Repurposing base editors for targeted knock-in and simultaneous multiplex knockouts to generate allo-CAR T cells with minimal translocations.

Authors

Glaser Viktor, Becker Lily Jo, Fuster-García Carla, Aird Eric Jackson, Huth Luis, Nitulescu Ana-Maria, Pu Yaolin, Kassing Isabell, Hartmann Laura Marie, Flugel Christian Luca, Kārkliņš Roberts, Shaji Shona, Pouzolles Marie, Stein Maik, Andrieux Geoffroy, Corn Jacob Ellery, Cathomen Toni, Volk Hans-Dieter, Reinke Petra, Kath Jonas, Wagner Dimitrios Laurin

Published in

Molecular therapy : the journal of the American Society of Gene Therapy. Jul 20, 2026. Epub Jul 20, 2026.

Abstract

Multiplex genome editing of cellular therapies frequently requires multiple DNA double-strand breaks (DSBs), which can induce genotoxicity through chromosomal rearrangements and large deletions. Base editors enable targeted sequence changes with minimal DSBs and are widely used for gene disruption, but their capacity for transgene insertion has remained unexplored. Here, we developed base editor-mediated knock-in (BEKI), a non-viral platform combining transgene insertion with multiplex gene disruption using a single enzyme. BEKI repurposes the base editor's Cas9 nickase domain to generate paired nicks (inducing a localized DSB) at the knock-in locus, while achieving multiplex knockouts through base editing. Optimized guide RNA orientation and spacing enabled efficient transgene insertion across multiple T cell-relevant genomic loci. DNA-PK inhibition enhanced knock-in efficiency but increased kilobase-scale deletions, which were mitigated by co-inhibition of Polθ. Compared with multiplex Cas9 editing, BEKI markedly reduced chromosomal translocations while preserving cell viability. BEKI supported targeted CAR knock-in alongside up to 10 simultaneous gene knockouts, enabling the generation of allogeneic CAR T cells with enhanced cytokine secretion and resistance to immunosuppressants and allo-rejection. Together, BEKI provides a streamlined and scalable strategy for multiplex CAR T-cell engineering with improved genomic stability, advancing safer next-generation cell therapies for cancer and autoimmune diseases.

PMID:
42478045
Bibliographic data and abstract were imported from PubMed on 21 Jul 2026.

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