Purpose: Using gene-disrupted allogeneic T cells as universal effector cells provides an alternative and potentially improves current chimeric antigen receptor (CAR) T-cell therapy against cancers and infectious diseases.Experimental Design: The CRISPR/Cas9 system has recently emerged as a simple and efficient way for multiplex genome engineering. By combining lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, β-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.Results: The CRISPR gene-edited CAR T cells showed potent antitumor activities, both in vitro and in animal models and were as potent as non-gene-edited CAR T cells. In addition, the TCR and HLA class I double deficient T cells had reduced alloreactivity and did not cause graft-versus-host disease. Finally, simultaneous triple genome editing by adding the disruption of PD1 led to enhanced in vivo antitumor activity of the gene-disrupted CAR T cells.Conclusions: Gene-disrupted allogeneic CAR and TCR T cells could provide an alternative as a universal donor to autologous T cells, which carry difficulties and high production costs. Gene-disrupted CAR and TCR T cells with disabled checkpoint molecules may be potent effector cells against cancers and infectious diseases. Clin Cancer Res; 23(9); 2255-66. ©2016 AACR.