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

作者：Hangu Nam, Keqiang Xie, Ishita Majumdar, Jiao Wang, Shaobo Yang, Jakob Starzyk, Danna Lee, Richard Shan, Jiahe Li, Hao Wu · 发表于：Nature Communications · 年份：2025 · DOI：10.1038/s41467-025-59790-3 · 被引用次数：6 · 研究领域：CRISPR and Genetic Engineering、Virus-based gene therapy research、RNA regulation and disease

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. Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination. Here the authors present the TESOGENASE system which enhances CRISPR-based gene integration by tethering circular single-stranded DNA to Cas9.