The genetic manipulation of Mycoplasma is notoriously difficult, as only a small number of tools are available. To bypass these limitations, a new strategy has been developed: (i) the Mycoplasma genome is transferred into a yeast cell and carried as an artificial chromosome, (ii) the cloned bacterial genome is modified using all the tools available in yeast, and later (iii) back transferred to a bacterial recipient cell to produce a mutant. This method has already been applied several mycoplasma species but shows two main limitations. First, the cloning step requires the integration of yeast elements in the bacterial genome to drive its replication and maintenance in yeast. Using the current methods, this insertion is random or depends on the presence of a unique restriction site in a non-essential gene, which can be difficult to find. Second, the succession of the cloning and engineering steps is time consuming.Here, we developed a new approach, based on the CRISPR-Cas9 system, to simultaneously clone and multi-edit a mycoplasma genome in yeast: the CReasPy-cloning. The yeast S. cerevisiae is co-transformed with four elements: the bacterial chromosome, two plasmids for the expression of a Cas9 nuclease and a guide RNA targeting a specific genome region, and a recombination template containing the yeast elements flanked by regions homologous to the insertion site. Upon transformation, the bacterial chromosome is cleaved at desired site(s) by the gRNA-Cas9 complex(es), and repaired by the yeast homologous recombination system, using the template(s) provided. With this method, we can precisely target the cleavage locus/loci on the bacterial genome allowing us to eliminate specific gene(s) or group of genes. Yeast transformants are screened by PCR to detect clones with the expected deletion(s), and PFGE and sequencing to check the completeness of the cloned and edited bacterial genome. In summary, the CReasPy-cloning method proved to be efficient for quick one-step engineering of bacterial chromosomes at large scale< Réduire