A genome-wide in vivo CRISPR screen identifies essential regulators of T cell migration to the CNS in a multiple sclerosis model

To study the effect of the Grk2 or Ets1 KOs in rat and human CD4+ T cells, in vivo or in vitro respectively, we generated KO CD4+ T cell lines by CRISPR-RNP nucleofection (with an gRNA targeting the gene locus) or control (with a non-targeting gRNA) 3' bulk RNAseq was conducted on CD4+ T-MBP rat cells isolated form spleen or spinal cord parenchyma three days after co-transfer of control and KO cells into the animal. Cells were FACS sorted based on their fluorescent protein reporters, BFP for ...

To identify genes driving encephalitogenic CD4+ T cell migration into the CNS, we performed a genome-wide CRISPR screen and a subsequent validation screen. For the genome-wide CRISPR screen, up to 4 sgRNA per gene and 800 non targeted controls were included, for a total of 87690 individual sgRNAs, and for the validation screen, up to 6 sgRNA per gene and 241 non targeted controls for a total of 12000 individual sgRNAs. Comparison of sgRNA distribution by pairwise comparisons across blood, spleen, ...

To study the regulation of candidate genes from our study in human cells, we analyzed CD4+ T cells from blood and CSF of MA patients and age and sex matched idiopathic intracranial hypertension controls We analyzed 40845 cells in control blood, 807 cells in control CSF, 29749 cells in MS blood and 15768 cells in MS CSF. Analysis of overlapping TCR sequences in blood and CSF CD4+ T cells of the same transcript, transcriptomic characterization of the cell clusters and analysis of the behaviour of ...