Directed evolution of hyperactive integrases for site specific insertion of transgenes
Year: 2024
Type: article
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.
Source: Nucleic Acids Research
DOI: 10.1093/nar/gkae534
Authors: Brian E Hew, Sabranth Gupta, Ryuei Sato, David F Waller, Ilko Stoytchev, James E Short, Lisa Sharek, Christopher T Tran, Ahmed H Badran, Jesse B Owens
Institutions: University of Hawaiʻi at Mānoa, Scripps Research Institute
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $4,192
Awards: Center for Translational Research on Aging, Institute for Biogenesis Research: COBRE, Development of a novel genome targeting technology using rational design and continuous evolution., Directed evolution of a sequence-specific targeting technology for therapeutic gene delivery to the human genome.
OpenAlex ID: W4400242416