Genome engineering of microorganisms has become a standard in microbial biotechnology. In 2010, promising synthetic biology technologies using yeast as a platform for the assembly and engineering of synthetic bacterial genomes followed by their transplantation into a recipient cell have emerged. These technologies have led to the creation of the first synthetic cells and opened new avenues towards the construction of cells with fully controlled biological properties.The transfer of these advanced tools to microorganisms of interest such as the Gram+ bacterium Bacillus subtilis (Bsu), a biotechnology workhorse, will constitute a central advance, particularly in the improvement of bacterial strains for industrial purposes. As a first step towards that goal, an INRAE consortium set out to clone the entire Bsu genome in yeast using CReasPy-Fusion, a newly developed method that relies on direct fusion between bacterial protoplasts and yeast spheroplasts preloaded with a CRISPR-Cas9 tool. Efforts to date have demonstrated: (1) cell-to-cell DNA transfer between Gram+ Bsu bacteria and yeast cells, a phenomenon never described before; (2) the efficiency of a CRISPR-Cas9 system carried by yeast cells to capture and modify a shuttle plasmid during Bsu/yeast fusion; and last (3) the efficiency of the same CRISPR-Cas9 system to capture a 130-kb fragment of the Bsu genome, thus validating the CReasPy-Fusion method for cloning the Bsu genome. Since then, larger Bsu genome fragments were cloned in yeast; their capture appears to be facilitated by the addition beforehand of yeast ARS (autonomously replicating sequence) elements along the Bsu chromosome. With this knowledge, we now aim to clone the complete ~3Mb genome of a genome-reduced Bsu strain in yeast.< Réduire