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PreprintCited by 2CRISPR and Genetic EngineeringVirus-based gene therapy researchRNA Interference and Gene Delivery

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

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Short, James E., Sharek, Lisa, Meckler, Joshua F., Stoytchev, Ilko, Tran, Christopher T., Errard, Clara, Hew, Brian E., Johnson, Brandon E., Waller, David F., Xie, Shanfu, Higgins, Joseph J., Tabibiazar, Raymond, Owens, Jesse B.·2025·Nucleic Acids Research
DOI 10.1101/2025.09.16.676703PMC PMC12684395

Preprint — published as 10.1093/nar/gkaf1316

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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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Metadata

Authors
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.
Affiliations
University of Hawaiʻi at Mānoa (US)
Corresponding author
Owens, Jesse B.
ORCID
Venue
Nucleic Acids Research
Publisher
openRxiv
Published
2025-09-17
Volume
53
Issue
22
ISSN
0305-1048 (ISSN-L)
DOI
10.1101/2025.09.16.676703
PMC
PMC12684395
OpenAlex
W4417183639
Cited by
2
References
1
Citations by year
2026: 2
FWCI
0.60
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by-nc)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, other-oa)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed Central (submittedVersion)
Funders
  • National Institute of Biomedical Imaging and Bioengineering (R01 EB031124)
  • National Institute of General Medical Sciences (R01 GM160155)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
IntAct, gene therapy, DNA-binding domain mutations, genome editing, human cells

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