Due to their evolution towards streamlined genomes, most mycoplasma species lack efficient recombination machineries, which hampers the development of genome engineering methods. Here we present the development of a toolbox for genome engineering in mycoplasma species of veterinary interest. First, we derived a counterselectable marker from the natural CRISPR system of Mycoplasma gallisepticum. This marker is functional in four mycoplasma species and was successfully used to cure the Mycoplasma capricolum subsp. capricolum genome from its 23 kbp Integrative Conjugative Element. The second tool is based on the recombination system RecET derived from an E. coli prophage. Using this imported recombination system, genome engineering including small deletion, gene replacement and insertion with antibiotic marker, were obtained in M. gallisepticum. The third tool is a cutting edge CRISPR-based editor system for precise base change, without mobilizing repair systems based on homologous recombination. It can be used to obtain gene knock-out by generating nonsense mutations. This system is versatile and its functionality was demonstrated with three major pathogenic mycoplasma species: M. gallisepticum, M. bovis and M. mycoides subsp. mycoides. The diversity of species used for evaluating these new tools suggests they may be easily adapted to other mollicutes species for which no method for targeted mutagenesis is currently available< Leer menos