Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration
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...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-05-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-59790-3 |
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| author | Hangu Nam Keqiang Xie Ishita Majumdar Jiao Wang Shaobo Yang Jakob Starzyk Danna Lee Richard Shan Jiahe Li Hao Wu |
| author_facet | Hangu Nam Keqiang Xie Ishita Majumdar Jiao Wang Shaobo Yang Jakob Starzyk Danna Lee Richard Shan Jiahe Li Hao Wu |
| author_sort | Hangu Nam |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-a196846003ca4aa89ea21cc99d1b559e |
| institution | DOAJ |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-a196846003ca4aa89ea21cc99d1b559e2025-08-20T03:07:56ZengNature PortfolioNature Communications2041-17232025-05-0116111410.1038/s41467-025-59790-3Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integrationHangu Nam0Keqiang Xie1Ishita Majumdar2Jiao Wang3Shaobo Yang4Jakob Starzyk5Danna Lee6Richard Shan7Jiahe Li8Hao Wu9Department of Bioengineering, Northeastern UniversityFull Circles Therapeutics, INC.Full Circles Therapeutics, INC.Full Circles Therapeutics, INC.Department of Bioengineering, Northeastern UniversityFull Circles Therapeutics, INC.Full Circles Therapeutics, INC.Full Circles Therapeutics, INC.Department of Biomedical Engineering, College of Engineering and School of Medicine, University of MichiganFull Circles Therapeutics, INC.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.https://doi.org/10.1038/s41467-025-59790-3 |
| spellingShingle | Hangu Nam Keqiang Xie Ishita Majumdar Jiao Wang Shaobo Yang Jakob Starzyk Danna Lee Richard Shan Jiahe Li Hao Wu Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration Nature Communications |
| title | Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration |
| title_full | Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration |
| title_fullStr | Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration |
| title_full_unstemmed | Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration |
| title_short | Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration |
| title_sort | engineering tripartite gene editing machinery for highly efficient non viral targeted genome integration |
| url | https://doi.org/10.1038/s41467-025-59790-3 |
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