A high-performance and versatile technology for precision microbiome engineering

项目摘要 哺乳动物胃肠道是复杂多样的微生物集合的家园， 在新陈代谢、宿主免疫和中枢神经系统功能中起关键作用。尽管增长 认识到平衡的微生物组对人类健康和行为的重要性， 我们研究和改变体内复杂微生物群落的能力 仍然非常有限。测序工作可以详尽地记录细菌的多样性和丰度， 仅提供观察信息;在小鼠中进行的gnotobiotic研究允许对殖民化进行严格控制，但 不能代表自然的宿主-微生物组相互作用;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，但不是在原生环境。解决这些 缺点，我们建议开发一种用于精确微生物组工程的创新平台技术 这将是第一次在体内实现基因和物种特异性编辑。我们的方法围绕两个 我们实验室最近取得的突破：一种使用CRISPR产生精确DNA插入的方法- 转座子系统（INTEGRATE技术），以及在肠道内动员遗传有效载荷的方法 使用宽宿主范围共轭载体（MAGIC技术）。通过结合和扩展这些工具，我们 将开发可编程的，自动驱动的元件，广泛传播，同时保留精致的核苷酸- 靶基因组的水平特异性。 我们的初步数据提供了强有力的证据，证实了我们的建议的基础，并表明 可行性在最近的合作努力中，我们开发了用于内切酶规模细菌基因组的INTEGRATE 通过系统评估一组指导RNA的全基因组插入特异性进行工程化，以及 在多种临床和工业相关细菌物种中显示出有效活性。在目标1中，我们 识别自主和增殖功能的过度活跃的INTEGRATE变体，并开发一种 综合指导RNA设计算法，其结合了经验脱靶数据和大型宏基因组 组装信息。在目标2中，我们将结合MAGIC和INTEGRATE来实现移动的传输， 在复杂的体外社区以及小鼠模型中的靶向整合。在目标3中，我们将 将我们的工具应用于体内功能获得和功能丧失研究：1）递送胆盐水解酶基因 并研究它们对微生物组组成和宿主代谢的相应影响， 和2）在肺炎克雷伯氏菌疾病模型中检测多药耐药基因。总体而言，我们 研究将推进强大的新合成生物学工具，可以广泛和灵活地应用于任何 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.