Collaborative Research: Synthetic CRISPR-Cas6 endonucleases for dynamic control of cellular phenotypes in yeast

合作研究：用于动态控制酵母细胞表型的合成 CRISPR-Cas6 核酸内切酶

基本信息

批准号：
2013991
负责人：
Wilfred Chen
金额：
$ 50万
依托单位：
University of Delaware
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-01 至 2025-05-31
项目状态：
未结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=2013991&HistoricalAwards=false
关键词：
Collaborative Research Synthetic CRISPR Cas6

项目摘要

The overall goal of this project is to use the power of synthetic biology to build microbial factories for the production of high value chemicals. The collaborative research team will use an innovative enzyme colocalization/assembly strategy to boost production of selected chemicals in yeast. This project will also facilitate outreach activities to local high school teachers and students through existing programs available at the University of Delaware and University of California, Irvine.In nature, dynamic interactions between proteins play a crucial role in defining many cellular functions such as metabolism, cell signaling, transcription regulation, apoptosis, cellular targeting, and protein degradation. By controlling the spatial and temporal organization of these supramolecular complexes using a protein scaffold, cellular functions can be modulated in a highly dynamic manner for optimum efficiency. Understanding how these proteins interact holds the key to deciphering their roles in native cellular function and in creating new cellular functions for synthetic biology applications. In this project, the researchers propose a new transformative framework that combines the predictability of RNA hybridization and the ease of RNA processing, while offering reversible protein assembly on demand, to create dynamic enzyme cascades for proteasome-targeted protein degradation and for metabolic pathway regulation in yeast. The proposed research relies on a new and potent approach that enables specific processing and high-affinity binding to RNA transcripts using the naturally occurring CRISPR/Cas6 system. While the native function of Cas6 is to generate CRISPR RNAs (crRNAs) to guide the cleavage of DNA targets, the researchers will repurpose the Cas6 family proteins as a generalizable platform for site-specific RNA binding/processing and will demonstrate its utility to assemble dynamic enzyme cascades for synthetic biology and metabolic engineering applications in yeast. The expertise of the Chen and Da Silva labs will be combined to develop and implement the metabolons. State of the art synthetic biology and genetic tools will be used to accomplish four overall aims: (1) Dynamic protein assembly and disassembly by strand displacement, (2) Dynamic protein degradation using orthogonal Cas6 proteins, (3) Dynamic assembly of metabolons for substrate channeling, and (4) Combined metabolon assembly and protein degradation for increased polyketide biosynthesis.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.

该项目的总体目标是利用合成生物学的力量来建立微生物工厂，以生产高价值化学物质。协作研究团队将使用创新的酶共定位/组装策略来增强酵母中所选化学品的生产。该项目还将通过特拉华大学和加利福尼亚大学的现有计划来促进当地高中教师和学生的外展活动。从本质上，蛋白质之间的动态相互作用在定义许多细胞功能（例如代谢，细胞信号，转录，跨牙剂，细胞靶标，细胞靶标，细胞靶标，蛋白质降解）方面起着至关重要的作用。通过使用蛋白质支架控制这些超分子复合物的空间和时间组织，可以以高度动态的方式调节细胞功能，以提高最佳效率。了解这些蛋白质的相互作用是解读其在天然细胞功能以及为合成生物学应用创建新的细胞功能方面的关键。在该项目中，研究人员提出了一个新的变革框架，结合了RNA杂交的可预测性和RNA加工的易于性，同时提供可逆的蛋白质组装按需，以创建动态酶cascades，以用于蛋白酶体靶向的蛋白质降解和酵母中的代谢途径。拟议的研究依赖于一种新的和有效的方法，该方法可以使用天然存在的CRISPR/CAS6系统具有特定的加工和高亲和力结合与RNA转录本。 While the native function of Cas6 is to generate CRISPR RNAs (crRNAs) to guide the cleavage of DNA targets, the researchers will repurpose the Cas6 family proteins as a generalizable platform for site-specific RNA binding/processing and will demonstrate its utility to assemble dynamic enzyme cascades for synthetic biology and metabolic engineering applications in yeast. 陈和达席尔瓦实验室的专业知识将合并为开发和实施变质。艺术合成生物学和遗传工具的状态将用于实现四个总体目标：（1）动态蛋白质组装和通过链位移的拆卸，（2）使用正交CAS6蛋白的动态蛋白质降解，（3）（3）用于增加底物通道和（4）组合素的化合物的动态组装的动态组装，并（4）组合蛋白质的蛋白质组装，并（4）蛋白质蛋白质组装的蛋白质蛋白质和（4）蛋白质的蛋白质组装，以及（4）蛋白质素质的组装蛋白质。生物合成。该奖项反映了NSF的法定任务，并被认为是值得通过基金会的知识分子优点和更广泛影响的评论标准来评估的。