CRISPR/Cas9-mediated Cxcr3 gene knock-out in unstimulated murine primary CD4+T cells

Background: Recognition of myelin basic protein (MBP) in hepatocytes of CRP-MBP mice induces tolerance of MBP-specific CD4+T cells and protection from MBP-driven neuroinflammatory disease. To investigate mechanisms, we aimed for a gene editing method targeting primary CD4 T cells, which are then followed in vivo after transfer to CRP-MBP mice. As current protocols rely on intermediate in vitro stimulation that can potentially confound the following in vivo experiment, we here establish a CRISPR/Cas9 protocol for unstimulated primary CD4+T cells, using the example of the liver-homing receptor Cxcr3.

Methods: CD4+T cells were isolated from murine spleens utilizing negative selection, subjected to electroporation with Cas9 and sgRNA, and immediately injected intravenously into CD45.1- CRP-MBP mice. Transferred cells were re-isolated after 7 days from blood, liver and spleen and analyzed by flow cytometry.

Results: Transferred T cells were vital after 7 days in vivo, and 80% of the cells manifested Cxcr3 gene knock-out, and undetectable Cxcr3 in flow-cytometry. Cxcr3 knock-out was ineffective in the other 20% of transferred T cells, which could thus serve as internal control. Transferred T cells with Cxcr3 knock-out manifested significantly decreased infiltration into the liver, as compared to transferred T cells without Cxcr3 knock-out.

Conclusion: The study demonstrates the feasibility of CRISPR/Cas9-mediated gene editing in unstimulated murine primary CD4+T cells for use in adoptive transfer experiments. Thus, this technique can be used for identification and functional validation of specific molecules in T cells in vivo without potentially confounding intermediate in vitro culture.

Publication History

Article published online:
20 January 2025