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Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System

hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorIPOUTCHA, Thomas
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorRIDEAU, Fabien
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorSIRAND-PUGNET, Pascal
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorGOURGUES, Geraldine
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorARFI, Yonathan
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorLARTIGUE, Carole
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorBLANCHARD, Alain
dc.date.issued2022
dc.identifier.issn0099-2240
dc.description.abstractEnMycoplasmas are minimal bacteria that infect humans, wildlife, and most economically relevant livestock species. Mycoplasma infections cause a large range of chronic inflammatory diseases, eventually leading to death in some animals. Due to the lack of efficient recombination and genome engineering tools for most species, the production of mutant strains for the identification of virulence factors and the development of improved vaccine strains is limited. Here, we demonstrate the adaptation of an efficient Cas9-Base Editor system to introduce targeted mutations into three major pathogenic species that span the phylogenetic diversity of these bacteria: the avian pathogen Mycoplasma gallisepticum and the two most important bovine mycoplasmas, Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides. As a proof of concept, we successfully used an inducible SpdCas9-pmcDA1 cytosine deaminase system to disrupt several major virulence factors in these pathogens. Various induction times and inducer concentrations were evaluated to optimize editing efficiency. The optimized system was powerful enough to disrupt 54 of 55 insertion sequence transposases in a single experiment. Whole-genome sequencing of the edited strains showed that off-target mutations were limited, suggesting that most variations detected in the edited genomes are Cas9-independent. This effective, rapid, and easy-to-use genetic tool opens a new avenue for the study of these important animal pathogens and likely the entire class Mollicutes. IMPORTANCE Mycoplasmas are minimal pathogenic bacteria that infect a wide range of hosts, including humans, livestock, and wild animals. Major pathogenic species cause acute to chronic infections involving still poorly characterized virulence factors. The lack of precise genome editing tools has hampered functional studies of many species, leaving multiple questions about the molecular basis of their pathogenicity unanswered. Here, we demonstrate the adaptation of a CRISPR-derived base editor for three major pathogenic species: Mycoplasma gallisepticum, Mycoplasma bovis, and Mycoplasma mycoides subsp. mycoides. Several virulence factors were successfully targeted, and we were able to edit up to 54 target sites in a single step. The availability of this efficient and easy-to-use genetic tool will greatly facilitate functional studies of these economically important bacteria.
dc.description.sponsorshipApproche rationnelle d'un vaccin Mycoplasma bovis - ANR-21-CE35-0008
dc.description.sponsorshipPlateforme d'Innovation " Forêt-Bois-Fibre-Biomasse du Futur " - ANR-10-EQPX-0016
dc.language.isoen
dc.publisherAmerican Society for Microbiology
dc.rights.urihttp://creativecommons.org/licenses/by/
dc.subject.enCRISPR-Cas9
dc.subject.enanimal pathogens
dc.subject.engenome editing
dc.subject.enminimal cell
dc.subject.enmycoplasma
dc.title.enGenome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System
dc.typeArticle de revue
dc.identifier.doi10.1128/aem.00996-22
dc.subject.halSciences du Vivant [q-bio]
dc.subject.halSciences du Vivant [q-bio]/Biologie animale/Médecine vétérinaire et santé animal
dc.subject.halSciences du Vivant [q-bio]/Médecine humaine et pathologie/Maladies infectieuses
bordeaux.journalApplied and Environmental Microbiology
bordeaux.pagee0099622
bordeaux.volume88
bordeaux.issue17
bordeaux.peerReviewedoui
hal.identifierhal-03763518
hal.version1
hal.popularnon
hal.audienceInternationale
hal.origin.linkhttps://hal.archives-ouvertes.fr//hal-03763518v1
bordeaux.COinSctx_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.jtitle=Applied%20and%20Environmental%20Microbiology&rft.date=2022&rft.volume=88&rft.issue=17&rft.spage=e0099622&rft.epage=e0099622&rft.eissn=0099-2240&rft.issn=0099-2240&rft.au=IPOUTCHA,%20Thomas&RIDEAU,%20Fabien&SIRAND-PUGNET,%20Pascal&GOURGUES,%20Geraldine&ARFI,%20Yonathan&rft.genre=article


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