Cited by 35CRISPR and Genetic EngineeringVirus-based gene therapy researchRNA Interference and Gene Delivery

Directed evolution of hyperactive integrases for site specific insertion of transgenes

Open access
Hew, Brian E, Gupta, Sabranth, Sato, Ryuei, Waller, David F, Stoytchev, Ilko, Short, James E, Sharek, Lisa, Tran, Christopher T +2 more·2024·Nucleic Acids Research

Published version of a preprint

Abstract

The ability to deliver large transgenes to a single genomic sequence with high efficiency would accelerate biomedical interventions. Current methods suffer from low insertion efficiency and most rely on undesired double-strand DNA breaks. Serine integrases catalyze the insertion of large DNA cargos at attachment (att) sites. By targeting att sites to the genome using technologies such as prime editing, integrases can target safe loci while avoiding double-strand breaks. We developed a method of phage-assisted continuous evolution we call IntePACE, that we used to rapidly perform hundreds of rounds of mutagenesis to systematically improve activity of PhiC31 and Bxb1 serine integrases. Novel hyperactive mutants were generated by combining synergistic mutations resulting in integration of a multi-gene cargo at rates as high as 80% of target chromosomes. Hyperactive integrases inserted a 15.7 kb therapeutic DNA cargo containing von Willebrand Factor. This technology could accelerate gene delivery therapeutics and our directed evolution strategy can easily be adapted to improve novel integrases from nature.

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Crossref · 10.1093/nar/gkae534

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Authors
Hew, B. E., Gupta, S., Sato, R., Waller, D. F., Stoytchev, I., Short, J. E., Sharek, L., Tran, C. T., Badran, A. H., & Owens, J. B.
Affiliations
  • University of Hawaiʻi at Mānoa (US)
  • Scripps Research Institute (US)
Corresponding author
Owens, Jesse B
Venue
Nucleic Acids Research
Published
2024-07-02
Volume
52
Issue
14
Pages
e64-e64
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
OpenAlex
W4400242416
Cited by
35
References
69
Citations by year
2026: 16 · 2025: 16 · 2024: 3
FWCI
4.75
Type
journal article
Language
en
Open access
Locations
  • Nucleic Acids Research (publishedVersion, cc-by-nc)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
Funders
National Institutes of Health (P20 GM103457, R21 GM132779, P20 GM125526, R01 EB031124)
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
serine integrase, site-specific DNA integration, directed evolution, phage-assisted continuous evolution, phiC31 integrase, Bxb1 integrase, att site, transgene delivery, von Willebrand factor, prime editing, gene therapy

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