Journal: Cancers

Article Title: The NOTCH-RIPK4-IRF6-ELOVL4 Axis Suppresses Squamous Cell Carcinoma

doi: 10.3390/cancers15030737

Figure Lengend Snippet: Transcriptome profiling of Ripk4 KO mouse keratinocytes. ( A ) Schematics showing the experimental workflow of RNA sequencing in Ripk4 knock-out keratinocytes and in vivo CRISPR screen of top 63 downregulated genes. LSL-Pik3ca H1047R ; LSL-Cas9-GFP embryonic (E9.5) mice were transduced with sgNTC (non-targeting control) or sgRipk4. At postnatal day P4, the transduced skin was harvested and processed for RNA sequencing. Differential gene expression analysis revealed 63 significantly downregulated genes in Ripk4 KO compared to sgNTC control mice. These 63 genes were selected to construct a targeted CRISPR library and screened in LSL-Pik3ca H1047R ; LSL-Cas9-GFP mice to identify the Ripk4 downstream genes that themselves act as tumor suppressor genes (image created with biorender.com). ( B ) Heat map showing the differential expression of genes in the sgNTC (control) versus sgRipk4 keratinocytes. ( C ) Gene set enrichment analysis (GSEA) revealed downregulation of the synthesis of very-long-chain fatty acids pathway in Pik3ca H1047R ; Ripk4 KO skin. ( D ) METASCAPE pathway analysis revealed several upregulated and downregulated pathways including epidermis development.

Article Snippet: Plasmid pDS1 (Addgene #158032) expressing U6-sgRNA stuffer-tracr cassette and hPGK-driven Cre recombinase (Cre) (19) was used in all the experiments involving CRISPR methodology. sgRNAs targeting genes of interest (63 downregulated gene library) and non-targeting sgRNAs (2), were ordered as a pooled oligo chip (CustomArray Inc., Redmond, WA, USA) and cloned into pDS1 using BsmBI restriction sites.

Techniques: RNA Sequencing Assay, Knock-Out, In Vivo, CRISPR, Transduction, Expressing, Construct

Journal: Cancers

Article Title: The NOTCH-RIPK4-IRF6-ELOVL4 Axis Suppresses Squamous Cell Carcinoma

doi: 10.3390/cancers15030737

Figure Lengend Snippet: Elovl4 is a tumor suppressor downstream of Ripk4. ( A ) Representative image of tumors in an LSL- Pik3ca H1047R ; LSL-Cas9-GFP mouse transduced with LV carrying Cre recombinase and sgRNAs targeting the genes downregulated in Ripk4-deficient keratinocytes. ( B ) Tumor-free survival for LSL-Pik3ca H1047R ; LSL-Cas9-GFP mouse transduced with downregulated genes library ( n ≥ 8 per group; p < 0.0001, log-rank test). ( C ) Pie chart showing the genes that have their corresponding sgRNAs enriched in the tumors collected from mice transduced with the CRISPR gene library targeting downregulated Ripk4 target genes. ( D ) Real-time PCR results showing relative expression of indicated genes in mouse keratinocytes carrying CRISPR/Cas9-mediated ablation of Ripk4 when compared to non-targeting control. Data are shown as means ± SEM ( n = 3). * Denotes p -value < 0.05. (E) Tumor-free survival for tumor-prone Pik3ca H1047R ;Cas9 mice transduced with an independent sgRNA against Elovl4. sgNTC served as a control ( n ≥ 5 per group; p < 0.0001, log-rank test). (F) Representative images of tumors in the head and neck region of Pik3ca H1047R ; Cas9 mice transduced with sgElovl4 LV-Cre.

Article Snippet: Plasmid pDS1 (Addgene #158032) expressing U6-sgRNA stuffer-tracr cassette and hPGK-driven Cre recombinase (Cre) (19) was used in all the experiments involving CRISPR methodology. sgRNAs targeting genes of interest (63 downregulated gene library) and non-targeting sgRNAs (2), were ordered as a pooled oligo chip (CustomArray Inc., Redmond, WA, USA) and cloned into pDS1 using BsmBI restriction sites.

Techniques: Transduction, CRISPR, Real-time Polymerase Chain Reaction, 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: 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