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<p>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 <Emphasis Type="Underline">g</Emphasis>enome engineering <Emphasis Type="Underline">catalyst</Emphasis> (GATALYST) for efficient and safe gene knock-in. Here, we introduce enGager, an <Emphasis Type="Underline">en</Emphasis>hanced <Emphasis Type="Underline">G</Emphasis>ATALYST <Emphasis Type="Underline">a</Emphasis>ssociated <Emphasis Type="Underline">g</Emphasis>enome <Emphasis Type="Underline">e</Emphasis>dito<Emphasis Type="Underline">r</Emphasis> 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 ‘<Emphasis Type="Underline">t</Emphasis>ripartite <Emphasis Type="Underline">e</Emphasis>ditor with <Emphasis Type="Underline">s</Emphasis>sDNA <Emphasis Type="Underline">o</Emphasis>ptimized <Emphasis Type="Underline">g</Emphasis>enome <Emphasis Type="Underline">en</Emphasis>gineering (TESOGENASE) offers a safer, more efficient alternative to viral vectors for therapeutic gene modification.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年5月16日

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  • Hangu Nam,
  • Keqiang Xie,
  • Ishita Majumdar,
  • Jiao Wang,
  • Shaobo Yang,
  • Jakob Starzyk,
  • Danna Lee,
  • Richard Shan,
  • Jiahe Li,
  • Hao Wu

摘要

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.