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High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration

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–as...

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Autores principales: 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.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Association for the Advancement of Science 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10005180/
https://www.ncbi.nlm.nih.gov/pubmed/36897939
http://dx.doi.org/10.1126/sciadv.ade1285
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author 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.
author_facet 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.
author_sort Elmore, Joshua R.
collection PubMed
description 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.
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spelling pubmed-100051802023-03-11 High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration 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. Sci Adv Biomedicine and Life Sciences 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. American Association for the Advancement of Science 2023-03-10 /pmc/articles/PMC10005180/ /pubmed/36897939 http://dx.doi.org/10.1126/sciadv.ade1285 Text en Copyright © 2023 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). https://creativecommons.org/licenses/by-nc/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license (https://creativecommons.org/licenses/by-nc/4.0/) , which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.
spellingShingle Biomedicine and Life Sciences
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
title High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration
title_full High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration
title_fullStr High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration
title_full_unstemmed High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration
title_short High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration
title_sort high-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration
topic Biomedicine and Life Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10005180/
https://www.ncbi.nlm.nih.gov/pubmed/36897939
http://dx.doi.org/10.1126/sciadv.ade1285
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