) and cloned as a pool into lentiCRISPRv2 vector.

Techniques: RNA Sequencing

Journal: Frontiers in Immunology

Article Title: Establishment of a CRISPR/Cas9 knockout library for screening type I interferon-inducible antiviral effectors in pig cells

doi: 10.3389/fimmu.2022.1016545

Figure Lengend Snippet: Expression levels of select ISGs in type I IFN-treated IBRS-2 cells. The font size of each ISG is directly proportional to its average fold change in type I IFN-treated IBRS-2 cells normalized to mock-treated cells. Of note, ISGs with biggest font size demonstrated highest expression levels with fold changes greater than 1000.

Article Snippet: Oligos encoding the sgRNA library with ~1908 specific sgRNA sequences targeting 359 ISGs were synthesized by a programmable microarray using the Synthesizer (GenScript, Wuhan) and cloned as a pool into lentiCRISPRv2 vector.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Establishment of a CRISPR/Cas9 knockout library for screening type I interferon-inducible antiviral effectors in pig cells

doi: 10.3389/fimmu.2022.1016545

Figure Lengend Snippet: ISG-targeting CRISPR screen identifies a subset of genes as potential key effectors of the IFN response to VSV-eGFP replication. (A) Schematic of ISG-targeting lentiCRISPR screen to identify antiviral effectors mediating IFN-α 2a-induced antiviral response to VSV-eGFP. (B) Amplification of sgRNA expression cassettes in eGFP-positive IBRS-2 knockout cells. (C) Bar plots show the top 25 most enriched hits in the context of IFN-α 1b, IFN-α 2a and IFN-β. (D) Overlap between the top 25 most enriched ISGs in the context of IFN-α 1b, IFN-α 2a and IFN-β.

Article Snippet: Oligos encoding the sgRNA library with ~1908 specific sgRNA sequences targeting 359 ISGs were synthesized by a programmable microarray using the Synthesizer (GenScript, Wuhan) and cloned as a pool into lentiCRISPRv2 vector.

Techniques: CRISPR, Amplification, Expressing, Knock-Out

Journal: Frontiers in Immunology

Article Title: Establishment of a CRISPR/Cas9 knockout library for screening type I interferon-inducible antiviral effectors in pig cells

doi: 10.3389/fimmu.2022.1016545

Figure Lengend Snippet: The effects of top four ISGs on VSV-eGFP replication. (A) Schematic representation of the gateway-compatible bicistronic lentiviral vectors used to stably overexpress ISGs. The viral backbone carries the ISG-IRES-TagRFP overexpression cassette under the CMV promoter. In parallel, control vector GFP-IRES-TagRFP was also designed. (B) Fluorescent micrographs of IBRS-2 cells in culture 24 h after transduction with the control vector GFP/TagRFP. (C) Fluorescent micrographs of IBRS-2 cells in culture 24 h after transduction with ISG/TagRFP vectors. (D) Schematic demonstration of workflow of transduction and virus infections. (E) The TCID 50 titration of VSV-eGFP titers in the vector control and ISG/TagRFP overexpression IBRS-2 cells. The experiment was repeated three times with replicate each. **P < 0.01.

Article Snippet: Oligos encoding the sgRNA library with ~1908 specific sgRNA sequences targeting 359 ISGs were synthesized by a programmable microarray using the Synthesizer (GenScript, Wuhan) and cloned as a pool into lentiCRISPRv2 vector.

Techniques: Stable Transfection, Over Expression, Control, Plasmid Preparation, Transduction, Virus, Titration