Chimeric antigen receptor T (CAR-T) cells engineered with viral vectors have been successfully applied to treat patients with B cell malignancy. However, viral integration has the potential risk of mutagenesis, and the efforts and cost of viral vector production are demanding. Using non-integrative episomal vector to generate integration free CAR-T cells is an attractive option. Herein we established a novel and fast method to generate minicircle vector without using bacteria (Bacteria-Free (BF) minicircle). Using BF minicircle vector, we generated integration-free CAR-T cells, which eliminated cancer cells efficiently both in vitro and in vivo. While the CAR expression of minicircle vector lasts shorter than that of integrated transgene, it provides an opportunity to avoid serious side effects such as cytokine storm and on-target off-tumor toxicity. (By Chen Cheng, https://jmg.bmj.com/content/early/2018/07/20/jmedgenet-2018-105405 )