Transfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR a and b genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR “single editing” (SE) approach, based on the disruption of the endogenous TCR a chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1 157-165 –specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1 pos targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy. © 2017 by The American Society of Hematology.