Generating universal anti-CD19 CAR T cells with a defined memory phenotype by CRISPR/Cas9 editing and safety evaluation of the transcriptome

Front Immunol. 2024 May 29:15:1401683. doi: 10.3389/fimmu.2024.1401683. eCollection 2024.


Introduction: Chimeric antigen receptor-expressing T cells (CAR T cells) have revolutionized cancer treatment, particularly in B cell malignancies. However, the use of autologous T cells for CAR T therapy presents several limitations, including high costs, variable efficacy, and adverse effects linked to cell phenotype.

Methods: To overcome these challenges, we developed a strategy to generate universal and safe anti-CD19 CAR T cells with a defined memory phenotype. Our approach utilizes CRISPR/Cas9 technology to target and eliminate the B2M and TRAC genes, reducing graft-versus-host and host-versus-graft responses. Additionally, we selected less differentiated T cells to improve the stability and persistence of the universal CAR T cells. The safety of this method was assessed using our CRISPRroots transcriptome analysis pipeline, which ensures successful gene knockout and the absence of unintended off-target effects on gene expression or transcriptome sequence.

Results: In vitro experiments demonstrated the successful generation of functional universal CAR T cells. These cells exhibited potent lytic activity against tumor cells and a reduced cytokine secretion profile. The CRISPRroots analysis confirmed effective gene knockout and no unintended off-target effects, validating it as a pioneering tool for on/off-target and transcriptome analysis in genome editing experiments.

Discussion: Our findings establish a robust pipeline for manufacturing safe, universal CAR T cells with a favorable memory phenotype. This approach has the potential to address the current limitations of autologous CAR T cell therapy, offering a more stable and persistent treatment option with reduced adverse effects. The use of CRISPRroots enhances the reliability and safety of gene editing in the development of CAR T cell therapies.

Conclusion: We have developed a potent and reliable method for producing universal CAR T cells with a defined memory phenotype, demonstrating both efficacy and safety in vitro. This innovative approach could significantly improve the therapeutic landscape for patients with B cell malignancies.

Keywords: CRISPR/Cas9; CRISPRroots; allogeneic CAR-T cells; anti CD 19 CAR-T cells; memory CAR-T cells.

MeSH terms

  • Antigens, CD19* / genetics
  • Antigens, CD19* / immunology
  • CRISPR-Cas Systems*
  • Cell Line, Tumor
  • Gene Editing* / methods
  • Humans
  • Immunologic Memory*
  • Immunotherapy, Adoptive* / adverse effects
  • Immunotherapy, Adoptive* / methods
  • Phenotype
  • Receptors, Chimeric Antigen* / genetics
  • Receptors, Chimeric Antigen* / immunology
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism
  • Transcriptome*


  • Antigens, CD19
  • Receptors, Chimeric Antigen

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This publication is based upon work from COST Action Gene Editing for the treatment of Human Diseases, CA21113 ( supported by COST (European Cooperation of Science and Technology). This project was funded by Spanish ISCIII Health Research Fund and the European Regional Development Fund (FEDER) through research grants PI18/01016 (CH) and PI18/00330 (KB). The CsyF of Junta de Andalucia FEDER/European Cohesion FUND (FSE) for Andalusia provided by the following research grant PE-0223-2018, and PECART-0027-2020. The study was also supported by Consejería de Universidad, Investigación e Innovación under Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020) (ProyExcel_00875), Junta de Andalucía. K.B. held Nicolas Monardes contract from Consejería de Salud y Consumo of the Junta de Andalucía. MDCL is funded by the grants PE-0223-2018 and RH-0058-2021 from CsyF of the Junta de Andalucia. This work was supported by the Independent Research Fund Denmark, FTP (9041-00317B to JG) and the Novo Nordisk Foundation (NNF21OC0068988 to JG). PHV is funded by Spanish Ministry of Education and Science through fellowships FPU20/03260. PHV is PhD student from the Biomedicine. Instituto de Salud Carlos III (ISCIII) and the European Regional Development Fund (FEDER): Research grants, PI21/00298 (FM); ISCIII – NextGenerationEU funds - actions of the Recovery and Resilience Mechanism: Red TerAv RD21/0017/0004. (FM). Ministerio de Ciencia e Innovación (MICIN). Plan de Recuperación, Transformación y Resiliencia, Centro para el Desarrollo Tecnológico Industrial (CDTI) and European Union-Next Generation EU: Research grants PMPTA22/00060 (FM). Consejería de Salud y Familias (CSyF) - Junta de Andalucía - FEDER/European Cohesion Fund (FSE) for Andalucía: Grants: CARTPI-0001-201 and PECART-0031-2020. Ministerio de Ciencia e Innovación (MICIN) – líneas estratégicas: Grant PLEC2021-008094 (FM). FM is financed by Maria Zambrano Senior Contract by the University of Granada.