Combining Fusion of Cells with CRISPR-Cas9 Editing for the Cloning of Large DNA Fragments or Complete Bacterial Genomes in Yeast

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.