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Related Experiment Video

Updated: Mar 7, 2026

Efficient and Rapid Generation of CAR-T and Cytokine-Induced Killer Cells in GMP-scalable Devices
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Efficient and Rapid Generation of CAR-T and Cytokine-Induced Killer Cells in GMP-scalable Devices

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A versatile system for rapid multiplex genome-edited CAR T cell generation.

Jiangtao Ren1, Xuhua Zhang1, Xiaojun Liu1

  • 1Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Oncotarget
|February 16, 2017
PubMed
Summary

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CRISPR gene editing enables rapid creation of universal CAR T cells for cancer immunotherapy. This one-shot protocol efficiently modifies T cells, enhancing their potential for cell therapy applications.

Area of Science:

  • Biotechnology
  • Immunology
  • Genetics

Background:

  • CRISPR gene editing shows therapeutic promise, particularly in cancer immunotherapy.
  • Genetically modified T cells, such as CAR T cells, are crucial for advanced cell therapies.
  • Targeting multiple gene loci for enhanced T cell function is a key strategy.

Purpose of the Study:

  • To develop a rapid and efficient multiplex genomic editing protocol using CRISPR.
  • To generate allogeneic universal CAR T cells resistant to immune evasion pathways.
  • To explore the potential of a one-shot CRISPR protocol for complex T cell engineering.

Main Methods:

  • Incorporation of multiple guide RNAs (gRNAs) into a CAR lentiviral vector.
  • Utilizing a one-shot CRISPR protocol for multiplex genomic editing.
Keywords:
CD95CRISPR/CAS9PD-1T lymphocyteschimeric antigen receptors

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  • Generating universal CAR T cells through gene disruption of endogenous TCR and HLA class I.

    • Main Results:

    • Achieved high-efficiency double knockout of endogenous TCR and HLA class I for universal CAR T cells.
    • Generated Fas-resistant universal CAR T cells via triple gene disruption.
    • Successfully attempted simultaneous editing of four gene loci for PD-1 and CTLA-4 deficient universal T cells.

    Conclusions:

    • The one-shot CRISPR protocol enables rapid and efficient multiplex genomic editing of T cells.
    • This strategy is effective for generating allogeneic universal CAR T cells with enhanced therapeutic potential.
    • Further optimization could lead to advanced cell therapies resistant to multiple inhibitory signals.