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 methylation, which in turn confers resistance to the aminoglycoside antimicrobial kasugamycin, enabling selection for ksgA mutants. Analyses of the complete sequence of the ksgA gene in 78 resistant transformants revealed various modifications of the target region, presumably caused by the directed CRISPR/Cas activity of M. gallisepticum. The analyses suggested that M. gallisepticum may utilize a non-homologous end joining (NHEJ) repair system, which can result in deletion or duplication of a short DNA segment in the presence of double-stranded breaks. This study has generated an improved understanding of the M. gallisepticum CRISPR/Cas system, and may also facilitate further development of tools to genetically modify this important pathogen.

需要新的，更有效的方法来促进支原体中基因功能的研究。CRISPR / Cas系统可为细菌提供对入侵核酸的免疫力，已被开发为多种生物进行基因组编辑的工具。我们探索了使用内源性鸡支原体CRISPR / Cas系统将靶向突变引入这一重要动物病原体染色体的潜力。组装携带ksgA基因中不同靶向区域的CRISPR阵列的三个构建体（pK1-CRISPR，pK-CRISPR-1和pK-CRISPR-2），并在oriC上引入鸡毒支原体质粒。KsgA的丢失阻止了核糖体甲基化，从而赋予了对氨基糖苷类抗菌剂春日霉素的抗性，从而可以选择ksgA突变体。对78个抗性转化子中ksgA基因的完整序列进行分析，揭示了靶区域的各种修饰，可能是由鸡毒支原体的定向CRISPR / Cas活性引起的。分析表明，鸡败血支原体可能利用了非同源末端连接（NHEJ）修复系统，在双链断裂的情况下，它可能导致短DNA片段的缺失或重复。这项研究使人们对鸡毒支原体有了更好的了解 CRISPR / Cas系统，也可能有助于进一步开发工具来对这种重要病原体进行基因修饰。