CReasPy-cloning: a method for simultaneous cloning and engineering of megabase-sized genomes in yeast using the CRISPR-Cas9 system

Abstract

Over the last 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 within 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.

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

“…The cloning of natural Mycoplasma genomes as yeast centromeric plasmids was a major breakthrough that seemed to overcome the scarcity of tools for this genus. 32 , 58 Indeed, complete genomes of several Mycoplasma species have been cloned and modified in yeast with a variety of genome-editing techniques such as TREC, 33 TREC-IN, 34 and CreasPy-cloning 35 that take advantage of the proficient recombination machinery found in S. cerevisiae . Perhaps the best example to illustrate the potential of this approach is the recent report of massive genome engineering in M. mycoides using the TREC technique that involved the multistep targeted deletion of up to 10% of the original genome.…”

“…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. 32 These genomes can later be comprehensively modified using the state-of-the-art editing tools available for yeast 33 − 35 before their reintroduction into a recipient Mycoplasma cell (i.e., genome transplantation). 36 This complete cycle of cloning, in-yeast modification, and genome transplantation, has led to the generation of a fully attenuated M. mycoides subsp.…”

Budding yeast as a factory to engineer partial and complete microbial genomes

“… Organism Size (Mbp) % G+C Genetic code Cloning strategy b Rescue strategy References a Mollicutes Mycoplasma genitalium 0,58 32 Non standard Synthesis & Assembly N/A [ 66 ] Mycoplasma mycoides subsp . capri 1,1 24 Non standard Cloning Transplantation [ 10 ] Mycoplasma pneumoniae 0,81 41 Non standard Cloning N/A [ 11 , 17 ] JCVI Syn 1.0 1,1 24 Non standard Synthesis & Assembly Transplantation [ 1 ] Acholeplasma laidlawii 1,5 32 Universal Cloning N/A [ 51 ] JCVI Syn 3.0 0,53 24 Non standard Synthesis & Assembly Transplantation [ 2 ] Mycoplasma mycoides subsp . mycoides 1,2 24 Non standard Cloning Transplantation [ 45 ] Mycoplasma capricolum subsp .…”

“…In this case, the genome is isolated, linearized in vitro by a restriction enzyme or using the CRISPR-Cas9 system and co-transformed into yeast together with a linear yeast vector containing homology sequences[ 13 , 14 , 15 , 16∗ ] ( Figure 1 B). A variation of this approach is CReasPy-Cloning which enables the simultaneous cloning and engineering of megabase-sized genomes in yeast[ 17 ] ( Figure 1 B). The TAR-cloning approach can be extended so that the yeast transformation is carried out with multiple overlapping fragments, either PCR-amplified, synthetic or previously TAR-cloned ( Figure 1 C), allowing for genome-wide engineering of microbial genomes.…”