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

作物品种的转基因是粮食安全和可持续农业的关键。CRISPR/CAS技术的出现极大地提高了我们设计基因组的能力，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。这一建议解决了农作物工程中最重要的问题，即基因组修改与用于编辑基因组的机制的无针对性和潜在的突变整合有关。这是有问题的，因为在随机整合的高背景下，识别目标转化子所需的筛查增加。此外，为了用于商业用途，必须从基因组中消除合成结构，这一过程对许多作物来说可能是漫长和昂贵的。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。这项技术建立在DNA聚合酶Theta(POLQ)介导的途径的识别基础上，该途径负责大多数转基因整合事件。我们将抑制这一途径，并研究其对基因靶向频率的影响。ABI1基因的定向突变将在拟南芥中提供原理证据，导致产生一种显性突变，允许在脱落酸存在的情况下萌发。这项工作将扩展到Brassica，以演示这项技术在作物物种上的应用。这个项目将大大提高我们利用基于知识的方法设计作物基因组的能力，并得益于申请者在植物转化和DNA重组机制方面的丰富经验。

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.