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SURE editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the Mycoplasma pneumoniae genome
The development of advanced genetic tools is boosting microbial engineering which can potentially tackle wide-ranging challenges currently faced by our society. Here we present SURE editing, a multi-recombinase engineering rationale combining oligonucleotide recombineering with the selective capacit...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9825166/ https://www.ncbi.nlm.nih.gov/pubmed/36215032 http://dx.doi.org/10.1093/nar/gkac836 |
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author | Piñero-Lambea, Carlos Garcia-Ramallo, Eva Miravet-Verde, Samuel Burgos, Raul Scarpa, Margherita Serrano, Luis Lluch-Senar, Maria |
author_facet | Piñero-Lambea, Carlos Garcia-Ramallo, Eva Miravet-Verde, Samuel Burgos, Raul Scarpa, Margherita Serrano, Luis Lluch-Senar, Maria |
author_sort | Piñero-Lambea, Carlos |
collection | PubMed |
description | The development of advanced genetic tools is boosting microbial engineering which can potentially tackle wide-ranging challenges currently faced by our society. Here we present SURE editing, a multi-recombinase engineering rationale combining oligonucleotide recombineering with the selective capacity of antibiotic resistance via transient insertion of selector plasmids. We test this method in Mycoplasma pneumoniae, a bacterium with a very inefficient native recombination machinery. Using SURE editing, we can seamlessly generate, in a single step, a wide variety of genome modifications at high efficiencies, including the largest possible deletion of this genome (30 Kb) and the targeted complementation of essential genes in the deletion of a region of interest. Additional steps can be taken to remove the selector plasmid from the edited area, to obtain markerless or even scarless edits. Of note, SURE editing is compatible with different site-specific recombinases for mediating transient plasmid integration. This battery of selector plasmids can be used to select different edits, regardless of the target sequence, which significantly reduces the cloning load associated to genome engineering projects. Given the proven functionality in several microorganisms of the machinery behind the SURE editing logic, this method is likely to represent a valuable advance for the synthetic biology field. |
format | Online Article Text |
id | pubmed-9825166 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-98251662023-01-09 SURE editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the Mycoplasma pneumoniae genome Piñero-Lambea, Carlos Garcia-Ramallo, Eva Miravet-Verde, Samuel Burgos, Raul Scarpa, Margherita Serrano, Luis Lluch-Senar, Maria Nucleic Acids Res Methods Online The development of advanced genetic tools is boosting microbial engineering which can potentially tackle wide-ranging challenges currently faced by our society. Here we present SURE editing, a multi-recombinase engineering rationale combining oligonucleotide recombineering with the selective capacity of antibiotic resistance via transient insertion of selector plasmids. We test this method in Mycoplasma pneumoniae, a bacterium with a very inefficient native recombination machinery. Using SURE editing, we can seamlessly generate, in a single step, a wide variety of genome modifications at high efficiencies, including the largest possible deletion of this genome (30 Kb) and the targeted complementation of essential genes in the deletion of a region of interest. Additional steps can be taken to remove the selector plasmid from the edited area, to obtain markerless or even scarless edits. Of note, SURE editing is compatible with different site-specific recombinases for mediating transient plasmid integration. This battery of selector plasmids can be used to select different edits, regardless of the target sequence, which significantly reduces the cloning load associated to genome engineering projects. Given the proven functionality in several microorganisms of the machinery behind the SURE editing logic, this method is likely to represent a valuable advance for the synthetic biology field. Oxford University Press 2022-10-10 /pmc/articles/PMC9825166/ /pubmed/36215032 http://dx.doi.org/10.1093/nar/gkac836 Text en © The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research. 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 non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Methods Online Piñero-Lambea, Carlos Garcia-Ramallo, Eva Miravet-Verde, Samuel Burgos, Raul Scarpa, Margherita Serrano, Luis Lluch-Senar, Maria SURE editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the Mycoplasma pneumoniae genome |
title | SURE editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the Mycoplasma pneumoniae genome |
title_full | SURE editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the Mycoplasma pneumoniae genome |
title_fullStr | SURE editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the Mycoplasma pneumoniae genome |
title_full_unstemmed | SURE editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the Mycoplasma pneumoniae genome |
title_short | SURE editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the Mycoplasma pneumoniae genome |
title_sort | sure editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the mycoplasma pneumoniae genome |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9825166/ https://www.ncbi.nlm.nih.gov/pubmed/36215032 http://dx.doi.org/10.1093/nar/gkac836 |
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