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T cell function can be compromised when continuously exposed to tumor antigens by immune checkpoint signaling. Immunotherapies with T cells and inhibitors of repressive checkpoint molecules such as PD-1, CTLA-4, etc. have been shown promising results in cancer clinical trials. Inhibitors of checkpoint molecules have been solely used to block or suppress the corresponding pathways to improve the functionality of T cells and enhance the anti-tumor immunity. However, few reports combined disruption of repressive checkpoint molecules with TCR-T cell therapy for the treatment of cancer patients. In our study, new TCR sequence was attained from T cells stimulated by Melan-A (aa27-35) peptide, but the cytotoxicity of MART-1-specific TCR-T cells was found to be inhibited by expression of PD-L1 in target cells. To overcome this PD(L)-1-mediated suppression, Cas9 mRNA and gRNA were electroporated into primary T cells infected by MART-1 TCR lentivirus. CRISPR/Cas9-mediated PD-1 disruption in MART-1 TCR-T cells recovered T cell cytotoxicity inhibited by PD-L1 overexpression in target cells. In addition, secretion of IFNγ and IL-2 was also increased in PD-1-damaged TCR-T cells. Furthermore, the differences between wild-type and PD-1-disrupted TCR-T cells were characterized by single-cell RNA (scRNA) sequencing. In summary, our study illustrates enhanced therapeutic efficacy and different features of PD-1-eliminated TCR-T cells compared to the control, providing an alternative strategy for current cell therapy. Citation Format: Qianqian Gao, Renpeng Ding, Huanyi Chen, Qumiao Xu, Linnan Zhu, Xuan Dong, Ying Gu, Cheng-chi Chao. Enhancing anti-tumor efficacy of MART-1-specific TCR-T cells by CRISPR-Cas9-based PD-1 disruption [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2204.