Collaborative Research: Redirecting cellular metabolism via synthetic toehold-gated dCas9 regulators

合作研究：通过合成的门控 dCas9 调节器重定向细胞代谢

• 批准号: 1817675
• 负责人: Wilfred Chen
• 金额: $ 55.2万
• 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: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1817675&HistoricalAwards=false
• 关键词: Collaborative; Research; Redirecting; cellular; metabolism

Cellular metabolism is capable of highly specific and efficient chemical synthesis at mild temperatures and pressures far beyond the capability of most synthetic chemical routes. Engineering specific pathways can be used to further improve the range of compounds that can be synthesized but it is a major challenge to achieve commercially viable productivity. To maximize productivity, it is crucial to fine-tune pathway fluxes. The goal of this project is to develop a new transformative approach to modulate cell metabolism based on endogenous cellular information. An emerging strategy is the use of regulators that provide dynamic control of pathway fluxes. A recently discovered modified CRISPR based tool offers a unique approach for DNA targeting and transcriptional regulation. These new generation of regulators can be used for dynamic gene repression and activation for many synthetic-biology and metabolic engineering applications. In addition to the scientific advancements, this project will help train graduate students through the integration of principles from protein engineering, synthetic biology, and cellular physiology. Outreach activities to local high school teachers and students through existing programs available at the University of Delaware and Rensselaer Polytechnic Institute are also planned.The goal of this project is to develop a new transformative approach to modulate cell metabolism based on endogenous cellular information. In particular, a new generation of toehold-gated dCas9 regulators governed by conditional sgRNA structures that are activated by toehold-mediated strand displacement will be created to provide simultaneous, orthogonal, and autonomous control of cellular metabolism. Because dCas9-based regulators are governed by a structurally defined single guide RNA (sgRNA) structure, it is easy to envision that conditional sgRNA structures can be created that are activated by endogenous mRNAs based on toehold-mediated strand displacement. This new framework to design toehold-gated dCas9 regulators responsive to any endogenous mRNA will lay the foundation as a new transformative approach for implementing dynamic control of cellular metabolism. The ability to modulate metabolism based on endogenous cellular information in optimizing the production of numerous products in yeast will be established. The long-term goal is to combine the knowledge gained from this project toward the design of dynamic and autonomous cellular control for any metabolic pathway of interest.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.

细胞代谢能够在温和的温度和压力下进行高度特异和高效的化学合成，远远超出了大多数合成化学路线的能力。工程上的特定途径可以用来进一步改进可合成化合物的范围，但要实现商业上可行的生产率，这是一个重大挑战。为了最大限度地提高生产率，微调途径通量至关重要。这个项目的目标是开发一种基于内源性细胞信息来调节细胞新陈代谢的新的变革性方法。一种新兴的策略是使用提供对途径通量的动态控制的调节器。最近发现的一个基于CRISPR的改进工具为DNA靶向和转录调控提供了一种独特的方法。这些新一代的调节剂可用于许多合成生物学和代谢工程应用中的动态基因抑制和激活。除了科学上的进步，这个项目还将通过整合蛋白质工程、合成生物学和细胞生理学的原理来帮助培养研究生。还计划通过特拉华大学和伦斯勒理工学院现有的项目向当地高中教师和学生开展外联活动。该项目的目标是开发一种新的变革性方法，基于内源性细胞信息来调节细胞新陈代谢。特别是，由脚趾介导的链置换激活的条件sgRNA结构控制的新一代脚趾门控dCas9调节器将被创建，以提供对细胞新陈代谢的同步、正交和自主控制。由于基于dCas9的调节子受结构定义的单引导RNA(SgRNA)结构的控制，因此很容易想象条件sgRNA结构可以由基于脚尖介导的链置换的内源mRNAs激活。这一新的框架设计了对任何内源mRNA做出反应的脚点门控dCas9调节器，将为实现细胞代谢动态控制的新的变革性方法奠定基础。在优化酵母中大量产品的生产中，将建立基于内源细胞信息来调节新陈代谢的能力。长期目标是将从这个项目中获得的知识结合起来，为任何感兴趣的代谢途径设计动态和自主的细胞控制。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

A modular approach for dCas9-mediated enzyme cascading via orthogonal bioconjugation

• DOI: 10.1039/d0cc04196c
• 发表时间: 2020-09-29
• 期刊: CHEMICAL COMMUNICATIONS
• 影响因子: 4.9
• 作者: Berckman, Emily A.;Chen, Wilfred
• 通讯作者: Chen, Wilfred

Dynamic modulation of enzyme activity by synthetic CRISPR–Cas6 endonucleases

• DOI: 10.1038/s41589-022-01005-7
• 发表时间: 2022-04
• 期刊: Nature Chemical Biology
• 影响因子: 14.8
• 作者: Alexander A Mitkas;M. Valverde;Wilfred Chen

A microRNA-gated thgRNA platform for multiplexed activation of gene expression in mammalian cells

用于哺乳动物细胞基因表达多重激活的 microRNA 门控 thgRNA 平台

• DOI: 10.1039/d2cc01478e
• 发表时间: 2022
• 期刊: Chemical Communications
• 影响因子: 4.9
• 作者: Hunt, Victoria M.;Chen, Wilfred
• 通讯作者: Chen, Wilfred