Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast

Guesdon Gabrielle, Gourgues Géraldine, Rideau Fabien, Ipoutcha Thomas, Manso-Silvan Lucia, Jules Matthieu, Sirand-Pugnet Pascal, Blanchard Alain, Lartigue Carole. 2023. Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast. ACS Synthetic Biology, 12 (11) : 3252-3266.

Url - jeu de données - Entrepôt autre : https://www.ncbi.nlm.nih.gov/bioproject/PRJNA910329/

Résumé : The genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeast Saccharomyces cerevisiae is considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of yeast cloning strategies, a new approach combining two already described methods, Fusion cloning and CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by the fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from several Mycoplasma species belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves the cloning efficacy.

Mots-clés Agrovoc : génie génétique, génome, levure, Saccharomyces cerevisiae, génomique, Mycoplasma

Mots-clés libres : CReasPy-Fusion, In-yeast genome cloning, CRISPR-Cas9, Cell fusion, Whole genome transfer, Genome fragment capture, Genome editing, Genome transplantation, Mycoplasma spp

Classification Agris : U60 - Sciences de la vie et de la Terre

Champ stratégique Cirad : CTS 7 (2019-) - Hors champs stratégiques

Agences de financement hors UE : Agence Nationale de la Recherche

Projets sur financement : (FRA) Construction et transplantation de génomes semi-synthétiques de Bacillus subtilis, vers le développement de la prochaine génération de châssis bactériens

Auteurs et affiliations

• Guesdon Gabrielle, INRAE (FRA)
• Gourgues Géraldine, INRAE (FRA)
• Rideau Fabien, INRAE (FRA)
• Ipoutcha Thomas, INRAE (FRA)
• Manso-Silvan Lucia, CIRAD-BIOS-UMR ASTRE (FRA) ORCID: 0000-0002-5539-3804
• Jules Matthieu, AgroParisTech (FRA)
• Sirand-Pugnet Pascal, Université de Bordeaux (FRA)
• Blanchard Alain, Université de Bordeaux (FRA)
• Lartigue Carole, INRAE (FRA) - auteur correspondant

Source : Cirad-Agritrop (https://agritrop.cirad.fr/608090/)

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