genomewide crispr knockout library  (Addgene inc)

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