Molecular therapy : the journal of the American Society of Gene TherapyJul 21, 2026

Using base editors to precisely add and remove genes at the same time to create allo-CAR T cells with fewer DNA rearrangements

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jul 22, 2026

Abstract

Base editor-mediated knock-in (BEKI) enables efficient transgene insertion alongside up to 10 simultaneous gene knockouts.

BEKI utilizes a single enzyme to achieve both transgene insertion and multiplex gene disruption.
The approach generates localized DNA breaks at the target site, reducing the risk of harmful chromosomal rearrangements.
Optimized guide RNA design significantly improves the efficiency of transgene insertion across relevant genomic regions.
Inhibition of DNA-PK enhances knock-in efficiency but is associated with increased large deletions, which can be reduced by co-inhibiting another enzyme.
Compared to traditional multiplex Cas9 editing, BEKI lowers the incidence of chromosomal translocations while maintaining cell viability.
This method supports the creation of allogeneic CAR T cells with improved cytokine release and resistance to immune rejection.

Simplified

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.