Development of screening in CD4+ T cells to identify drug targets for immune-mediated inflammatory diseases


G Bergamin1; R M Trigg1; L L Brayshaw1; B E Saunders1; R I Jepras1; L Suckling1; M New1; R Kasprowicz1; W Li1; K Tvermosegaard1; D Tough1; R A Randle1; H D Lewis1; K Maratou1


Immune-mediated inflammatory diseases (IMIDs) are a group of disorders characterised by tissue inflammation as a result of dysregulated immune responses. Enhancing the production of anti-inflammatory cytokines from T cells may balance the inflammatory state of IMIDs and provide therapeutic benefit. To this end, we developed a genome-wide CRISPR screen in primary CD4+ T cells to identify genes which, when knocked out, lead to increased release of an anti-inflammatory cytokine. We extensively optimised several elements of the screening workflow including lentiviral transduction, lentivirus production, Cas9 electroporation in bulk cell populations and FACS sorting, and ran a focused screen based on a mini library of ~1,000 gRNAs to assess the performance of the workflow before conducting a genome-wide screen.

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