Abstract Background Although successful discovery and applications of genome editing system, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/associated nuclease 9), has been reported in basic science research, it has been unclear what happen to patients when moving CRISPR/Cas9 technology to bedside. We performed the first-in-man clinical trial (NCT02793856), phase I, to assess the safety of CRISPR/Cas9-mediated knockout of the programmed cell death 1 (PD-1) gene in autologous T lymphocytes in patients with metastatic non-small cell lung cancer (NSCLC). Methods Using a 3 + 3 + 3 dose-escalation study, we assigned patients with advanced NSCLC with PD-L1 expression positive on tumor cells that had progressed after the 3rd line standard therapeutic regimens. Pre-A cohort was used to enroll 2 patients who received PD-1 deficient gene-edited T cells (PD-1- T) with 2x107 cells/kg in one cycle via CRISPR/Cas9 for safety concern. Then 3 cohorts (A, B, C) enrolled 3 patients receiving total dose of PD-1- T cells with 1 ×107/kg, 2 x 107/kg, 4×107/kg each cycle every 4 weeks, respectively. The primary outcome was safety. Secondary end points were objective response rate, 8 weeks progression-free survival. In exploratory analyses, next-generation sequencing (NGS) was performed on PD-1 editing region of T cell and the CDR3 region of the T-cell receptor beta chain. Results 8 patients received totally 16 cycles of PD-1- T cell infusion in cohort Pre-A, A and B. 13 adverse events (AEs) related to the PD-1- T cell infusion occurred (84.6% of grade 1 and 15.4% of grade 2). Most common AEs were acute fever and hepatic dysfunction (both 15.4%). No DLT and 3-5 AEs were found. 92.3% (12/13) AEs presented within 1st cycle. 7 patients were response evaluable. 2/4 of patients receiving 2 x 107/kg PD-1- T cell experienced SD while other 3/3 patients with lower dose had disease progression. 8-week PFS were 28.6%. Of exploratory data, numerous novel T-cell clonotypes were detected in PD-1- T cell products but not in peripheral bloods before cell infusion. Intriguingly, the persisting novel T cell clones were found in patient peripheral bloods with the frequency of 0.005%-3.05% during the cell therapy, indicating the existence of potential responsive T cell clones. Conclusion The patients in this trial seemed safety when conducing the PD-1 deficient engineered T cells with CRISPR/Cas9 system. Further study should be performed to explore the effective dose and the related immune response. Citation Format: You Lu, Meijuan Huang, Tao Deng, Xiaojuan Zhou, Kun Yu, Maozhi Liang, Lei Deng, Jianxin Xue, Xin Yi, Zhenyu Ding, Youling Gong, Jiang Zhu, Yongsheng Wang, Yuqi Wang, Jin Song, Ruizhan Tong, Li Li, Jingwen Huang, Feifei Na, Min Zhao, Chong Chen, Yuquan Wei, Weimin Li. A phase I trial of PD-1 deficient engineered T cells with CRISPR/Cas9 in patients with advanced non-small cell lung cancer with PD-L1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT133.