Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration

Hangu Nam, Keqiang Xie, Ishita Majumdar, Jiao Wang, Shaobo Yang, Jakob Starzyk, Danna Lee, Richard Shan, Jiahe Li () and Hao Wu ()
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Hangu Nam: Northeastern University
Keqiang Xie: Full Circles Therapeutics, INC.
Ishita Majumdar: Full Circles Therapeutics, INC.
Jiao Wang: Full Circles Therapeutics, INC.
Shaobo Yang: Northeastern University
Jakob Starzyk: Full Circles Therapeutics, INC.
Danna Lee: Full Circles Therapeutics, INC.
Richard Shan: Full Circles Therapeutics, INC.
Jiahe Li: University of Michigan
Hao Wu: Full Circles Therapeutics, INC.

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: Abstract Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination (HR), with efficiency improved by CRISPR/Cas9 technology. Circular single-stranded DNA (cssDNA) has been used as a genome engineering catalyst (GATALYST) for efficient and safe gene knock-in. Here, we introduce enGager, an enhanced GATALYST associated genome editor system that increases transgene integration efficiency by tethering cssDNA donors to nuclear-localized Cas9 fused with single-stranded DNA binding peptide motifs. This approach further improves targeted integration and expression of reporter genes at multiple genomic loci in various cell types, showing up to 6-fold higher efficiency compared to unfused Cas9, especially for large transgenes in primary cells. Notably, enGager enables efficient integration of a chimeric antigen receptor (CAR) transgene in 33% of primary human T cells, enhancing anti-tumor functionality. This ‘tripartite editor with ssDNA optimized genome engineering (TESOGENASE) offers a safer, more efficient alternative to viral vectors for therapeutic gene modification.

Date: 2025
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DOI: 10.1038/s41467-025-59790-3

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