Targeting Tissue-Specific T Cell Plasticity by Pooled Single-Cell CRISPR Screening in Preclinical Mouse Models

Background: Treatment of autoimmune diseases demands a shift from unspecific immunosuppression towards targeted therapies. This could be achieved by turning proinflammatory T helper cells into anti-inflammatory subsets. However, the molecular pathways involved in T cell plasticity and stability are not fully understood. Single cell CRISPR-screens are a powerful tool to simultaneously analyze the impact of multiple genes on cellular phenotypes. Methods: By combining single cell gene expression analysis and T cell receptor sequencing, we uncover Th17 to Th1 cell plasticity in the human kidney in renal autoimmunity. To investigate the molecules involved in T cell plasticity in disease settings, we established in vivo single cell CRISPR droplet sequencing (iCROP-seq). Results: By applying this technique to in vivo models of inflammatory diseases in the kidney and intestine, we demonstrate that CRISPR-induced alterations in T cell polarization can be identified and ranked according to corresponding transcriptional perturbations. In particular, we targeted pro-inflammatory Th17 cells in models of immune-mediated diseases and quantified polarization biases into Th1 and regulatory T cells. Conclusions: iCROP-seq will facilitate the identification of therapeutic targets by highly efficient functional stratification of genes and pathways in a disease- and tissuespecific manner. Funding: Government Support - Non-U.S.