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Updated: Mar 7, 2026
Efficient and Rapid Generation of CAR-T and Cytokine-Induced Killer Cells in GMP-scalable Devices
Published on: December 5, 2025
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.
Abstract:
The therapeutic potential of CRISPR system has already been demonstrated in many instances and begun to overlap with the rapidly expanding field of cancer immunotherapy, especially on the production of genetically modified T cell receptor or chimeric antigen receptor (CAR) T cells. Efficient genomic disruption of multiple gene loci to generate universal donor cells, as well as potent effector T cells resistant to multiple inhibitory pathways such as PD-1 and CTLA4 is an attractive strategy for cell therapy. In this study, we accomplished rapid and efficient multiplex genomic editing, and re-directing T cells with antigen specific CAR via a one-shot CRISPR protocol by incorporation of multiple gRNAs in a CAR lentiviral vector. High efficient double knockout of endogenous TCR and HLA class I could be easily achieved to generate allogeneic universal CAR T cells. We also generated Fas-resistant universal CAR T cells by triple gene disruption. Simultaneous gene editing of four gene loci using the one-shot CRISPR protocol to generate allogeneic universal T cells deficient of both PD1 and CTLA-4 was also attempted.
Insights
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.
- 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.
