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

BTT EAGER：通过使用非整合 T-DNA 技术进行清洁基因组编辑

基本信息

批准号：
1848434
负责人：
Stanton Gelvin
金额：
$ 30万
依托单位：
Purdue University
依托单位国家：
美国
项目类别：
Continuing Grant
财政年份：
2019
资助国家：
美国
起止时间：
2019-02-15 至 2022-01-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1848434&HistoricalAwards=false
关键词：
BTT EAGER Clean genome editing

项目摘要

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.

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。该项目与英国利兹大学的Christopher West博士合作，解决了工程作物中最重要的问题，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有关。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，合成构建体必须在一个过程中从基因组中消除，该过程对于许多作物，特别是具有长世代时间的那些（例如树种）和那些繁殖的蔬菜（例如马铃薯、甘薯、香蕉等）来说可能是漫长和昂贵的。该项目将根据申请人在植物转化和DNA重组机制方面的丰富经验，开发一种基于抑制随机转基因整合的清洁遗传工程方法。大多数目前的植物基因组工程技术平台需要或具有非预期的后果，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DNA聚合酶Theta（PolQ）介导的途径的鉴定上，该途径负责大多数农杆菌介导的转基因整合事件。使用作为可表达的T-DNA编码基因或直接作为肽引入植物中的DNA聚合酶θ的显性负性片段来抑制该途径，将减轻T-DNA整合到宿主基因组中。异位T-DNA整合的抑制将增强检测由同源依赖性修复介导的基因靶向事件的能力。原理的证明将在拟南芥中通过同源定向修复的ABI 1基因的靶向突变提供，导致产生允许在脱落酸存在下萌发的显性突变。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利用知识为基础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知识价值和更广泛的影响审查标准进行评估，被认为值得支持。