A toolbox for manipulating the genome of the major goat pathogen, subsp. .

subspecies () is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease listed by the World Organisation for Animal Health (WOAH) as a notifiable disease and threatening goat production in Africa and Asia. Although a few commercial inactivated vaccines are available, they do not comply with WOAH standards and there are serious doubts regarding their efficacy. One of the limiting factors to comprehend the molecular pathogenesis of CCPP and develop improved vaccines has been the lack of tools for genome engineering. In this work, key synthetic biology techniques recently developed for closely related mycoplasmas were adapted to . CReasPy-Cloning was used to simultaneously clone and engineer the genome in yeast, prior to whole-genome transplantation into subsp. recipient cells. This approach was used to knock out an S41 serine protease gene recently identified as a potential virulence factor, leading to the generation of the first site-specific mutants. The Cre-lox recombination system was then applied to remove all DNA sequences added during genome engineering. Finally, the resulting unmarked S41 serine protease mutants were validated by whole-genome sequencing and their non-caseinolytic phenotype was confirmed by casein digestion assay on milk agar. The synthetic biology tools that have been successfully implemented in allow the addition and removal of genes and other genetic features for the construction of seamless targeted mutants at ease, which will pave the way for both the identification of key pathogenicity determinants of and the rational design of novel, improved vaccines for the control of CCPP.