PNAS|重磅,全球首次利用CRISPR技术,在珊瑚里面进行基因操作

iPlants：2018年4月25日，德克萨斯大学奥斯汀分校Matz研究组在PNAS杂志发表了题为“CRISPR/Cas9-mediated genome editing in a reef-building coral”的研究论文，该论文首次使用CRISPR技术， 靶向GFP和RFP等基因，取得了预期的研究结果，这同时也显示CRISPR / Cas9在珊瑚中允许反向遗传方法的潜力。这对于进一步研究珊瑚礁的生物学功能提供了一种新型的方法，这会极大的推动该领域发展。

珊瑚礁珊瑚是集生态，经济和美学重要的物种，为海洋提供重要的栖息地，初级生产和生物多样性【1,2】。珊瑚在营养贫瘠的水域生长并沉积碳酸钙基礁石材料的能力取决于它们与Symbiodinium中的光合作用甲藻藻类的共生作用，它们提供大部分必需能量【3-5】。由于人类环境压力，目前珊瑚受到全球威胁【6】。这些全球珊瑚礁危机极大促使了对珊瑚生物学更深入理解的需求，导致最近的研究激增，包括大量的基因组学和转录组学研究【7-16】。特别是，珊瑚和其他海洋生物中基因表达的研究已经提出了许多关于控制基础过程的基因和分子途径的合理假设，例如共生体建立期间的伙伴选择，共生伴侣之间的代谢交换，生物矿化，局部适应和生理可塑性，以及对压力的回应。然而，支持这种假设的证据在很大程度上仍然是相关的，因为缺乏能够进行更严格测试的遗传工具。

产生目标基因敲除或敲低的反向遗传方法已成功阐明了许多模型和非模型生物的基因功能。最近，由于CRISPR / Cas9基因组编辑技术的出现，这种方法的范围和能力已经大大扩展，该技术可以应用于多种生物体，并且不仅促进了功能缺失突变的产生，而且促进了引入更多微妙修饰的基因，蛋白质标签和大规模基因组重组【17-19】。将这种技术应用于珊瑚的一个障碍是配子生成受精卵的有限可用性。大多数珊瑚礁珊瑚通过广播产卵季节性繁殖，每年释放一次或几次配子，以响应温度【20-27】。尽管如此，对于许多珊瑚物种来说，有很好的方法来获得配子，实现受精，在实验室培养幼虫，诱导幼虫沉降和变态【26,28】。这些方法使得用基因组修饰试剂显微注射单细胞合子和分析所得表型成为可能，尽管这无疑带来了后勤方面的挑战。

在最初尝试使用CRISPR / Cas9在珊瑚中产生功能缺失突变时，德克萨斯大学奥斯汀分校Matz研究组针对编码成纤维细胞生长因子1a（FGF1a），绿色荧光蛋白（GFP）和红色荧光蛋白（RFP）的弯曲杆菌基因【29-31】。 GFP和RFP基因在该物种中都是多拷贝的;它们在幼虫中高度表达并且对环境扰动有反应【32-38】。

在将CRISPR / Cas9核糖核蛋白复合物微注射到受精卵后，德克萨斯大学奥斯汀分校Matz研究组使用限制性片段长度，Sanger测序和高通量Illumina测序的变化检测了靶基因中的诱导性突变。在约50％的个体中观察到突变，并且这些个体中野生型和各种突变基因拷贝的比例，表明突变诱导在注射后至少持续数个细胞周期持续。尽管编码绿色荧光蛋白的多个旁系同源基因存在于A. millepora中，但是适当的sgRNA设计使Matz研究组能够在多于一个旁系同源物中同时诱导突变。因为A.millepora幼虫可以在实验室中诱导沉降并开始形成菌落，所以基于CRISPR / Cas9的基因编辑应该允许在幼虫和成年珊瑚中对基因功能进行严格测试。

原文链接：

http://www.pnas.org/content/early/2018/04/19/1722151115

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