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Updated: Sep 9, 2025
Production of Human CRISPR-Engineered CAR-T Cells
Published on: March 15, 2021
Orthogonal CRISPR systems for targeted integration and multiplex base editing enable nonviral engineering of
Nanna S Mikkelsen1, Sujan Ravendran1, Amalie D Broksø1
1Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark.
Abstract:
Multiple genomic modifications, including targeted transgene integrations and knockouts, may be required to develop potent, allogeneic chimeric antigen receptor (CAR)-T cell therapies. Conventional CRISPR-Cas systems generate double-strand breaks (DSBs) associated with genomic rearrangements and genotoxicities. DSB-free base editing reduces these risks. Here, we facilitate multiplex editing by combining Staphylococcus aureus Cas9 (SaCas9) mRNA base editors for DSB-free knockout of B2M and REGNASE-1 with Streptococcus pyogenes Cas9 nucleases for targeted integration of an anti-CD19 CAR transgene at the T cell receptor α constant locus. Combined, these edits have been reported to generate safer allogeneic CAR-T cells with enhanced activity and persistence. We demonstrate multiplex gene editing in primary human T cells with B2M and REGNASE-1 base editing frequencies reaching 66% and 84%, respectively, while integrating the anti-CD19 CAR transgene in up to 36% or 71% of cells using nonviral single-stranded DNA repair templates or viral vector templates (AAV6), respectively. Importantly, no detrimental effects on CAR-T cell function were observed in vitro or in vivo, and knockout by base editing reduced rates of balanced chromosomal translocations by 210-fold. This orthogonal CRISPR-Cas engineering approach represents a novel and safer strategy for nonviral, multiplexed genetic engineering of CAR-T cells.
Insights
This study introduces a safer, double-strand break-free CRISPR base editing method for engineering chimeric antigen receptor (CAR) T cells, enhancing their potential for cancer therapy.
Area of Science:
- Gene editing technologies
- Cellular immunotherapy
- Genomic engineering
Background:
- Developing potent allogeneic chimeric antigen receptor (CAR) T cell therapies requires complex genomic modifications.
- Conventional CRISPR/Cas systems carry risks of genomic rearrangements and genotoxicity due to double-strand breaks (DSBs).
Purpose of the Study:
- To develop a safer, multiplex gene editing strategy for CAR T cell therapy by utilizing DSB-free base editing.
- To combine base editing for gene knockouts with targeted transgene integration for enhanced CAR T cell development.
Main Methods:
- Utilized S. aureus Cas9 (SaCas9) mRNA base editors for DSB-free knockout of B2M and REGNASE-1.
- Employed S. pyogenes Cas9 (SpCas9) nucleases for targeted anti-CD19 CAR transgene integration at the TRAC locus.
- Performed multiplex gene editing in primary human T cells using nonviral ssDNA or viral vector (AAV6) templates.
Main Results:
- Achieved high base editing frequencies for B2M (66%) and REGNASE-1 (84%).
- Successfully integrated the anti-CD19 CAR transgene in up to 71% of cells.
- Demonstrated a 210-fold reduction in balanced chromosomal translocations compared to traditional methods, with no observed detrimental effects on CAR T cell function.
Conclusions:
- This orthogonal CRISPR/Cas approach offers a novel and safer strategy for nonviral, multiplexed genetic engineering of CAR T cells.
- DSB-free base editing significantly reduces genotoxic risks associated with CAR T cell development.
- The developed method enhances the potential for creating safer and more effective allogeneic CAR T cell therapies.
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