Post-transcriptional genome regulation in bacteria with next generation CRISPR-Cas tools

使用下一代 CRISPR-Cas 工具进行细菌转录后基因组调控

• 批准号: 2225632
• 负责人: Jesse Zalatan
• 金额: $ 67.16万
• 依托单位: University of Washington
• 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: 2022-08-15 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2225632&HistoricalAwards=false
• 关键词: Post; transcriptional; genome; regulation; bacteria

This project seeks to develop new tools to regulate bacterial gene expression for biosynthesis. Dramatic successes in metabolic engineering have been achieved through laborious efforts to optimize gene expression for the production of high-value chemicals. Programmable, DNA-targeting CRISPR-Cas tools can be used to rapidly implement complex genetic programs, but in bacteria these systems have limitations in their ability to precisely control individual genes. To improve the ability to precisely control cellular behavior, the investigators will develop RNA-targeting CRISPR-Cas systems that act post-transcriptionally and may overcome the limitations of the DNA-targeting systems. The sophisticated control systems developed in this project will be useful for practical biosynthesis, bacterial engineering, and basic research in bacteria. These findings will be incorporated into educational materials and courses taught to chemistry and engineering students. This project will also provide opportunities for underrepresented students at the high school and undergraduate levels to participate in laboratory research.Bacterial metabolic pathways perform complex chemical transformations with high specificity to produce biosynthetic products. Introducing heterologous genes allows metabolism to be diverted to new synthetic targets, but optimizing the function of these engineered strains is challenging. The goal of this proposal is to develop a new class of bacterial RNA-targeting tools to systematically regulate multi-gene expression programs, and to identify regulatory architectures that can improve the output of biosynthetic pathways. Previously, DNA-targeting CRISPR-Cas transcriptional regulatory circuits have been successfully assembled into sophisticated multi-gene regulatory programs. Despite their enormous potential, DNA-targeting CRISPR-Cas systems have important limitations in their ability to precisely up- or down-regulate individual bacterial gene targets. For this project, the investigators will create dCas13-based RNA-targeting tools, which act post-transcriptionally and may overcome the limitations of DNA-targeting CRISPR-Cas systems. The immediate goal of the project is to develop new capabilities for gene repression and activation at the translational level. First, the investigators will use Agile BioFoundry (ABF) capabilities to learn global rules for dCas13-mediated translational regulation of gene expression and metabolic flux in bacteria. Next, they will systematically compare dCas13-mediated translational regulation to dCas9-mediated transcriptional control to determine whether these systems can produce distinct functional effects. Finally, they will demonstrate the utility of this knowledge by implementing translational control systems in Design-Build-Test-Learn (DBTL) cycles applied to engineer aromatic biosynthesis in a non-model microbe that can be used for industrial bioproduction. These systems will further expand the toolkit for exploring the large space of regulatory architectures to optimize bacterial 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.

该项目旨在开发新的工具来调节细菌生物合成的基因表达。代谢工程的巨大成功是通过艰苦的努力来优化基因表达以生产高价值化学品而取得的。可编程的DNA靶向CRISPR-Cas工具可用于快速实施复杂的遗传程序，但在细菌中，这些系统精确控制单个基因的能力有限。为了提高精确控制细胞行为的能力，研究人员将开发RNA靶向CRISPR-Cas系统，该系统在转录后发挥作用，并可能克服DNA靶向系统的局限性。本计画所开发的精密控制系统，将可应用于细菌的实际生物合成、细菌工程及基础研究。这些发现将被纳入化学和工程专业学生的教材和课程。该项目还将为高中和本科阶段代表性不足的学生提供参与实验室研究的机会。细菌代谢途径执行具有高度特异性的复杂化学转化以产生生物合成产物。引入异源基因可以使代谢转向新的合成靶标，但优化这些工程菌株的功能具有挑战性。该提案的目标是开发一类新的细菌RNA靶向工具，以系统地调节多基因表达程序，并确定可以改善生物合成途径输出的调控结构。此前，靶向DNA的CRISPR-Cas转录调控电路已成功组装成复杂的多基因调控程序。尽管具有巨大的潜力，但DNA靶向CRISPR-Cas系统在精确上调或下调单个细菌基因靶标的能力方面存在重要的局限性。对于这个项目，研究人员将创建基于dCas 13的RNA靶向工具，这些工具在转录后发挥作用，并可能克服DNA靶向CRISPR-Cas系统的局限性。该项目的近期目标是在翻译水平上开发基因抑制和激活的新能力。首先，研究人员将使用Agile BioFoundry（ABF）的能力来学习细菌中dCas 13介导的基因表达和代谢通量的翻译调控的全局规则。接下来，他们将系统地比较dCas 13介导的翻译调控与dCas 9介导的转录调控，以确定这些系统是否可以产生不同的功能效应。最后，他们将通过在设计-构建-测试-学习（DBTL）循环中实施翻译控制系统来展示这一知识的实用性，该循环应用于可用于工业生物生产的非模型微生物中的芳香族生物合成工程。这些系统将进一步扩展工具包，以探索监管架构的大空间，以优化细菌生物合成。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。