Data_Sheet_1_Multiple Origins and Specific Evolution of CRISPR/Cas9 Systems in Minimal Bacteria (Mollicutes).docx

CRISPR/Cas systems provide adaptive defense mechanisms against invading nucleic acids in prokaryotes. Because of its interest as a genetic tool, the Type II CRISPR/Cas9 system from Streptococcus pyogenes has been extensively studied. It includes the Cas9 endonuclease that is dependent on a dual-guide RNA made of a tracrRNA and a crRNA. Target recognition relies on crRNA annealing and the presence of a protospacer adjacent motif (PAM). Mollicutes are currently the bacteria with the smallest genome in which CRISPR/Cas systems have been reported. Many of them are pathogenic to humans and animals (mycoplasmas and ureaplasmas) or plants (phytoplasmas and some spiroplasmas). A global survey was conducted to identify and compare CRISPR/Cas systems found in the genome of these minimal bacteria. Complete or degraded systems classified as Type II-A and less frequently as Type II-C were found in the genome of 21 out of 52 representative mollicutes species. Phylogenetic reconstructions predicted a common origin of all CRISPR/Cas systems of mycoplasmas and at least two origins were suggested for spiroplasmas systems. Cas9 in mollicutes were structurally related to the S. aureus Cas9 except the PI domain involved in the interaction with the PAM, suggesting various PAM might be recognized by Cas9 of different mollicutes. Structure of the predicted crRNA/tracrRNA hybrids was conserved and showed typical stem-loop structures pairing the Direct Repeat part of crRNAs with the 5′ region of tracrRNAs. Most mollicutes crRNA/tracrRNAs showed G + C% significantly higher than the genome, suggesting a selective pressure for maintaining stability of these secondary structures. Examples of CRISPR spacers matching with mollicutes phages were found, including the textbook case of Mycoplasma cynos strain C142 having no prophage sequence but a CRISPR/Cas system with spacers targeting prophage sequences that were found in the genome of another M. cynos strain that is devoid of a CRISPR system. Despite their small genome size, mollicutes have maintained protective means against invading DNAs, including restriction/modification and CRISPR/Cas systems. The apparent lack of CRISPR/Cas systems in several groups of species including main pathogens of humans, ruminants, and plants suggests different evolutionary routes or a lower risk of phage infection in specific ecological niches.

CRISPR/Cas系统（CRISPR/Cas systems）可为原核生物提供针对入侵核酸的适应性防御机制。鉴于其作为基因编辑工具的重要研究价值，来自化脓性链球菌（Streptococcus pyogenes）的II型CRISPR/Cas9系统（Type II CRISPR/Cas9 system）已被广泛研究。该系统包含依赖于由反式激活CRISPR RNA（tracrRNA）和CRISPR RNA（crRNA）组成的双向导RNA（dual-guide RNA）的Cas9核酸内切酶（Cas9 endonuclease）。靶标识别依赖于crRNA的退火作用以及前间隔序列邻近基序（protospacer adjacent motif, PAM）的存在。柔膜菌纲（Mollicutes）是目前已报道存在CRISPR/Cas系统的基因组最小的细菌类群。其中多数类群可感染人类与动物（如支原体、脲原体）或植物（如植原体及部分螺原体）。本研究开展了一项全局性调查，以鉴定并比较这些基因组极小的细菌中存在的CRISPR/Cas系统。在52种代表性柔膜菌物种中，有21种的基因组中发现了完整或降解型的II-A型CRISPR/Cas系统，极少数为II-C型。系统发育重建分析显示，所有支原体的CRISPR/Cas系统具有共同起源，而螺原体的CRISPR/Cas系统则至少存在两种起源。柔膜菌的Cas9蛋白与金黄色葡萄球菌Cas9蛋白结构相似，但参与结合PAM的PI结构域（PI domain）存在差异，这提示不同柔膜菌的Cas9可能识别不同的PAM序列。预测得到的crRNA/tracrRNA杂合结构保守性较强，呈现典型的茎环结构，该结构通过crRNA的直接重复序列区域与tracrRNA的5'区域互补配对形成。多数柔膜菌的crRNA/tracrRNA的G+C含量显著高于其基因组整体水平，这表明存在维持这些二级结构稳定性的选择压力。研究还发现了与柔膜菌噬菌体匹配的CRISPR间隔序列，其中一个典型案例是犬支原体（Mycoplasma cynos）菌株C142，其自身不含前噬菌体（prophage）序列，但携带的CRISPR/Cas系统的间隔序列可靶向另一株无CRISPR系统的犬支原体菌株基因组中的前噬菌体序列。尽管柔膜菌的基因组尺寸极小，但它们仍保留了针对入侵DNA的防御系统，包括限制-修饰系统与CRISPR/Cas系统。在多个物种类群中（包括主要感染人类、反刍动物与植物的病原体类群）未发现CRISPR/Cas系统，这提示它们可能经历了不同的进化路径，或是在特定生态位中面临噬菌体感染的风险较低。