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

Abstract

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 in-yeast cloning strategies, a new approach combining two already described methods, the Fusion cloning and the CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by 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 cloning efficacy.

A toolbox for manipulating the genome of the major goat pathogen, Mycoplasma capricolum subsp. capripneumoniae

“…genome. This is not unusual when attempting to clone or edit bacterial genomes in yeast using the CRISPR/ Cas9 editing system and may be explained by the fact that the gRNAs cloned into pgRNA plasmids to specifically target a selected locus are not always as effective as predicted [77]. In both the aforementioned and current study, gRNA selection was performed using the CRISPR Guide RNA design tool implemented in the cloud-based platform Benchling (https://www.benchling.com/).…”

“…A first proof of this has already been obtained in our laboratory. The in-yeast genome engineering process was indeed recently applied to two other Mccp strains from Niger and Tanzania, and the corresponding mutants were obtained with the same efficacy as presented here [77]. genome.…”

A synthetic biology approach to assemble and reboot clinically-relevantPseudomonas aeruginosatailed phages

“…Yeast has been extensively used as a cloning platform for high-length DNA molecules since 1980 64 . Different methods have been developed to clone genomic fragments and full-length virus or bacterial genomes in yeast 47,[65][66][67][68] , and each of these methods requires the addition of yeast elements (Ars, Cen, Trp) to maintain the DNA molecule in yeast over time. These methods have allowed the cloning of genomes up to 1.8 Mb 29,66 , and as expected for small phage genomes, we successfully obtained several yeast clones containing stable JG024 genome and DMS3 genomes (Figure 3, 4).…”