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bioRxiv · 10.1101/2025.08.06.668737

Efficient gene disruption with CRISPR-Cas3 in human T cells

Mashimo, T.·Fujii, T.·Sakoda, Y.·Yoshimi, K.·Takeshita, K.·Yokoyama, K.·Watanabe, S.·Tamada, K.
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Abstract

The CRISPR-Cas9 system has been widely adopted as a genome editing tool due to its high efficiency and versatility, contributing to the development of various therapeutic strategies. However, its clinical application remains limited by safety concerns, including off-target effects and large-scale chromosomal rearrangements such as translocations and inversions. Recently, the CRISPR-Cas3 system, a Class 1 CRISPR effector complex with unidirectional DNA degradation activity, has gained attention as a potential alternative, offering reduced off-target activity. In this study, we applied the CRISPR-Cas3 system to human T cells and successfully disrupted two clinically relevant genes, T cell receptor alpha constant (TRAC) and beta-2 microglobulin (B2M). These gene deletions were associated with a reduction in both graft-versus-host disease (GVHD) risk and host immune rejection. Importantly, no off-target mutations were detected in CRISPR-Cas3-edited cells, in contrast to the off-target effects observed with CRISPR-Cas9. Furthermore, CAR-T cells generated by deleting TRAC or B2M using CRISPR-Cas3 maintained their antigen-specific cytotoxicity against tumor cells, while exhibiting reduced alloreactivity. These results suggest that CRISPR-Cas3 provides a safer and promising platform for genome editing in T cell engineering, with potential applications in the development of next-generation allogeneic T cell therapies.

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Mashimo, T., Fujii, T., Sakoda, Y., Yoshimi, K., Takeshita, K., Yokoyama, K., Watanabe, S., Tamada, K.. 2025-08-08. Efficient gene disruption with CRISPR-Cas3 in human T cells. https://doi.org/10.1101/2025.08.06.668737

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