Mycoplasmas are minimal bacteria that infect humans, wildlife, and economically relevant livestock. Mycoplasma infections cause chronic inflammatory diseases, which can lead to death in some animals. However, the lack of efficient recombination and genome engineering tools limits the production of mutant strains for the identification of virulence factors and the development of improved vaccine strains. This has hampered functional studies of many species and left several questions about the molecular basis of their pathogenicity unanswered. To address this issue, we adapted an efficient Cas9-Base Editor system to introduce targeted mutations into four major pathogenic species that span the phylogenetic diversity of these bacteria: the avian pathogen Mycoplasma gallisepticum, the small ruminant’s pathogen Mycoplasma agalactiae and the two most important bovine mycoplasmas, Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides. Using an inducible SpdCas9-pmcDA1 cytosine deaminase system, we disrupted several major virulence factors in these pathogens. We evaluated various induction times and inducer concentrations to optimize editing efficiency. The optimized system was powerful enough to disrupt 54 of 55 insertion sequence transposases in a single experiment. Lastly, we successfully disrupted two different genes in Mycoplasma bovis in a single assay, using an all-in-one multiple gRNA system. These systems are designed to generate mutants eligible for vaccine development without any antibiotic resistance markers in their genome by undergoing a curing process. Whole-genome sequencing of the edited strains showed that off-target mutations were limited, suggesting that most variations detected in the edited genomes are Cas9-independent. Our findings present, for the first time, an effective, rapid, and easy-to-use genetic tool for the study of these important animal pathogens and likely the entire class Mollicutes.< Leer menos