Querying functional drivers in primary murine naïve and memory T cells using RNP-mediated CRISPR-Cas9 Gene Deletion

ABSTRACT Cytotoxic CD8 T lymphocytes (CTL) are critical for the clearance of pathogenic cells via antigen-dependent cell lysis, thus providing protection against infectious diseases and cancers. CTLs represent one of the key targets of immunotherapies and vaccine design. While microarray and single-cell RNA sequencing of effector and memory CD8 T cells have identified several promising gene targets that may modulate CD8 T cell responses, their development is slowed down by the financial and time constraints related to the generation of germline knockout mice for further validation studies. Here we present a protocol for conducting efficient deletion of genes from activated effector, as well as resting naïve and memory primary murine CD8 T cells using RNP-based Crispr/Cas9 technology. This CRISPR modification of CD8 T cells was then adapted to study the effects of gene deletion in the context of acute memory differentiation as well as chronic exhaustion of CD8 T cells. To this end, we have titrated the necessary dose of antigen-specific CD8 T cells to study their differentiation in acute and chronic infections and confirmed the model by demonstrating rapid expansion of CRISPR mediated PD-1 ablated CD8 T cells in a chronic viral infection. Finally, by combining this methodology with a murine model of subcutaneous tumor challenge, this study provides a unique screening system for genes critical for mediating clearance of malignant tumor cells by CTLs. This study expands current technical capabilities for rapid evaluation of functional role of any candidate gene in CTL responses to infections and cancer through targeted gene deletion at any stage of CD8 T cell differentiation without the need for germline gene deletion mouse models. Full Text Availability The license terms selected by the author(s) for this preprint version do not permit archiving in PMC. The full text is available from the preprint server.