genome-wide brunello single guide rna (sgrna) library  (CustomArray Inc)

Journal: Cell Reports Medicine

Article Title: Potentiating anti-tumor immunity by re-engaging immune synapse molecules

doi: 10.1016/j.xcrm.2025.101975

Figure Lengend Snippet: ICAM-1 is co-expressed with a wide range of pro-inflammatory genes and is epigenetically regulated in tumor cells (A) Workflow of CRISPR screen to identify regulators of ICAM-1 expression. Cas9-expressing A549 cells were transduced with a genome-wide sgRNA library. CRISPR-edited A549 cells were then sorted into ICAM-1 high and ICAM-1 low fractions, followed by genomic DNA extraction and sequencing to determine the sgRNA abundance. (B) Volcano plot showing the log 2 fold change and p values of ICAM-1 regulators identified from CRISPR screen. The left graph shows the depleted hits (KO of the gene reduced ICAM-1 expression) and the right graph shows the enriched hits (KO of the gene enhanced ICAM-1 expression). Annotated genes represent the NF-κB pathway (blue) and epigenetic regulators (red). (C) Log 2 fold change of sgRNAs against indicated genes in ICAM-1 high A549 cells as compared with control. Depleted sgRNA (KO leads to reduced ICAM-1) and enriched sgRNAs (KO leads to enhanced ICAM-1) are labeled in blue and red bars, respectively. The control sgRNAs are indicated by gray bars. (D) Gene ontology (GO) analysis in top 100 enriched hits from ICAM-1 high A549 cells of CRISPR screen. (E) FACS analysis of ICAM-1 level on A549-Cas9 cells expressing control sgRNA or sgRNAs targeting UHRF1, DNMT1, EED, BPTF, and STAG2. The same control sample was used for all comparisons shown in the panel. Data are representative of two independent experiments (E).

Article Snippet: Human sgRNA library Brunello in lentiGuide-Puro , Addgene , RRID: Addgene_73178.

Techniques: CRISPR, Expressing, Transduction, Genome Wide, DNA Extraction, Sequencing, Control, Labeling

Journal: Cell Reports Medicine

Article Title: Potentiating anti-tumor immunity by re-engaging immune synapse molecules

doi: 10.1016/j.xcrm.2025.101975

Figure Lengend Snippet: Reconstitution of ICAM-1/LFA-1 signaling through fusion protein Cet×ICAM1-D1 (A) Schematic structure of Cet×ICAM1-D1 fusion protein (left) and working hypothesis (right). Cet×ICAM1-D1 is composed of Fab fragment of cetuximab and murine natural D1 domain of ICAM-1, fused to a “LALA-PG” human Fc fragment. Working hypothesis: in the absence of ICAM-1, the fusion protein could interact and activate with LFA-1 signaling through the ICAM-1 D1 domain. (B) Binding affinity of cetuximab and Cet×ICAM1-D1 to EGFR in MC38 cells ( n = 3). (C and D) OT-1 T cells were co-cultured with MC38 (C) and B16F10 (D) tumor cells with serial dilutions of Cet×ICAM1-D1 or cetuximab. FACS analysis showing the percentage of intracellular IFN- γ -producing OT-I T cells ( n = 3). (E) OT-1 cells were co-cultured with SIINFEKL-pulsed or unpulsed MC38 tumor cells in the presence of 10 nM Cet×ICAM1-D1. FACS analysis showing the percentage of intracellular IFN- γ -producing OT-I T cells ( n = 3). (F) OT-I T cells were transduced with a control sgRNA (sgControl) or sgRNA targeting Cd11a . Representative FACS plot (left) and summary of mean fluorescence intensity (MFI) (right) demonstrate high knockout efficiency of Cd11a , as determined by CD11a staining ( n = 3). (G) CD11a KO or control OT-1 cells were co-cultured with MC38 tumor cells in the presence of 10 nM Cet×ICAM1-D1 or cetuximab. Representative FACS (left) and summary (right) showing the percentage of intracellular IFN- γ -producing OT1 cells in the indicated conditions ( n = 3). (H) Structure of the human version of the “LFA-1 engager,” Cet×hICAM1-D1 fusion protein. Cet×hICAM1-D1 comprises the Fab fragment of Cetuximab and the human natural D1 domain of ICAM-1, fused to a mutant “LALA-PG” human Fc fragment. (I and J) FACS analysis of intracellular IFN- γ production in NY-ESO-1-specific CTLs upon co-culture with A498 (I) and SW480 (J) tumor cells with serial dilutions of Cet×hICAM1-D1 or cetuximab ( n = 3). Data are presented as means ± SEM (B–G and I and J). ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001 by two-way ANOVA (B–D, G, and I and J) and unpaired Student’s t test (E and F). ns, not significant. Data are representative of at least two independent experiments (B–G and I and J).

Article Snippet: Human sgRNA library Brunello in lentiGuide-Puro , Addgene , RRID: Addgene_73178.

Techniques: Binding Assay, Cell Culture, Transduction, Control, Fluorescence, Knock-Out, Staining, Mutagenesis, Co-Culture Assay

Journal: Cell Reports Medicine

Article Title: Potentiating anti-tumor immunity by re-engaging immune synapse molecules

doi: 10.1016/j.xcrm.2025.101975

Figure Lengend Snippet:

Article Snippet: Human sgRNA library Brunello in lentiGuide-Puro , Addgene , RRID: Addgene_73178.

Techniques: Control, Recombinant, Virus, Modification, Expressing, Lysis, Protease Inhibitor, Saline, Isolation, Cell Isolation, Plasmid Preparation, Transfection, Cell Viability Assay, Staining, Protein Extraction, Bicinchoninic Acid Protein Assay, Methylation, Cloning, Knock-In, Software