Abstract T cell therapies for cancer have shown striking clinical efficacy in different tumor settings. Integration of a transgene cassette expressing an antigen receptor into the genome enables T cells to recognize specific tumor antigens. Viral vectors and transposons can introduce transgenes into primary human T cells. However these approaches cannot control genomic location and have limited control over the copy number of cassette insertions. Here, we used epigenetic analysis, transcriptional profiling, high-throughput gene-editing and T cell functional experiments to identify the optimal genomic locus amenable to non-viral genome targeting. First we analyzed the epigenetic landscape of primary human T cells to identify regions (“gene deserts”) that are accessible but devoid of annotated regulatory elements or coding sequences. Candidate loci were validated using CRISPR/Cas9 genome editing to directly cut and a marker gene cassette was inserted into these sites. Knock-in efficiency and transgene expression stability in primary human T cells were evaluated for all loci. Evaluation of 40 loci demonstrated substantial variability (10 fold) in transgene expression and confirmed that not all sites were equivalent in their ability to sustain transgene expression. Next, we evaluated 8 most promising candidate loci for compatibility with complex T cell programs embodied by integrated circuits, containing a priming receptor (PrimeR) that triggers expression of a CAR in response to a priming antigen. We identified one integration site (termed “GS94”) that supported: 1) stable and high PrimeR expression; 2) high and inducible CAR expression; and 3) a superior T cell cytotoxic and cytokine secretion profile. Finally, we assessed off-target cutting mediated by the CRISPR sgRNA targeting GS94. The sgRNA is the most specific out of the candidates evaluated with iGUIDE and targeted PCR. We were unable to detect any measurable off-target activity by targeted PCR on predicted sites. The GS94 site is a component of the integrated circuit T cell (ICT) therapy, AB-1015, which is now under development for the treatment of high-grade serous carcinoma ovarian tumors. AB-1015 includes an inserted cassette that contains a logic gate and two shRNAs that enhance the potency and solid tumor microenvironment resistance of the AB-1015 ICT cells. These computational and experimental approaches can be generalized to identify new safe harbor sites for different cell types, expanding the genome engineering toolkit for diverse cell therapy applications. Citation Format: Grace X.Y. Zheng, Somya Khare, Brendan Galvin, Robby Moot, Aaron Cooper, Michelle Nguyen, Michelle Tan, Shan Sabri, Audre May, Jun Fung, Anzhi Yao, Andrea Liu, Matt Drever, Steve Santoro, W Nicholas Haining, Tarjei Mikkelsen. Identification of a safe harbor CRISPR-Cas9 integration site for improved cell therapy safety and potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2840.