Mycoplasma pneumoniae genome editing based on oligo recombineering and Cas9-mediated counterselection

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• dc.contributor.author Piñero-Lambea, Carlos
• dc.contributor.author García Ramallo, Eva
• dc.contributor.author Martínez, Sira
• dc.contributor.author Delgado Blanco, Javier
• dc.contributor.author Serrano Pubull, Luis, 1982-
• dc.contributor.author Lluch-Senar, Maria 1982-
• dc.date.accessioned 2020-10-13T05:59:19Z
• dc.date.available 2020-10-13T05:59:19Z
• dc.date.issued 2020
• dc.description.abstract Mycoplasma species share a set of features, such as lack of a cell wall, streamlined genomes, simplified metabolism, and the use of a deviant genetic code, that make them attractive approximations of what a chassis strain should ideally be. Among them, Mycoplasma pneumoniae arises as a candidate for synthetic biology projects, as it is one of the most deeply characterized bacteria. However, the historical paucity of tools for editing Mycoplasma genomes has precluded the establishment of M. pneumoniae as a suitable chassis strain. Here, we developed an oligonucleotide recombineering method for this strain based on GP35, a ssDNA recombinase originally encoded by a Bacillus subtilis-associated phage. GP35-mediated oligo recombineering is able to carry out point mutations in the M. pneumoniae genome with an efficiency as high as 2.7 × 10-2, outperforming oligo recombineering protocols developed for other bacteria. Gene deletions of different sizes showed a decreasing power trend between efficiency and the scale of the attempted edition. However, the editing rates for all modifications increased when CRISPR/Cas9 was used to counterselect nonedited cells. This allowed edited clones carrying chromosomal deletions of up to 1.8 kb to be recovered with little to no screening of survivor cells. We envision this technology as a major step toward the use of M. pneumoniae, and possibly other Mycoplasmas, as synthetic biology chassis strains.
• dc.description.sponsorship This project has received funding from the European Union’s Horizon 2020 research and innovation program under Grant 634942 (MycoSynVac) and was also financed by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under Grant 670216 (MYCOCHASSIS) and the FEDER project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016) (reference CP16/00094). We also acknowledge support of the Spanish Ministry of Science and Innovation, to the EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Programme/Generalitat de Catalunya. Finally, we would like to thank Dr. Sarah A. Head for critical manuscript revision.
• dc.format.mimetype application/pdf
• dc.identifier.citation Piñero-Lambea C, Garcia-Ramallo E, Martinez S, Delgado J, Serrano L, Lluch-Senar M. Mycoplasma pneumoniae genome editing based on oligo recombineering and Cas9-mediated counterselection. ACS Synth Biol. 2020; 9(7):1693-704. DOI: 10.1021/acssynbio.0c00022
• dc.identifier.doi http://dx.doi.org/10.1021/acssynbio.0c00022
• dc.identifier.issn 2161-5063
• dc.identifier.uri http://hdl.handle.net/10230/45462
• dc.language.iso eng
• dc.publisher American Chemical Society (ACS)
• dc.relation.ispartof ACS Synth Biol. 2020; 9(7):1693-704
• dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/634942
• dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/670216
• dc.rights This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html), which permitscopying and redistribution of the article or any adaptations for non-commercial purposes.
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.rights.uri http://pubs.acs.org/page/policy/authorchoice_termsofuse.html
• dc.subject.keyword Sensors
• dc.subject.keyword Bacteria
• dc.subject.keyword Genetics
• dc.subject.keyword Genomics
• dc.subject.keyword Biopolymers
• dc.title Mycoplasma pneumoniae genome editing based on oligo recombineering and Cas9-mediated counterselection
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion