Design Principles for Complex and Dynamically-Regulated CRISPR-Cas Gene Expression Programs in Bacteria

该项目寻求开发新的工具来调节细菌的基因表达，以支持生物合成过程。通过努力优化多个基因的表达以生产高价值的化学物质，代谢工程已经取得了巨大的成功。该项目将开发可编程的CRISPR-CAS系统，以同时针对多个基因进行激活和抑制。这将在没有广泛基因组工程的情况下迅速实施复杂的遗传计划。主要项目目标包括开发对细菌中合成转录激活剂的可靠控制。这些调控策略的实施将有助于基因表达的动态调控。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水平的代表性不足的学生提供参与实验室研究的机会。该计划的目标是开发新的工具，系统地和动态地调节多基因表达计划，并确定能够改善生物合成途径输出的调节架构。CRISPR-Cas转录调控电路将用于通过基于CRISPRi的基因抑制和基于CRISPRa的合成激活的组合，同时上调和下调内源和外源基因。目前，可用于细菌系统的人工转录激活剂很少，这项提议的一个关键组成部分将是设计新的激活域。CRISPR-Cas系统将由可诱导和/或动态响应的启动子控制，以调节多基因表达程序的时间。为了确定提高生物合成产量的转录程序，代谢产品的基于RNA的生物传感器将被用于高通量筛选工程菌株的文库。将CRISPR-CAS用于复杂控制多基因表达程序的工具与筛选许多变体的能力相结合，将使系统地探索大片动态响应的调控体系结构，以优化细菌生物合成。该奖项反映了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 to support biosynthetic processes. Dramatic successes in metabolic engineering have been achieved through laborious efforts to optimize the expression of multiple genes to produce high-value chemicals. This project will develop programmable CRISPR-Cas systems to simultaneously target multiple genes for activation and repression. This will rapidly implement complex genetic programs without extensive genome engineering. Key project goals include the development of reliable control of synthetic transcriptional activators in bacteria. The implementation of these control strategies will help to dynamically regulate gene expression. 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.The goal of this proposal is to develop new tools to systematically and dynamically regulate multi-gene expression programs, and to identify regulatory architectures that can improve the output of biosynthetic pathways. CRISPR-Cas transcriptional regulatory circuits will be used to simultaneously up- and down-regulate both endogenous and heterologous genes using a combination of CRISPRi-based gene repression and CRISPRa-based synthetic activation. There are currently very few synthetic transcriptional activators available for bacterial systems, and a key component of this proposal will be to engineer new activation domains. The CRISPR-Cas system will be controlled by inducible and/or dynamically-responsive promoters to regulate the timing of multi-gene expression programs. To identify transcriptional programs that improve biosynthetic yields, RNA-based biosensors for metabolic products will be used to enable high-throughput screening of libraries of engineered strains. Combining CRISPR-Cas tools for sophisticated control of multi-gene expression programs with the ability to screen many variants will enable a systematic exploration of a large space of dynamically-responsive 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.