We first reproduced the inhibition of Ag-specific CD4 T cells proliferation in the presence of a first cohort with in vitro generated memory Marilyn cells, allowing us to perform the genome wide inactivation of these cells transduced with the lentiviral gRNA libraries. To overcome the delivery challenges of Cas9, we have crossed Marilyn CD45.1 mice to Cas9-expressing mice. In the hypothesis that genes involved in the activation or survival of memory CD4 T cells are also involved in their proliferation as well as their retention in the lymph node, we will also use a Tamoxifen-inducible strategy (Rosa26-LSL-Cas9 mice crossed to Tam inductible-Cre-ERT2 Marilyn mice, allowing for temporally controlled gene inactivation. We have already optimized Marilyn CD4 T cells transduction. We have also validated the coverage of sgRNAs in both amplified libraries by deep sequencing. We will perform the pool screening several times and 10 recipient mice will be used to generate sufficient numbers of T cells for DNA isolation and deep sequencing, as well as to reduce potential variability. Likewise, a large number of control sgRNAs within each pool are present to assess the frequency of off-target effects. Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) method will be performed to robustely identify the targets of the in vivo positive screen. New gRNAs corresponding to the identified hits will be designed and validated in OT2 model prior to a depth functional annotation of immunosuppressive pathways study by RNAseq with Nextera protocol and HiSeq sequencer.
