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bioRxiv · 10.1101/2025.09.16.676703

Gene-sized DNA insertion at genomic safe harbors in human cells using a site-directed transposase

Short, J. E.·Sharek, L.·Meckler, J. F.·Stoytchev, I.·Tran, C. T.·Errard, C.·Hew, B. E.·Johnson, B. E.·Waller, D. F.·Xie, S.·Higgins, J. J.·Tabibiazar, R.·Owens, J. B.
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Abstract

Achieving precise and efficient integration of gene-sized DNA sequences into the human genome remains a major obstacle to gene therapy. Existing approaches depend on double-strand DNA breaks, which can lead to unintended genome alterations. Many monogenic diseases arise from diverse patient-specific mutations, making individualized correction impractical and underscoring the need for universal full-gene replacement strategies. We developed INsertion by Targeted Anchoring and Conditional Transposition (INTACT) to enable targeted insertion at genomic safe harbor loci. We engineered a mammalian transposase with mutations in its DNA-binding domain to reduce off-target integration. Site specificity was then restored by linking programmable sequence-specific DNA-binding proteins to the transposase. Systematic optimization of INTACT revealed key determinants of precision, including non-covalent linkage between the transposase and DNA-binding protein, strict spacing between the binding site and the TTAA insertion sequence, and linkage of the DNA-binding protein to an internal position within the transposase. On-target insertion was achieved across multiple loci, with optimized INTACT averaging 1.2 targeted insertions per cell. An off-target assay confirmed that DNA-binding domain mutations substantially reduced unwanted integration events to near-background levels. Our site-directed transposase enables precise, efficient genomic insertion of >4kb DNA without double-strand breaks, offering a powerful new tool for genome engineering.

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Short, J. E., Sharek, L., Meckler, J. F., Stoytchev, I., Tran, C. T., Errard, C., Hew, B. E., Johnson, B. E., Waller, D. F., Xie, S., Higgins, J. J., Tabibiazar, R., Owens, J. B.. 2025-09-17. Gene-sized DNA insertion at genomic safe harbors in human cells using a site-directed transposase. https://doi.org/10.1101/2025.09.16.676703

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