Background: The efficacy of tumor-infiltrating lymphocyte (TIL) therapy is limited by the rarity of anti-tumor TCRs and exhausted status of the product. Its improvement should focus on a high frequency of diverse anti-tumor TCRs with stem-like phenotype, which can be achieved synthetically by identifying multiple anti-tumor TCRs from the patient's intra-tumor TCR repertoire and subsequently engineering them into the patient’s peripheral T cells. Such a ‘synthetic TIL’ is cost-prohibitive using existing technologies. To enable a synthetic TIL product, we developed ultra-high throughput TCR gene synthesis and screening methods to synthesize and screen > 50,000 TCRs from scRNA-Seq data from > 30 patients and identified > 100 tumor/NeoAg-reactive TCRs. The gene expression patterns of the T cells carrying such experimentally validated tumor/NeoAg-reactive TCRs gave rise to an algorithm called Interpreter to predict potentially anti-tumor TCRs from scRNA-Seq data. Methods: To translate synthetic TIL to the clinic, we developed a method to generate up to 100 sequence-verified TCR-encoding plasmids (selected by Interpreter) from fresh tumor biopsy in just 3 weeks. Our GMP-grade production uses a collective knock-in approach, where a mixture of up to 100 plasmids (each encoding a distinct TCR) at equal proportion are packaged into AAV which serve as template to precisely insert the TCR genes into a primarily monoallelic locus and simultaneously inhibit the endogenous TCR. This gene editing is characterized by a modified scRNA-Seq method called EntrySeq. In first-in-human, N-of-1 trials, 10⁹ to 6x10⁹ synthetic TIL cells were administered to solid tumor patients with or without lymphodepletion and IL2. An investigator-initiated Phase 1 trial was then opened to evaluate safety, preliminary efficacy, and cellular activity of synthetic TIL and to determine RP2D and MTD. Subjects with relapsed, unresectable locally advanced solid tumors who received at least 1 standard systemic therapy are allowed to enroll in this study. Those patients with no standard treatment regimens available, or those who are unable to obtain effective treatment due to economic conditions are also permitted. Results: EntrySeq confirmed that > 80% of the T cells express only 1 TCR, avoiding mispairing of different TCRs. The frequencies of different TCRs were largely uniform and were stable through plasmid, AAV and T-cell mixtures. Up to 1.5x10¹⁰ synthetic TIL cells were produced from a patient’s leukopak. 40 to 60% of the T cells in the product for infusion express an exogenous TCR. In first-in-human trials, the administered T cells showed favorable persistence and safety profile. The Phase 1 trial is ongoing. Conclusions: We established a fast, affordable, and GMP-compliant process to manufacture synthetic TIL products, enabling a Phase 1 trial.