Journal: Journal of experimental & clinical cancer research : CR
Article Title: A CRISPR activation screen identifies MUC-21 as critical for resistance to NK and T cell-mediated cytotoxicity.
doi: 10.1186/s13046-023-02840-9
Figure Lengend Snippet: Fig. 1 Identification of candidate genes conferring resistance to NK cell cytotoxicity through a surfaceome CRISPR activation screen. (A) Overview of the surfaceome-focused CRISPRa screen used in this study. A lentiviral library comprising 58,071 sgRNAs targeting the promoter regions of 6213 cell surface protein genes and 500 control sgRNAs was employed. K562-V2M cells were transduced with the sgRNA library and exposed to NK-92 cells for two days. Genomic DNA was extracted from the cells that survived, and gene abundance was determined using next-generation sequencing. (B) MAGeCK Analysis of the enrichment of sgRNA sequences in the surviving K562-V2M cells. X-Axis: MAGeCK Gene Score; Y-Axis: inverse log P value. (C) Representative FACS analysis of surface MUC21 expression in K562-tet-MUC21 cells after 24 h of culture with or without Dox (1 µg/mL). Two different clones of antibodies (clones heM21C and heM21D) targeting human MUC21 were used. (D-E) K562-tet-MUC21 cells were cultured in the presence of Dox (1 µg/mL) for 24 h, and co-incubated with NK-92 cells for 6 h. (D) NK cell-mediated killing activity was evaluated by measuring the luciferase activity of the surviving K562 cells. Parental K562 cells were used as a positive control. Ratio of effector to target cells (E:T). (E) Representative FACS analysis (above panel) of the surface CD107a and intracellular IFN-γ expression in NK-92 cells. A summary graph (below panel) showing the percentage of CD107a and IFN-γ expressing NK-92 cells in two independent experiments. (F) Dox-treated K562-tet-MUC21 cells were exposed to primary NK cells at a 1:1 E:T ratio for six hours. NK cell killing was evaluated by measuring luciferase activity in surviving K562-tet-MUC21 cells. (G) Immunoblotting for phospho-AKT expression in lysates from NK-92 cells after stimulation with paraformaldehyde-fixed K562-tet-MUC21 cells for 0 and 10 min. β-actin was used as a loading control. Statistical significance was determined by two-tailed unpaired t-tests; **P < 0.01, ***P < 0.001, ****P < 0.001
Article Snippet: The Wright Human Membrane Protein Activation Library (Addgene plasmid #113345), a gift from Dr. Gavin Wright, contains 58,570 sgRNAs targeting the promoter regions of 6,213 membrane proteins and 500 non-targeting sgRNAs [12].
Techniques: CRISPR, Activation Assay, Control, Transduction, Next-Generation Sequencing, Expressing, Clone Assay, Cell Culture, Incubation, Activity Assay, Luciferase, Positive Control, Western Blot, Two Tailed Test
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