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

最近的技术发展使人们有可能以故意和精确的方式改变植物的基因组成，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以对植物育种和农业产生变革性影响，因为它可以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，通过改变它们的DNA来防御感染因子。采用这种细菌技术的巨大优势在于，一旦完全开发，该技术将不会像以前的技术那样在编辑过的植物中留下外源基因。基因组编辑技术仍然存在挑战。然而，该项目将开发新的方法来促进植物基因组编辑。这些方法利用植物幼嫩部分中的一组细胞，这些细胞保留了发育新器官的潜力，类似于动物干细胞的相同特性。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。高风险实验将对推进新的基因组编辑技术产生重大影响。该项目将创建一个番茄转基因品系，该品系携带Cas9内切核酸酶和由茎分生组织特异性启动子驱动的条件（在底物存在下致死）反选择标记（CSM）。后者代表一个条件性的和组织特异性的分生组织自杀基因。通过农杆菌介导的CSM转基因番茄幼苗的感染，将靶向CSM和感兴趣的基因的sgRNA递送到幼茎。sgRNA在分生组织中的表达将导致CSM和靶基因的失活。然后将注射的植物在接种部位处断头以诱导新的芽再生。CSM将仅在茎尖分生组织中表达。因此，选择性底物的应用将导致未突变的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、靶编辑的和底物抗性的芽。这种方法可以以前所未有的速度显著加快基因组编辑，此外还可以使基因组编辑成为传统方法难以转化和再生的作物物种的现实。

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.