BACKGROUND: To date, the non-viral vector Chimeric Antigen Receptor (CAR) T cell preparation platform, exemplified by transposons, has demonstrated significant potential in tumor immunotherapy and yielded positive results in multiple clinical trials. Nonetheless, non-methylated CpG sequences within plasmid DNA can elicit an inflammatory response via Toll-like receptor 9 (TLR9) during CAR-T cell preparation, adversely affecting transgene expression. Additionally, de novo DNA methylation programs promote T cell exhaustion, which poses a significant limitation for CAR-T cell therapy applications. METHODS: High-throughput liquid protein chip and CBA analyses were utilized to determine the expression levels of inflammatory factors. Flow cytometry and luciferase reporter assays were employed for mutation screening. BALB/c mice and M-NSG mice were used to evaluate the inflammatory response and efficacy of LCG CAR-T RESULTS: In this study, we modified the newly discovered Passer (JL) transposon to construct a low-CpG content transposon for CAR-T cell (LCG CAR-T cell) preparation. CONCLUSIONS: Collectively, our results demonstrate the high safety and efficacy of non-viral, low CpG Passer transposon CAR-T cells, offering new avenues for improving CAR-T cell efficacy while minimizing