Targeted mutagenesis of Mycoplasma gallisepticum using its endogenous CRISPR/Cas system

Abstract

New, more efficient methods are needed to facilitate studies of gene function in the mycoplasmas. CRISPR/Cas systems, which provide bacteria with acquired immunity against invading nucleic acids, have been developed as tools for genomic editing in a wide range of organisms. We explored the potential for using the endogenous Mycoplasma gallisepticum CRISPR/Cas system to introduce targeted mutations into the chromosome of this important animal pathogen. Three constructs carrying different CRISPR arrays targeting regions in the ksgA gene (pK1-CRISPR, pK-CRISPR-1 and pK-CRISPR-2) were assembled and introduced into M. gallisepticum on an oriC plasmid. The loss of KsgA prevents ribosomal methylati..