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Directed evolution of hyperactive integrases for site specific insertion of transgenes
Open accessPreprint — published as 10.1093/nar/gkae534 · Paper page
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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Highly accurate protein structure prediction with AlphaFold
Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., et al. (2021) · Nature · doi:10.1038/s41586-021-03819-2 · Cited by 43,764
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Metadata
- 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
- Publisher
- openRxiv
- Published
- 2024-06-11
- Volume
- 52
- Issue
- 14
- Pages
- e64-e64
- ISSN
- 0305-1048 (ISSN-L)
- PMC
- PMC11317131
- OpenAlex
- W4400242416
- Cited by
- 35
- References
- 1
- Citations by year
- 2026: 16 · 2025: 16 · 2024: 3
- FWCI
- 4.76
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by-nc
- 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
- National Institute of General Medical Sciences (P20GM125526, P20GM103457, R21GM132779)
- National Institute of Biomedical Imaging and Bioengineering (R01EB031124)
- 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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