Integrases on Demand
Kelly Williams1*(email@example.com), Jesse Cahill1, Lauren Clark2, Catherine Mageeney1, Laura Quinto4, Michael Jewett2,3, Farren Isaacs4, and Joe Schoeniger1
1Sandia National Laboratories; 2Northwestern University; 3Stanford University; and 4Yale University
The Intrinsic Control for Genome and Transcriptome Editing in Communities (InCoGenTEC) project sponsored by the BSSD Secure Biosystems Design initiative conducts mechanistic studies of gene flows between bacteria encompassing broad phylogenetic diversity and evolutionary time, focusing on mechanisms that permit natural gene delivery and gene integration. In prokaryotes DNA rearrangements are physiologically and ecologically important and an important potential source of genome instability and loss of biocontainment of engineered features. Goals include comprehensively mapping and classifying bacterial genomic islands, analyzing mechanisms of mobility and identifying routes of horizontal gene transfer. The team will also use this information to identify prophages suitable as vectors for editing microbial community members and to improve design of synthetic genetic elements that can be integrated into target organisms.
Recent advances in genome editing have stimulated a renewed interest in microbial DNA integrases. Previously the team published bioinformatic methods for precise high-throughput definition of bacterial and archaeal genomic islands that contain integrases (Mageeney, et al. 2020). These methods enable pairing of integrases with their specific attachment site (Att) sequences. As part of InCoGenTEC, the team has mapped ~one million genomic islands in ~350,000 bacterial genomes, yielding tens of thousands of unique integrase-Att pairs.
Researchers have implemented assays for high-throughput in vitro and in vivo characterization of integrase activity to verify integrase activity at predicted Att sites as well as investigate integrase dependence on host species factors that might limit the range of horizontal gene transfer. Considerable work has been published on the use of serine integrases for synthetic biology and genome editing of target organisms, typically requiring first the introduction of a heterologous Att site (“landing pad”) into the target genome. Less attention has been paid to the much more abundant tyrosine integrases because of their putative dependences on microbial host factors. Researchers have used the project’s assays to demonstrate activity in E. coli of both serine and tyrosine integrases drawn from broad phylogenetic spans. On the other hand, the team also demonstrated that its methods can be used to identify from near neighbors of a target strain integrases that utilize native Att sites in the target genome, simplifying and potentially greatly increasing the efficiency of bacterial genome editing. Finally, team members have used modified versions of its assays to mine difficult to identify directionality factors (excisionases) from genomic islands. These methods have the potential to significantly improve genome editing efficiency and enable layered genome restructuring in diverse organisms, while improving understanding of the biochemical determinants of the specificity of integrase activity and mobile element host range.
Mageeney, C. M., et al. 2020. “New Candidates for Regulated Gene Integrity Revealed Through Precise Mapping of Integrative Genetic Elements,” Nucleic Acids Research 48(8), 4052–65.
Funding was provided by the U. S. Department of Energy, Office of Science, through the Genomic Science Program, Office of Biological and Environmental Research, under the Secure Biosystems Design Initiative project Intrinsic Control for Genome and Transcriptome Editing in Communities (InCoGenTEC); Sandia National Laboratories is managed by National Technology and Engineering Solutions of Sandia, LLC, a wholly owned subsidiary of Honeywell International, Inc., for the U.S. Department of Energy [DE-NA-0003525].