A high-performance and versatile technology for precision microbiome engineering

项目总结 哺乳动物的胃肠道是复杂多样的微生物集合的家园，这些微生物 在新陈代谢、宿主免疫和中枢神经系统功能中起着至关重要的作用。尽管不断增长的 认识到平衡的微生物群对人类健康和行为的重要性，以及广泛的 可能由生物失调引起的疾病，我们研究和修改体内复杂微生物群落的能力 仍然受到严格的限制。测序工作可以详尽地记录细菌的多样性和丰度，但 只提供观察性信息；在小鼠身上进行的诺生菌研究允许严格控制殖民，但 不能代表自然的宿主-微生物群相互作用；基因工程可以用来操纵 特定的基因或途径在选定的微生物中，但不在自然环境中。要解决这些问题 针对这些不足，我们提出开发一种用于精密微生物组工程的创新平台技术 这将首次实现体内特定于基因和物种的编辑。我们的方法主要围绕两个方面 我们实验室最近取得的突破：一种使用CRISPR生成精确DNA插入的方法- 转座子系统(集成技术)以及一种在肠道内动员遗传有效载荷的方法 使用广泛的宿主范围共轭向量(魔术技术)。通过组合和扩展这些工具，我们 将开发可编程的自动驾驶元件，在广泛传播的同时保留精致的核苷酸- 目标基因组的水平特异性。 我们的初步数据为我们的建议提供了强有力的证据，并证明了 可行性。在最近的一次合作中，我们开发了千碱基规模的细菌基因组整合 通过系统地评估一组引导RNA的全基因组插入特异性来进行工程，以及 在多种临床和工业相关的细菌物种中表现出有效的活性。在目标1中，我们将 识别自主和增殖功能的高度活跃的整合变体，并开发出 结合经验脱靶数据和大型元基因组的综合指导RNA设计算法 程序集信息。在目标2中，我们将把魔术和集成结合起来，以实现移动传输和 在复杂的体外群落中以及在小鼠模型中进行定向整合。最后，在目标3中，我们将 将我们的工具用于体内功能获得和功能丧失的研究：1)传递胆盐水解酶基因 并研究它们对微生物群组成和宿主代谢的相应影响， 2)在肺炎克雷伯菌病模型中灭活多药耐药基因。总的来说，我们的 研究将推动强大的新合成生物学工具，这些工具可以广泛而灵活地应用于任何 复杂的感兴趣的细菌群落，用于基础研究和最终的治疗应用。

PROJECT SUMMARY The mammalian gastrointestinal tract is home to a complex and diverse collection of microorganisms that play crucial roles in metabolism, host immunity, and central nervous system function. Despite a growing appreciation for the importance of a balanced microbiome on human health and behavior, and the wide range of diseases that can result from dysbiosis, our ability to study and modify complex microbial communities in vivo remains severely limited. Sequencing efforts can exhaustively catalog bacterial diversity and abundance, but offer only observational information; gnotobiotic research in mice allows for tight control over colonization, but fails to represent natural host-microbiome interactions; and genetic engineering can be used to manipulate specific genes or pathways in select microbes, but not within native environments. To address these shortcomings, we propose to develop an innovative platform technology for precision microbiome engineering that will, for the first time, enable gene- and species-specific editing in vivo. Our approach centers around two recent breakthroughs made in our laboratories: a method for generating precise DNA insertions using CRISPR- transposon systems (INTEGRATE technology), and a method for mobilizing genetic payloads within the gut using broad-host-range conjugative vectors (MAGIC technology). By combining and expanding these tools, we will develop programmable, self-driving elements that disseminate broadly while retaining exquisite nucleotide- level specificity for target genomes. Our preliminary data provide strong evidence to substantiate the basis of our proposal and demonstrate feasibility. In a recent collaborative effort, we developed INTEGRATE for kilobase-scale bacterial genome engineering by systematically assessing genome-wide insertion specificity across a panel of guide RNAs, and demonstrating efficient activity in multiple clinically and industrially relevant bacterial species. In Aim 1, we will identify hyperactive INTEGRATE variants that function autonomously and proliferatively, and develop a comprehensive guide RNA design algorithm that incorporates empirical off-target data and large metagenome assembly information. In Aim 2, we will combinate MAGIC with INTEGRATE to enable mobile transmission and targeted integration within complex in vitro communities, as well as in a mouse model. Finally, in Aim 3, we will apply our tool for both gain-of-function and loss-of-function studies in vivo: 1) to deliver bile salt hydrolase genes in the murine gut and investigate their corresponding effects on microbiome composition and host metabolism, and 2) to inactivate multidrug resistance genes in a Klebsiella pneumoniae disease model. Collectively, our studies will advance powerful new synthetic biology tools that can be broadly and flexibly applied within any complex bacterial community of interest, for both basic research and eventual therapeutic applications.