Combining Fusion of Cells with CRISPR-Cas9 Editing for the Cloning of Large DNA Fragments or Complete Bacterial Genomes in Yeast
MANSO-SILVÁN, Lucía
Animal, Santé, Territoires, Risques et Ecosystèmes [UMR ASTRE]
Département Systèmes Biologiques [Cirad-BIOS]
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Animal, Santé, Territoires, Risques et Ecosystèmes [UMR ASTRE]
Département Systèmes Biologiques [Cirad-BIOS]
Langue
en
Article de revue
Ce document a été publié dans
ACS Synthetic Biology. 2023-10-16, vol. 12, n° 11, p. 3252-3266
American Chemical Society
Résumé en anglais
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 ...Lire la suite >
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.< Réduire
Mots clés en anglais
CRISPR-Cas9
CReasPy-Fusion
Mycoplasma spp
Saccharomyces cerevisiae
cell fusion
genome editing
genome fragment capture
genome transplantation
in-yeast genome cloning
membrane nuclease MnuA
whole genome transfer
Project ANR
Construction et transplantation de génomes semi-synthétiques de Bacillus subtilis, vers le développement de la prochaine génération de châssis bactériens - ANR-18-CE44-0003
Origine
Importé de halUnités de recherche