Flugel, C. L. | bioRxiv Science

bioRxiv2025

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

The CRISPR-Cas system enables precise genome engineering of cell therapies. For allogeneic applications, multiplex editing is frequently required to improve efficacy, persistence, and safety. However, strategies involving multiple DNA double-strand breaks (DSBs) induce genotoxicity by provoking chromosomal aberrations. Base editors, which enable sequence changes without generating DSBs, are widely used for gene disruption, but their capacity for gene insertion remains unexplored. Here, we developed Base editor-mediated knock-in (BEKI), a non-viral platform that allows targeted transgene insertion in parallel with multiplex gene disruption using a single base editor. Repurposing the Cas9 nickase domain of base editors generates paired nicks, inducing homology-directed repair (HDR). In human T cells, optimized guide RNA orientation and nick distance, together with HDR-enhancing modulators, enabled efficient transgene knock-in at the TRAC, CD3{zeta}, B2M, and CD3{varepsilon} loci. Simultaneous base editing of multiple additional genes produced chimeric antigen receptor (CAR) T cells with increased cytokine secretion, drug resistance, and resistance to allo-rejection. Compared to multiplex editing with Cas9, BEKI markedly reduced chromosomal translocations. BEKI therefore provides a streamlined, scalable strategy for multiplex CAR T-cell engineering with a single enzyme, offering a safer route to clinical-grade manufacturing of off-the-shelf therapies for cancer and autoimmune diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/676172v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@f5f6e5org.highwire.dtl.DTLVardef@246411org.highwire.dtl.DTLVardef@125a478org.highwire.dtl.DTLVardef@510278_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells

I.Multiple genetic modifications may be required to develop potent off-the-shelf chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knock-out (KO) or targeted transgene knock-in (KI). However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells. Here, we combine a non-viral CRISPR-Cas9 nuclease-assisted KI and Cas9-derived base editing technology for DSB free KOs within a single intervention. We demonstrate efficient insertion of a CAR into the T cell receptor alpha constant (TRAC) gene, along with two KOs that silence major histocompatibility complexes (MHC) class I and II expression. This approach reduced translocations to 1.5% of edited cells. Small insertions and deletion at the base editing target sites indicated guide RNA exchange between the editors. This was overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR KI and a Cas9-derived base editor enabled the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. Resulting T cell receptor- (TCR-) and MHC-negative CAR T cells resisted allogeneic T cell targeting in vitro. Thus, we demonstrate a solution for safer multiplex-edited cell products and a path towards off-the-shelf CAR therapeutics.

bioengineering↗