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// Jiangtao Ren 1 , Xuhua Zhang 1 , Xiaojun Liu 1 , Chongyun Fang 1 , Shuguang Jiang 1 , Carl H. June 1, 2 , Yangbing Zhao 1, 2 1 Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 2 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Correspondence to: Yangbing Zhao, email: yangbing@upenn.edu Keywords: CRISPR/CAS9, T lymphocytes, chimeric antigen receptors, PD-1, CD95 Received: November 18, 2016 Accepted: January 27, 2017 Published: February 09, 2017 ABSTRACT The therapeutic potential of CRISPR system has already been demonstrated in many instances and begun to overlap with the rapidly expanding field of cancer immunotherapy, especially on the production of genetically modified T cell receptor or chimeric antigen receptor (CAR) T cells. Efficient genomic disruption of multiple gene loci to generate universal donor cells, as well as potent effector T cells resistant to multiple inhibitory pathways such as PD-1 and CTLA4 is an attractive strategy for cell therapy. In this study, we accomplished rapid and efficient multiplex genomic editing, and re-directing T cells with antigen specific CAR via a one-shot CRISPR protocol by incorporation of multiple gRNAs in a CAR lentiviral vector. High efficient double knockout of endogenous TCR and HLA class I could be easily achieved to generate allogeneic universal CAR T cells. We also generated Fas-resistant universal CAR T cells by triple gene disruption. Simultaneous gene editing of four gene loci using the one-shot CRISPR protocol to generate allogeneic universal T cells deficient of both PD1 and CTLA-4 was also attempted.