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

该项目旨在开发新的工具来调节细菌基因的表达，以进行生物合成。代谢工程已经取得了巨大的成功，通过艰苦的努力来优化基因表达以生产高价值的化学品。可编程的、DNA靶向的CRISPR-CAS工具可以用于快速实施复杂的遗传程序，但在细菌中，这些系统在精确控制单个基因的能力方面存在局限性。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靶向系统的限制。该项目开发的复杂控制系统将有助于实际的生物合成、细菌工程和细菌基础研究。这些发现将被纳入化学和工程专业学生的教材和课程中。该项目还将为高中和本科生中代表性不足的学生提供参与实验室研究的机会。细菌代谢途径进行具有高度特异性的复杂化学转化，以产生生物合成产品。引入异源基因可以将代谢转移到新的合成靶点，但优化这些工程菌株的功能是具有挑战性的。该提案的目标是开发一类新的细菌RNA靶向工具，以系统地调控多基因表达程序，并识别能够提高生物合成途径输出的调控体系。此前，以DNA为靶点的CRISPR-Cas转录调控电路已成功组装成复杂的多基因调控程序。尽管有巨大的潜力，但以DNA为靶点的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限制。对于这个项目，研究人员将创建基于dCas13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系统的限制。该项目的直接目标是开发在翻译水平上抑制和激活基因的新能力。首先，研究人员将使用Agile BioFoundry(ABF)功能来了解dCas13介导的细菌中基因表达和代谢流量的翻译调节的全球规则。接下来，他们将系统地比较dCas13介导的翻译调控和dCas9介导的转录调控，以确定这些系统是否能够产生不同的功能效果。最后，他们将通过在设计-建造-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，该循环应用于在可用于工业生物生产的非模型微生物中设计芳香生物合成。这些系统将进一步扩展工具包，探索监管架构的巨大空间，以优化细菌生物合成。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。

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.