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bioRxiv · 10.64898/2026.01.20.700476

Site-specific integration of transposon via engineered piggyBac transposase

Xu, N.·Han, L.·Hu, X.·Fang, Y.·Wu, L.·Wang, X.·Tu, H.·Cong, W.·Sun, K.·Jin, Y.·Wu, X.
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Abstract

The precise integration of large DNA fragments into the human genome holds significant therapeutic potential. Here, we demonstrate that combining engineered piggyBac (PB) transposase with CRISPR/Cas9 enables targeted integration of PB transposons into specified genomic loci. Our engineered PB transposase (PBase) retains high excision activity while substantially reducing endogenous integration activity. In the developed Cas9-PBase fusion system, PBase excises the transposon to generate linear DNA fragments, while Cas9 introduces site-specific double-strand breaks (DSBs), facilitating insertion of the excised fragment at the target locus. The optimized tool achieves 6.1-7.3 kb transposon integration at multiple genomic sites with 10-15% efficiency, demonstrating 60-80% targeted integration specificity. As a proof of concept, we inserted a 7.1 kb transposon encoding three genes into the {beta}2M locus of human induced pluripotent stem cells (iPSCs), conferring protection against allogeneic natural killer (NK) cell-mediated cytotoxicity in derived iNK cells. These results establish Cas9-PBase as a precise and programmable platform for large DNA sequence insertion with potential clinical applications.

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Xu, N., Han, L., Hu, X., Fang, Y., Wu, L., Wang, X., Tu, H., Cong, W., Sun, K., Jin, Y., Wu, X.. 2026-01-21. Site-specific integration of transposon via engineered piggyBac transposase. https://doi.org/10.64898/2026.01.20.700476

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