TRTech-PGR: Development of highly efficient and unconstrained CRISPR systems for plant functional genomics

新兴的植物基因组工程技术是旨在扩大基础研究和转化研究的工具。近年来，不同的基因组编辑工具被开发出来，用于在植物基因组中产生单碱基精度的靶向突变。由于农艺性状是由突变或DNA变异引起的，基因组编辑技术加速了高产、营养丰富、抗病和气候适应型作物的育种，以满足不断增长的世界人口和不断变化的饮食需求。基因组编辑技术通常用于使基因失活或改变基因功能。相比之下，基因激活技术可以增强基因功能，为代谢工程和作物改良提供新的手段。目前，这些基因组工程工具的靶向范围有限，使得许多基因组或基因序列无法进行编辑或激活。为了克服这一主要瓶颈，我们将开发一套改进的基因组工程工具，以提供更大的灵活性和更高的效率和精度。此外，我们将把这些新工具转换成一个综合的瑞士刀式工具箱，以便用户可以选择适合不同植物基因组工程应用的工具。这样的工具箱不仅可以适应一次只针对一个基因的小规模使用，而且还可以促进模式植物和作物中性状发现的大规模全基因组筛选。培训未来的科学家，特别是来自历史上代表性不足的群体的科学家，是这个研究项目的核心任务。植物研究人员从快速发展的CRISPR-Cas9、Cas12a、C-to-T碱基编辑和A-to-G碱基编辑系统中受益匪浅。CRISPR-Cas9也被改造成高效的基因激活系统，在植物基因组学、代谢工程和作物改良方面具有很大的前景。然而，有两个主要的限制。第一个限制是由Cas9或Cas12a的原间隔邻近基序（PAMs）定义的基因组靶向限制。第二个限制是缺乏为植物全基因组遗传筛选量身定制的多功能CRISPR载体系统。为了克服这两大局限，本项目旨在(1)利用CRISPR- cas12a改进植物中无pam的基因组编辑，(2)利用CRISPR- cas9改进植物中无pam的基因组编辑，(3)改进植物中无pam的碱基编辑系统，(4)将新的CRISPR系统转化为一体化载体，在植物中大规模筛选。这些新的基因组工程工具将在单子叶植物（如水稻）和双子叶植物（如番茄）上进行测试，以确保这些技术适用于所有植物。这些用户友好的CRISPR载体将通过Addgene （www.addgene.org）提供给其他研究人员，以帮助推进植物的基础和转化研究。该项目更广泛的影响包括对马里兰大学帕克分校和华盛顿霍华德大学（主要的HBCU）的本科生进行培训。该项目还为蒙哥马利布莱尔高中和埃莉诺罗斯福高中（ERHS）的学生提供STEM培训，这两所高中的非裔美国人占60-62%，西班牙裔美国人占9-12%。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。

Emerging plant genome engineering technologies are tools designed to expand basic research and translational research. In recent years, different genome editing tools have been developed for generating targeted mutations in the plant genome with single base precision. Since agronomic traits are caused by mutations or DNA variation, genome editing technologies accelerate the breeding of high yield, nutritious, disease resistant, and climate resilient crops to meet the demand of an increasing world population and evolving diet. Genome editing technologies are often used to inactivate or alter gene function. By contrast, gene activation technologies can enhance gene function, which provide new means for metabolic engineering and crop improvement. Currently, these genome engineering tools have limited targeting scope, making many genomic or gene sequences inaccessible for editing or activation. To overcome this major bottleneck, we will develop a suite of improved genome engineering tools that provide more flexibility for targeting and are of high efficiency and precision. Furthermore, we will convert these new tools into a comprehensive all-in-one swiss knife-like toolbox so that users can pick appropriate tools for different genome engineering applications in plants. Such a toolbox will not only accommodate small scale usage when targeting only one gene at a time, but also facilitate large-scale genome-wide screens for trait discovery in model plants and crops. Training future scientists, especially from historically under-represented groups, is a core mission of this research project. Plant researchers have greatly benefited from the rapidly evolving CRISPR-Cas9, Cas12a, C-to-T base editing and A-to-G base editing systems. CRISPR-Cas9 has also been repurposed and engineered into efficient gene activation systems which holds great promise in plant genomics, metabolic engineering, and crop improvement. However, there are two major limitations. The first limitation is genome targeting restriction defined by the protospacer adjacent motifs (PAMs) of Cas9 or Cas12a. The second limitation is a lack of versatile CRISPR vector systems tailored for genome-wide genetic screens in plants. To overcome these two major limitations, this project aims to (1) improve PAM-relaxed genome editing by CRISPR-Cas12a in plants, (2) Improve PAM-less genome editing by CRISPR-Cas9 in plants, (3) Improve PAM-less base editing systems in plants, and (4) Convert new CRISPR systems into all-in-one vectors for large-scale screens in plants. These new genome engineering tools will be tested in both monocot (e.g., rice) and dicot (e.g., tomato) plants, which ensure the technologies’ applicable to all plants. These user-friendly CRISPR vectors will be made available to other researchers through Addgene (www.addgene.org), to help advance basic and translational research in plants. The broader impacts of this project include training for undergraduate students at University of Maryland at College Park and Howard University at Washington (a premier HBCU). The project also brings STEM training to students from Montgomery Blair High School and Eleanor Roosevelt High School (ERHS), which enrolls 60-62% African Americans and 9-12% Hispanics.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.