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

Mycoplasmas 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...

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Autores principales: Ipoutcha, Thomas, Rideau, Fabien, Gourgues, Geraldine, Arfi, Yonathan, Lartigue, Carole, Blanchard, Alain, Sirand-Pugnet, Pascal
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9469718/
https://www.ncbi.nlm.nih.gov/pubmed/36000854
http://dx.doi.org/10.1128/aem.00996-22
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author Ipoutcha, Thomas
Rideau, Fabien
Gourgues, Geraldine
Arfi, Yonathan
Lartigue, Carole
Blanchard, Alain
Sirand-Pugnet, Pascal
author_facet Ipoutcha, Thomas
Rideau, Fabien
Gourgues, Geraldine
Arfi, Yonathan
Lartigue, Carole
Blanchard, Alain
Sirand-Pugnet, Pascal
author_sort Ipoutcha, Thomas
collection PubMed
description Mycoplasmas 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.
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spelling pubmed-94697182022-09-14 Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System Ipoutcha, Thomas Rideau, Fabien Gourgues, Geraldine Arfi, Yonathan Lartigue, Carole Blanchard, Alain Sirand-Pugnet, Pascal Appl Environ Microbiol Genetics and Molecular Biology Mycoplasmas 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. American Society for Microbiology 2022-08-24 /pmc/articles/PMC9469718/ /pubmed/36000854 http://dx.doi.org/10.1128/aem.00996-22 Text en Copyright © 2022 Ipoutcha et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Genetics and Molecular Biology
Ipoutcha, Thomas
Rideau, Fabien
Gourgues, Geraldine
Arfi, Yonathan
Lartigue, Carole
Blanchard, Alain
Sirand-Pugnet, Pascal
Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System
title Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System
title_full Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System
title_fullStr Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System
title_full_unstemmed Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System
title_short Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System
title_sort genome editing of veterinary relevant mycoplasmas using a crispr-cas base editor system
topic Genetics and Molecular Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9469718/
https://www.ncbi.nlm.nih.gov/pubmed/36000854
http://dx.doi.org/10.1128/aem.00996-22
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