Background- Due to the lack of efficient recombination and genome engineering tools, the production of mutants for functional genomics and applications such as the development of vaccine strains remains a bottleneck for most mycoplasma species. Methods- Over the years, our team has developed many tools for the genome engineering of mycoplasmas. Some are designed for a direct use in mycoplasma cells and some others were developed following pioneered studies performed at the J. Craig Venter Institute, allowing the cloning and engineering of mycoplasma genomes in yeast before a whole genome transplantation into a suitable recipient mycoplasma.Results- Among the tools we recently developed, the RecET system, derived from a Bacillus subtilis prophage, is an imported recombination system that was used to introduce small deletion, gene replacement and insertion in Mycoplasma gallisepticum. The recombinase-assisted genomic engineering (RAGE), was shown to be efficient for gene inactivation or large DNA replacement in Mycoplasma pneumoniae. The CRISPR-derived Base-Editor system was used to obtain gene knock-out by generating nonsense mutations in four major pathogenic mycoplasma species: Mycoplasma M. gallisepticum, Mycoplasma agalactiae, Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides. Regarding in-yeast genome engineering, 21 mollicutes genomes have already been cloned in yeast and the back-genome transplantation to generate a living bacterium has been achieved for 7 species, all related to the M. mycoides cluster.Conclusion- Overall, our recent studies indicate that genome engineering in mycoplasmas is now feasible in many significant species for which there is a need for improved control, including by building innovative vaccines.< Réduire