Recent clinical applications for allogenic lymphocytes with chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs), have opened the door for curative immunotherapies. With this ground-breaking technology established, lymphocytes with engineered receptors can n ow be screened for their recognition to numerous disease-associated antigens in vitro. Here, we deploy pre-designed TRAC- and TRBC-targeting EDIT-R synthetic guides (sgRNAs) that serve as potent and effective TCR knock-out controls prior to screening the specificity of engineered receptors to an antigen of interest. Briefly in this application, we demonstrate greater-than 95% RNP-based knock-out of the endogenous αβ T cell receptor (TCR) expressed on primary CD8+ T cells utilizing four unique sgRNAs individually. The resulting TCR-negative CD8+ T cells did not retain endogenous cytotoxic killing function, whereas TCR-negative CD8+ T cells engineered with anti-CD19 CAR lentivirus showed CD19-specific killing. Revvity pre-designed TRAC- and TRBC-targeting EDIT-R sgRNAs are a valuable control that can seamlessly be added to the CAR-T screening workflow.
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