CReasPy-Cloning: A Method for Simultaneous Cloning and Engineering of Megabase-Sized Genomes in Yeast Using the CRISPR-Cas9 System

Abstract

Over the past decade, a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or "cloning") their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeast Saccharomyces cerevisiae has been used to clone and engineer genomes from viruses, bacteria, and algae. The cloning step requires the insertion of yeast genetic elements in the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast's highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome of Mycoplasma pneumoniae as a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (<50) number of clones, allowing for significantly shortened genome engineering cycle times.

Exploring Selective Pressure Trade-Offs for Synthetic Addiction to Extend Metabolite Productive Lifetimes in Yeast

“…Our previous study in engineered Escherichia coli showed that the common error mode was disruption by mobile elements. , These results imply that one should consider a common error mode of engineered microbes to find an optimal biosensor design. Strong recombination activity in yeast can be applied for various purposes − but can also cause instability in the engineered strain . We expect that deleting the potential homology region ( GFA1 locus on the genome in our case) will further improve the production stability; therefore, understanding the common error mode will be critical to operating long-term cultivation with engineered hosts.…”

Mycoplasma pneumoniae Genome Editing Based on Oligo Recombineering and Cas9-Mediated Counterselection

“…Strikingly, the genome of M. pneumoniae has also been cloned as a yeast centromeric plasmid and successfully edited using the CreasPy-cloning technique. 35 In spite of this, the reintroduction of this in-yeast engineered M. pneumoniae genome into a Mycoplasma acceptor cell (i.e., genome transplantation) has never been reported. Therefore, although the M. pneumoniae genome can be edited in yeast, the generation of M. pneumoniae edited cells remains unsolved.…”

“…Alternatively, a more affordable strategy is to clone naturally existing Mycoplasma genomes as yeast circular centromeric plasmids . These genomes can later be comprehensively modified using the state-of-the-art editing tools available for yeast − before their reintroduction into a recipient Mycoplasma cell (i.e., genome transplantation) . This complete cycle of cloning, in-yeast modification, and genome transplantation, has led to the generation of a fully attenuated M. mycoides subsp.…”

Rapid method for generating designer algal mitochondrial genomes

“…Alternatively, once the genome is cloned in yeast, the plasmid backbone can be moved to a different location by co-transformation of a plasmid containing homology hooks to the new location and another fragment that will delete the plasmid in the original location and restore the interrupted gene. Finally, a CRISPR/Cas9 system can be devised to produce appropriate cut-sites at the desired location [39,40]. The linearized algal mitochondria genome was captured by transforming it into S. cerevisiae along with a PCR-amplified plasmid backbone containing homology on each end to the organellar regions flanking the cut-site.…”