BTT EAGER: 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。该项目与英国利兹大学的克里斯托弗·韦斯特博士合作，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，特别是那些世代时间长的作物(如树种)和那些无性繁殖的作物(如土豆、红薯、香蕉等)。该项目以申请者在植物转化和DNA重组机制方面的丰富经验为基础，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。大多数目前的植物基因组工程技术平台需要或意外地将引入的基因组工程试剂整合到宿主染色体中。将在该项目中测试的技术建立在识别DNA聚合酶Theta(POLQ)介导的途径的基础上，该途径负责农杆菌介导的大多数转基因整合事件。利用DNA聚合酶theta的显性负片段作为可表达的T-DNA编码基因或直接作为肽引入植物中，抑制这一途径，将减轻T-DNA整合到寄主基因组中的程度。抑制异位T-DNA整合将增强检测同源依赖修复介导的基因靶向事件的能力。通过同源定向修复对ABI1基因进行定向突变，从而产生显性突变，从而在脱落酸存在的情况下萌发，将在拟南芥中提供原理证明。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。该项目将极大地提高我们使用基于知识的方法设计作物基因组的能力。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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 project, in collaboration with Dr. Christopher West, University of Leeds, UK, 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 because of 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, especially those with long generation times (such as tree species) and those propagated vegetatively (such as potato, sweet potato, banana, etc.). This project, informed by the applicants' considerable experience in plant transformation and DNA recombination mechanisms, will develop a clean genetic engineering methodology based on the suppression of random transgene integration. Most current plant genome engineering technology platforms require, or have as an unintended consequence, the integration of introduced genome engineering reagents into the host chromosomes. The technology that will be tested in this project builds on the identification of a DNA Polymerase Theta (PolQ)-mediated pathway that is responsible for the majority of Agrobacterium-mediated transgene integration events. Suppression of this pathway, using dominant negative fragments of DNA polymerase theta introduced into the plant either as expressible T-DNA-encoded genes or directly as peptides, will mitigate T-DNA integration into the host genome. Inhibition of ectopic T-DNA integration will enhance the ability to detect gene targeting events mediated by homology-dependent repair. Proof of principle will be provided in Arabidopsis through targeted mutation of the ABI1 gene through homology-directed repair, 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.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.