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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技术的出现为我们的基因组工程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，解决了作物工程中最重要的问题，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，需要增加筛选以确定目标转化体。此外，为了商业用途，合成结构必须从基因组中去除，这一过程对许多作物来说可能是漫长而昂贵的，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）。本项目将基于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基因工程方法。目前大多数植物基因组工程技术平台都需要将引入的基因组工程试剂整合到宿主染色体中，或者产生了意想不到的后果。本项目将测试的技术建立在鉴定DNA聚合酶Theta （PolQ）介导的途径的基础上，该途径负责大多数农杆菌介导的转基因整合事件。抑制这一途径，将DNA聚合酶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，将减轻T-DNA整合到宿主基因组中。抑制异位T-DNA整合将增强检测同源依赖性修复介导的基因靶向事件的能力。通过同源定向修复ABI1基因的靶向突变，将在拟南芥中提供原理证明，从而产生一个显性突变，允许在脱落酸存在下发芽。这项工作将扩展到芸苔属，以证明该技术在作物品种上的应用。这个项目将显著提高我们利用基于知识的方法设计作物基因组的能力。该奖项反映了美国国家科学基金会的法定使命，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。