PURPOSE: Autologous chimeric antigen receptor T (CAR-T) cell therapy is an effective treatment for relapsed/refractory acute lymphoblastic leukemia (r/r ALL). However, certain characteristics of autologous CAR-T cells can delay treatment availability. Relapse caused by antigen escape after single-targeted CAR-T therapy is another issue. Therefore, we aim to develop CRISPR-edited universal off-the-shelf CD19/CD22 dual-targeted CAR-T cells as a novel therapy for r/r ALL. EXPERIMENTAL DESIGN: In this open-labeled dose-escalation phase I study, universal CD19/CD22-targeting CAR-T cells (CTA101) with a CRISPR/Cas9-disrupted TRAC region and CD52 gene to avoid host immune-mediated rejection were infused in patients with r/r ALL. Safety, efficacy, and CTA101 cellular kinetics were evaluated. RESULTS: CRISPR/Cas9 technology mediated highly efficient, high-fidelity gene editing and production of universal CAR-T (UCAR-T) cells. No gene editing-associated genotoxicity or chromosomal translocation was observed. Six patients received CTA101 infusions at doses of 1 (3 patients) and 3 (3 patients) _ 10(6) CAR-positive T cells/kg body weight. Cytokine release syndrome occurred in all patients. No dose limit toxicity, graft-versus-host disease, neurotoxicity, or genome editing-associated adverse events have occurred to date. The complete remission (CR) rate was 83.3% on D28 after CTA101 infusion. With a median follow-up of 4.3 months, three of the five patients who achieved CR/CRi remained MRD negative. CONCLUSIONS: CRISPR/Cas9-engineered universal CD19/CD22 CAR-T cells exhibited a manageable safety profile and prominent anti-leukemia activity. Universal dual-targeted CAR-T cell therapy may offer an alternative therapy for patients with r/r ALL.