PlantSynBio/TR-Tech-PGR: Targeted Integration of User-Defined DNA in Plants

将新的DNA植入植物基因组既效率低又不精确。这种低效率阻碍了所有开发新的和增强作物现有农业性状的方法。CRISPR/Cas9核酸酶等新技术就像分子“剪刀”一样，在基因组的特定位置切割DNA，提高了植物基因组工程的精度。然而，在切割DNA之后，新DNA的精确添加仍然是一个巨大的挑战。该项目确定了分子“剪刀”的缺失对应物-在Cas9切割的位点精确添加DNA所需的分子“胶水”。该项目的目标包括开发将DNA精确添加到作物基因组中，以及测试如何使用这种新的基因组工程技术来快速使单个基因或整个植物对胁迫做出反应。该项目还将测试管理这种靶向DNA添加系统的关键规则，例如可以引入的DNA的大小和类型。这种使能技术有可能改变植物基因组工程的研究和工业。最后，该项目将激发中学生从事STEM职业的兴趣，并教育尚未获得其他研究经验机会的本科生。该奖项的总体目标是制作一个可用且可访问的技术工具包，使未来的植物合成生物学方法能够将新能力引入关键农业植物。首先，该项目旨在将新DNA整合到发现植物拟南芥和作物玉米和大豆的基因组中。第二，该项目旨在探索可以被递送到基因组特定区域的合成货物DNA的类型和限制。这些包括工程表达盒的递送，与蛋白质编码基因一起框内插入表位标签，以及在基因编码区上游添加新的增强子元件。第三，该项目旨在展示这种靶向DNA整合系统如何用于重新连接转录反应，并设计植物表型，以在单个基因水平或整个基因组上克服环境胁迫。最后，该项目旨在使“任何人都可以成为科学家”外展计划适应COVID和后COVID时代，并根据其中一项科学目标开发基于课程的本科生研究体验，为学生提供第一次动手的真实生物研究体验。所有项目成果，包括遗传和分子资源，将根据要求和通过长期存放库提供。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

The placement of new DNA into plant genomes is both inefficient and imprecise. This inefficiency hampers all approaches to develop new and enhance existing agricultural traits in crops. New technology such as CRISPR/Cas9 nucleases act like molecular “scissors” to cut DNA at specific locations in the genome, improving the precision of plant genome engineering. However, after cutting the DNA, the precise addition of new DNA has remained a grand challenge. This project identifies the missing counterpart to the molecular “scissors”--the molecular “glue” needed to precisely add DNA at sites cleaved by Cas9. Project aims include developing the precise addition of DNA into crop genomes, as well as test how this new genome engineering technology can be used to quickly make a single gene or entire plant stress-responsive. This project will also test key rules governing this system of targeted DNA addition, such as the size and type of the DNA that can be introduced. This enabling technology has the potential to transform the engineering of plant genomes for both research and industry. Lastly, this project will excite middle school students in STEM careers and educate undergraduate students that have not yet had other research experience opportunities.The overarching goal of this award is the production of a usable and accessible toolkit of technology that enables future plant synthetic biology approaches to introduce new abilities into key agricultural plants. First, this project aims to target the integration of new DNA into the genome of the discovery plant Arabidopsis thaliana and crop plants maize and soybean. Second, this project aims to explore the types and limitations of the synthetic cargo DNA that can be delivered to specific regions of the genome. These include the delivery of engineered expression cassettes, inserting epitope tags in-frame with protein-coding genes, and adding new enhancer elements upstream of gene coding regions. Third, this project aims to demonstrate how this system of targeted DNA integration can be used to rewire the transcriptional response and engineer the plant phenotype to overcome environmental stress on either a single gene level or for the entire genome. Lastly, this project aims to adapt the “Anyone Can Be A Scientist” outreach program to the COVID and post-COVID eras and develop a course-based undergraduate research experience based on one of the scientific aims to provide students with their first hands-on authentic biological research experience. All project outcomes including genetic and molecular resources will be made available upon request and through long-term repositories.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.