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Karklins, R.

Publications and source records attributed to Karklins, R..

2 recordsLinked to original sources
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↗
bioRxiv2025

Versatile and efficient non-viral integration of large transgenes in human T cells via CRISPR knock-in and engineered integrases

Current gene transfer methods often lack the precision, versatility, or efficiency when integrating large transgenes, limiting the ability to engineer therapeutic T-cells with more complex payloads. Here, we report one-pot PASTA (Programmable and Site-specific Transgene Addition), a non-viral genome engineering strategy for large gene insertion that combines CRISPR-Cas-mediated homology-directed repair (HDR) and site-specific recombination via serine integrases. Using one-pot PASTA with the Bxb1 integrase, we demonstrate efficient integration of transgenes at multiple genomic loci relevant for T-cell engineering (e.g., TRAC, B2M, CD3E, CD3Z, GAPDH). For constructs > 8 kb, one-pot PASTA outperforms conventional HDR by 19-fold on average and prime-editing-assisted site-specific integrase gene editing (PASSIGE) by 5-fold. This enables the delivery of multi-cistronic cargo to generate dual-antigen targeting CAR T-cells with a safety-switch that overcome antigen escape in lymphoma models. Finally, one-pot PASTA can be further optimized with improved integrase enzymes, such as engineered variants of Pa01 or Bxb1, and plasmids with minimized backbones. In summary, one-pot PASTA represents a versatile and scalable platform for precise, non-viral gene insertion in T-cells.

synthetic biology↗