CRISPR/Cas systems are adaptive defense mechanisms against invading nucleic acids that are widely found in prokaryotes. Mollicutes are currently the bacteria with the smallest genomes in which CRISPR/Cas systems have been reported. Indeed, complete or degraded systems were found in the genome of 21 out of 52 representative Mollicutes species, and most of them belong to the Type II-A while others to the Type II-C. In this study, we characterized a representative CRISPR/Cas9 system of mycoplasmas previously identified in the bird pathogen Mycoplasma gallisepticum (Mgal). In silico analysis provided evidence of CRISPR system evolution between the strain S6 and CA06 isolated in chicken and North American songbirds (house finch), respectively. In vitro cleavage assays were undertaken to determine the specificity of MgalCas9 from the two Mgal strains regarding the Protospacer Adjacent Motif (PAM), an element that is critical for the self/non-self recognition of the defense system. Cleavage of a plasmid library with a target and a degenerated 7 nt PAM sequence resulted in a 5-position PAM consensus of NNAAT and NNGAD for strains S6 and CA06, respectively. In vivo interference assays confirmed this difference of specificity using plasmid transformation experiments. Phylogenetic analyses using different strains of Mgal showed a correlation between the intraspecific evolution of MgalCas9 and the host shift that happened in 1995 when Mgal jumped from poultry to house finch. We are currently trying to understand more precisely this evolution of specificity and the potential role of this in the emergence of Mgal in a new host.< Réduire