Gene editing is being used to improve cell-based therapies, including disruption of checkpoint protein genes to create engineered cells refractory to tumor immunosuppression or knockdown of endogenous TCRs to reduce mispairing of endogenous and engineered TCRs and/or to create allogeneic donor cells. The work published in Mol Ther., 24(9), 1570-1580, 2016 and summarized above augmented their previous virus- based CRISPR-mediated delivery method with the MaxCyte GTx®. Activated T cells were first electroporated with multiple mRNAs encoding Cas9 and AAV helper proteins. Electroporated cells were then transduced with AAV for delivery of CRISPR gRNA targeting Tim3 alone or Tim3 & CD3. The dual gRNA targeting both Tim3 and CD3 lead to >60% knockdown of both proteins as assessed via FACS. See publication for detailed methods.