In recent years, chimeric antigen receptor (CAR) T cell therapy has shown great promise in treating hematological malignancies. However, CAR T cell therapy currently has several limitations. Here we successfully developed a two-in-one approach to generate non-viral genome specific targeted CAR T cells through CRISPR/Cas9. Based on the optimized protocol, the feasibility was preliminarily demonstrated by a preclinical study inserting an anti-CD19 CAR cassette into the AAVS1 safe harbor locus. We found that non-viral AAVS1-knockin CAR T cells behave comparably to those conventionally produced by lentivirus. Furthermore, an innovative type of anti-CD19 CAR T cells with PD1-integration was constructed and shows a superior ability to eradicate tumor cells with high PD-L1 expression. In adoptive therapy for relapsed/refractory (r/r) aggressive B-cell non-Hodgkin lymphoma (B-NHL), we observed a high rate (87.5%) of complete remission (CR) and durable responses without serious adverse events in eight patients after treatment. Notably, these enhanced CAR T cells were effective even at a low infusion dose and with a low CAR percentage, which indicated that they have higher potency. No off-target events were found in the infusion product. Single-cell RNA sequencing analysis further validated the advantage of PD1 interference that results in fewer dysfunctional CAR T cells through this treatment. Collectively, our results demonstrate the outstanding safety and efficacy of non-viral genome specific integrated CAR T cells, thus providing a revolutionary technology for CAR T cell therapy.