Targeting T cell plasticity by pooled single cell CRISPR-screening in preclinical models of kidney and gut inflammation - Colitis experiment
Treatment of autoimmune diseases demands a shift from unspecific immunosuppression towards targeted therapies. This could be achieved by turning pro-inflammatory T helper (Th) 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. To investigate the molecules involved in T cell plasticity in disease settings, we established in vivo single cell CRISPR droplet sequencing (iCROP-seq). By applying this technique to in vivo models of inflammatory diseases in the 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. These findings uncover Th17 to Th1 cell plasticity in the human kidney in the context of renal autoimmunity. iCROP-seq will facilitate the identification of therapeutic targets by highly efficient functional stratification of genes and pathways in a disease- and tissue-specific manner. This series contains the alternate sequencing of the original sample from the iCROP-seq colitis experiment. Original processed data which was used in the analysis presented in the publication can be found at Figshare (https://doi.org/10.6084/m9.figshare.28547528.v2). Overall design: Splenocytes were obtained from Il17a-Cre x Cas9-GFP mice and enriched for CD4+ T cells. With these cells a Th17 polarization was performed. After polarization the cells were transduced with lentiviral particles containing vectors for guide-RNAs and blue fluorescent protein (eBFP2). Transduced cells were then injected in Rag-1-deficient mice. For the disease model of experimental colitis, 14 days prior the injection of the transducted cells into  Rag-1-deficient mice, the mice were gavaged with colitogenic bacteria. Intestinal lymphozytes were then isolated and analysed using scRNAseq.