18-BTT Clean genome editing through the use of nonintegrating T-DNA technology

18-BTT 通过使用非整合 T-DNA 技术进行清洁基因组编辑

• 批准号: BB/S020225/1
• 负责人: Christopher West
• 金额: $ 25.7万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FS020225%2F1
• 关键词: 18; BTT; Clean; genome; editing

Genetic modification of crop species is the key to both food security and sustainable agriculture. The advent of CRISPR/Cas technology has provided a great advance in our ability to engineer genomes, but barriers remain to the routine employment of these methods in the most important agricultural species. This proposal addresses the most significant problem in engineering crop plants, that genome modification is associated with untargeted and potentially mutagenic integration of the machinery used to edit the genome. This is problematic due to the increased screening required to identify targeted transformants against the high background of random integrations. In addition, for commercial use the synthetic constructs must be eliminated from the genome in a process that can be lengthy and expensive for many crops. This project will develop a clean genetic engineering methodology based on the suppression of random transgene integration. This technology builds on the identification of a DNA Polymerase Theta (PolQ)-mediated pathway that is responsible for the majority of transgene integration events. We will suppress this pathway and investigate the effect on gene targeting frequencies. Proof of principle will be provided in Arabidopsis through targeted mutation of the ABI1 gene, resulting in the production of a dominant mutation that allows germination in the presence of abscisic acid. This work will be extended to Brassica to demonstrate the application of this technology to crop species. This project will significantly advance our ability to engineer crop genomes using a knowledge-based approach and informed by the applicants' considerable experience in plant transformation and DNA recombination mechanisms.

作物品种的基因改造是粮食安全和可持续农业的关键。CRISPR/Cas技术的出现为我们设计基因组的能力提供了巨大的进步，但在最重要的农业物种中常规使用这些方法仍然存在障碍。该提议解决了工程作物植物中最重要的问题，即基因组修饰与用于编辑基因组的机器的非靶向和潜在诱变整合相关。这是有问题的，因为在随机整合的高背景下鉴定靶向转化体所需的筛选增加。此外，对于商业用途，必须在对许多作物来说可能是漫长和昂贵的过程中从基因组中消除合成构建体。该项目将开发一种基于抑制随机转基因整合的清洁基因工程方法。该技术建立在鉴定DNA聚合酶Theta（PolQ）介导的途径的基础上，该途径负责大多数转基因整合事件。我们将抑制这一途径，并研究对基因靶向频率的影响。通过ABI 1基因的靶向突变将在拟南芥中提供原理证明，导致产生允许在脱落酸存在下发芽的显性突变。这项工作将扩展到芸苔属，以证明这项技术在作物物种中的应用。该项目将大大提高我们使用基于知识的方法设计作物基因组的能力，并通过申请人在植物转化和DNA重组机制方面的丰富经验获得信息。