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

TRTech-PGR：开发用于植物功能基因组学的高效且无约束的 CRISPR 系统

• 批准号: 2132693
• 负责人: Yiping Qi
• 金额: $ 120.58万
• 依托单位: University of Maryland, College Park
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2132693&HistoricalAwards=false
• 关键词: TRTech; PGR; Development; highly; efficient

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.

新兴的植物基因组工程技术是旨在扩大基础研究和翻译研究的工具。近年来，已经开发出不同的基因组编辑工具，以产生具有单碱基精度的植物基因组中的靶向突变。由于农艺性状是由突变或DNA变异引起的，因此基因组编辑技术可以加快产量，营养，抗病性和耐候作物的高产量，以满足对世界增加的人群的需求和不断发展的饮食。基因组编辑技术通常用于灭活或改变基因功能。相比之下，基因激活技术可以增强基因功能，这为代谢工程和作物改善提供了新的手段。当前，这些基因组工程工具的靶向范围有限，使许多基因组或基因序列无法访问用于编辑或激活。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，这些工具为靶向提供了更大的灵活性，并且具有很高的效率和精度。此外，我们将将这些新工具转换为全面的类似瑞士刀的工具箱，以便用户可以为植物中的不同基因组工程应用选择适当的工具。这样的工具箱一次仅靶向一个基因时，不仅可以容纳小规模的用法，而且还促进了大规模的全基因组筛选，以在模型植物和农作物中发现特质。培训未来的科学家，尤其是从历史上代表性不足的群体中培训这项研究项目的核心使命。植物研究人员从迅速发展的CRISPR-CAS9，CAS12A，C-T碱基编辑和A-G-G基础编辑系统中受益匪浅。 CRISPR-CAS9也已被重新利用并设计到有效的基因激活系统中，该系统在植物基因组学，代谢工程和作物改善方面具有巨大的希望。但是，有两个主要限制。第一个限制是基因组靶向限制，由Cas9或cas12a的原始基序（PAM）定义。第二个限制是缺乏针对植物中全基因组遗传筛查的多功能CRISPR矢量系统。 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.这些新的基因组工程工具将在单子植物（例如大米）和二氧化碳（例如番茄）植物中进行测试，这些工具可确保该技术适用于所有植物。这些用户友好的CRISPR向量将通过Addgene（www.addgene.org）提供给其他研究人员，以帮助推进植物的基本和翻译研究。该项目的更广泛影响包括马里兰州大学公园大学和华盛顿大学霍华德大学的本科生培训（主要HBCU）。该项目还为蒙哥马利·布莱尔高中和埃莉诺·罗斯福高中（ERHS）的学生带来了STEM培训，该学生招募了60-62％的非洲裔美国人和9-12％的西班牙裔美国人。这奖反映了NSF的法定任务，并通过该基金会的知识优点和广泛的影响来通过评估来诚实地通过评估来进行评估，以诚实地进行评估。

项目成果

Genome-edited foods

• DOI: 10.1038/s44222-023-00115-8
• 发表时间: 2023-10
• 期刊: Nature Reviews Bioengineering
• 作者: Aytug Tuncel;Changtian Pan;T. Sprink;Ralf Wilhelm;R. Barrangou;Li Li-Li;Patrick M. Shih;Rajeev K. Varshney;Leena Tripathi;Joyce Van Eck;K. Mandadi;Yiping Qi

Prime editor integrase systems boost targeted DNA insertion and beyond

• DOI: 10.1016/j.tibtech.2022.05.002
• 发表时间: 2022-08-01
• 期刊: TRENDS IN BIOTECHNOLOGY
• 影响因子: 17.3
• 作者: Eid, Ayman;Qi, Yiping
• 通讯作者: Qi, Yiping