T-cell immunotherapy is showing great promise and therefore undergoing intensive developments for cancer treatment. In this study, we applied liposome-encapsulated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9) genome editing tool to specifically knock out the programmed death-1 (PD-1) gene from T cells (PD-1- T cells). We then activated these cells by dendritic/tumor fusion cells (FCs) and examined their anti-cancer potential. Results showed that, following the antigen presentation and activation by DC/HepG2 FCs, PD-1- T cells showed a significantly higher ability than PD-1+ T cells to proliferate, secrete pro-inflammatory cytokine IFN-γ, and kill HepG2 cells in vitro. Consistently, in vitro activated PD-1- T cells inhibited proliferation and induced apoptosis in HepG2 xenografts in vivo, leading to significantly suppressed tumor growth and improved mouse survival. Liposome-encapsulated CRISPR/Cas9 genome editing technology effectively knocked out PD-1 gene in T cells, stimulating T cell activation in response to DC/tumor FCs and affording T cell-mediated cancer immunotherapy. Our study provides evidence to target checkpoint receptors in adoptively transfected T cells, as a novel therapeutic modality for adoptive T cell transfer.