EAGER: Development of a Novel Method for Rapid and Efficient Genome Editing in Tomatoes

EAGER：开发一种快速高效的番茄基因组编辑新方法

• 批准号: 1636397
• 负责人: Neelima Sinha
• 金额: $ 30万
• 依托单位: University of California-Davis
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1636397&HistoricalAwards=false
• 关键词: EAGER; Development; Novel; Method; Rapid

Recent technological developments have made it possible to alter the genetic makeup of plants in deliberate and precise ways that were unthinkable just a few years ago. The new technology, called genome editing, can have a transformative impact on plant breeding and agriculture because it will be possible to introduce favorable genetic variants to increase yields and food quality. Genome editing techniques are based on a bacterial mechanism that is used for defense against infectious agents by changing their DNA. The great advantage of adapting this bacterial technique is that once fully developed, the technology will not leave foreign genes in the edited plant like previous technologies did. There are remaining challenges to the genome editing technique. However, this project will develop new methods to facilitate genome editing in plants. These methods make use of a group of cells in young plant parts that retain the potential to develop new organs, similar to the same property of animal stem cells. The research will test new ways of introducing the molecules that carry out genome editing in tomato stem cells. The high risk experiments will have a major impact on advancing the new genome editing technology.This project will create a tomato transgenic line carrying both the Cas9 endonuclease and a conditional (lethal in the presence of a substrate) counter-selectable marker (CSM) driven by a shoot meristem-specific promoter. The latter represents a conditional and tissue-specific meristem suicide gene. A sgRNA targeting both the CSM and a gene of interest will be delivered to young stems by Agrobacterium-mediated infection of CSM-transgenic tomato seedlings. Expression of the sgRNA in the meristem will lead to inactivation of both CSM and target genes. The injected plants will then be decapitated at the inoculation site to induce new shoot regeneration. The CSM will be expressed exclusively in shoot apical meristems. Thus, application of the selective substrate will lead to meristem death in non-mutated regenerating meristems, but will allow the stem of the plant to continue support of newly formed, target-edited, and substrate-resistant shoots. This approach can significantly expedite genome editing at an unprecedented rate, and in addition can make genome editing a reality for crop species that are recalcitrant to transformation and regeneration with traditional methodologies.

最近的技术发展使以深思熟虑和精确的方式改变植物的基因构成成为可能，而这在几年前是不可想象的。这项名为基因组编辑的新技术可能会对植物育种和农业产生革命性的影响，因为它将有可能引入有利的遗传变异来提高产量和食品质量。基因组编辑技术是基于一种细菌机制，这种机制用于通过改变感染病原体的DNA来防御它们。采用这种细菌技术的最大好处是，一旦完全开发，这项技术就不会像以前的技术那样在编辑后的植物中留下外来基因。基因组编辑技术仍然面临着挑战。然而，该项目将开发新的方法来促进植物的基因组编辑。这些方法利用了幼年植物部分的一组细胞，这些细胞保留了发展新器官的潜力，类似于动物干细胞的相同属性。这项研究将测试在番茄干细胞中引入进行基因组编辑的分子的新方法。这些高风险的实验将对推进新的基因组编辑技术产生重大影响。本项目将创建一个同时携带Cas9内切酶和由茎分生组织特异启动子驱动的条件性(在底物存在下是致命的)反选择标记(CSM)的番茄转基因品系。后者代表一种条件性和组织特异性分生组织自杀基因。通过农杆菌介导法将针对CSM和目的基因的sgRNA导入转CSM基因的番茄幼苗。SgRNA在分生组织中的表达将导致CSM和靶基因的失活。然后，注射的植株将在接种地点被斩首，以诱导新的芽再生。CSM将仅在顶端分生组织中表达。因此，选择性底物的应用将导致非突变再生分生组织中的分生组织死亡，但将允许植物的茎继续支持新形成的、靶标编辑的和底物抗性的新芽。这种方法可以以前所未有的速度显著加快基因组编辑，此外，还可以使那些不愿用传统方法进行转化和再生的作物物种的基因组编辑成为现实。