A Fully Optimized CRISPR Workflow for Drug Discovery in T Cells
Immune-mediated inflammatory diseases (IMIDs) are characterized by excessive tissue inflammation, uncontrolled antibody production and an imbalance of immunoregulatory cytokines. Drugs that enhance production of anti-inflammatory cytokines could provide therapeutic benefit for patients with IMID and other autoimmune disorders. Here we present a pilot study for the end-to-end, whole-genome CRISPR knockout screening of primary CD4+ T cells to discover new therapeutic targets. High-efficiency MaxCyte® electroporation of Cas9 protein and optimized cytokine-positive cell enrichment were combined with the SLICE (sgRNA lentiviral infection with Cas9 protein electroporation) screening protocol. This framework reproduced validated hits and was used to identify new genetic targets with a view to drug development.
Lentiviral CRISPR screening in primary T cells presents significant challenges due to the low transduction efficiency of lenti-Cas9 vectors and short T cell lifespan ex vivo. The SLICE protocol was previously described as a solution for whole-genome screens in human T cells with a combined approach of lentiviral gRNA library transduction followed by electroporation of Cas9 protein. Here, SLICE screening was adapted for use with CD4+ T cells with MaxCyte electroporation and optimized cytokine assays and transduction methods from GSK®. The MaxCyte ExPERTTM platform provided scalability up to 3 x 108 cells and high-efficiency gene editing with low cell toxicity, all critical parameters for handling sensitive primary T cells in a genome-wide CRISPR screen.
Analysis of gRNA Enrichment
- The SLICE protocol with cytokine assays were fully optimized in a mini-screen approach.
- T cell characteristics were verified and maintained by immunophenotyping at multiple steps during the screen.
- MaxCyte electroporation enables optimal editing efficiency and scalability to 3 x 108 cells in a single tranfection for whole-genome primary T cell screens.
- MaxCyte electroporation ensures high cell viability for sensitive cell populations.
- Potential future experiments:
- Genome-wide CRISPR screen for targets that regulate cytokine expression
- CRISPR RNP-based validation of individual hits
- In-depth interrogation of target druggability, gene expression, gene pathways and toxicity
- Potential application to a wide range of future CRISPR screens in primary T cells
- Webinar: Development of a genome-wide CRISPR screen in CD4+ T cells to identify drug targets for immune-mediated inflammatory diseases
- Shifrut E, Carnevale J, Tobin V, et al. Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function. Cell. 2018;175(7):1958-1971.e15. doi:10.1016/j.cell.2018.10.024