High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration
Abstract
Efficient genome engineering is critical to understand and use microbial functions. Despite recent development of tools such as CRISPR-Cas gene editing, efficient integration of exogenous DNA with well-characterized functions remains limited to model bacteria. Here, we describe serine recombinase–assisted genome engineering, or SAGE, an easy-to-use, highly efficient, and extensible technology that enables selection marker–free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. SAGE uses no replicating plasmids and thus lacks the host range limitations of other genome engineering technologies. We demonstrate the value of SAGE by characterizing genome integration efficiency in five bacteria that span multiple taxonomy groups and biotechnology applications and by identifying more than 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate that SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.
- Authors:
-
[1];
[2];
[1];
[3];
[1];
[2];
[2];
[5];
[2];
[1]
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Biological Science Division
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Univ. of Tennessee, Knoxville, TN (United States)
- Univ. of California, Berkeley, CA (United States)
- Univ. of California, Berkeley, CA (United States); US Dept. of Agriculture (USDA), Albany, CA (United States). Agricultural Research Service (ARS)
- Publication Date:
- Research Org.:
- Pacific Northwest National Laboratory (PNNL), Richland, WA (United States); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Energy Efficiency and Renewable Energy (EERE)
- OSTI Identifier:
- 1962851
- Alternate Identifier(s):
- OSTI ID: 1962852; OSTI ID: 1997679
- Report Number(s):
- PNNL-SA-181397
Journal ID: ISSN 2375-2548
- Grant/Contract Number:
- AC05-76RL01830; AC05-00OR22725
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Science Advances
- Additional Journal Information:
- Journal Volume: 9; Journal Issue: 10; Journal ID: ISSN 2375-2548
- Publisher:
- AAAS
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
Elmore, Joshua R., Dexter, Gara N., Baldino, Henri, Huenemann, Jay D., Francis, Ryan, Peabody, George L., Martinez-Baird, Jessica, Riley, Lauren A., Simmons, Tuesday, Coleman-Derr, Devin, Guss, Adam M., and Egbert, Robert G. High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration. United States: N. p., 2023.
Web. doi:10.1126/sciadv.ade1285.
Elmore, Joshua R., Dexter, Gara N., Baldino, Henri, Huenemann, Jay D., Francis, Ryan, Peabody, George L., Martinez-Baird, Jessica, Riley, Lauren A., Simmons, Tuesday, Coleman-Derr, Devin, Guss, Adam M., & Egbert, Robert G. High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration. United States. https://doi.org/10.1126/sciadv.ade1285
Elmore, Joshua R., Dexter, Gara N., Baldino, Henri, Huenemann, Jay D., Francis, Ryan, Peabody, George L., Martinez-Baird, Jessica, Riley, Lauren A., Simmons, Tuesday, Coleman-Derr, Devin, Guss, Adam M., and Egbert, Robert G. Fri .
"High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration". United States. https://doi.org/10.1126/sciadv.ade1285. https://www.osti.gov/servlets/purl/1962851.
@article{osti_1962851,
title = {High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration},
author = {Elmore, Joshua R. and Dexter, Gara N. and Baldino, Henri and Huenemann, Jay D. and Francis, Ryan and Peabody, George L. and Martinez-Baird, Jessica and Riley, Lauren A. and Simmons, Tuesday and Coleman-Derr, Devin and Guss, Adam M. and Egbert, Robert G.},
abstractNote = {Efficient genome engineering is critical to understand and use microbial functions. Despite recent development of tools such as CRISPR-Cas gene editing, efficient integration of exogenous DNA with well-characterized functions remains limited to model bacteria. Here, we describe serine recombinase–assisted genome engineering, or SAGE, an easy-to-use, highly efficient, and extensible technology that enables selection marker–free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. SAGE uses no replicating plasmids and thus lacks the host range limitations of other genome engineering technologies. We demonstrate the value of SAGE by characterizing genome integration efficiency in five bacteria that span multiple taxonomy groups and biotechnology applications and by identifying more than 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate that SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.},
doi = {10.1126/sciadv.ade1285},
journal = {Science Advances},
number = 10,
volume = 9,
place = {United States},
year = {Fri Mar 10 00:00:00 EST 2023},
month = {Fri Mar 10 00:00:00 EST 2023}
}
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