A high-performance and versatile technology for precision microbiome engineering

用于精密微生物组工程的高性能、多功能技术

• 批准号: 10278809
• 负责人: Samuel Henry Sternberg
• 金额: $ 68.85万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10278809
• 关键词: Address; Algorithm Design; Area; Automobile Driving; Bacteria; Bacterial Genes; Bacterial Genome; Basic Science; Biological; Biological Models; CRISPR/Cas technology; Candidate Disease Gene; Catalogs; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Communities; Complex; Coupled; Custom; DNA; DNA Double Strand Break; Data; Data Pooling; Data Set; Directed Molecular Evolution; Disease; Disease model; Drug resistance; Elements; Engineering; Environment; Escherichia coli; Exhibits; Foundations; Future; Gastrointestinal tract structure; Gene Silencing; Genes; Genetic Engineering; Genome; Genome engineering; Gnotobiotic; Guide RNA; Health behavior; Home; Horizontal Gene Transfer; Human; Human Engineering; Hydrolase; Hyperactivity; Immunity; In Situ; In Vitro; Industrialization; Insertional Mutagenesis; Integrase; Klebsiella oxytoca; Klebsiella pneumoniae; Knock-out; Laboratories; Mammalian Cell; Metabolism; Metagenomics; Methods; Microbe; Modification; Multidrug Resistance Gene; Mus; Mutation; Names; Nervous System Physiology; Neuraxis; Nucleotides; Orthologous Gene; Outcome; Pathway interactions; Performance; Physiological; Plasmids; Play; Pseudomonas putida; RNA library; Regulatory Element; Research; Role; Scientist; Specificity; System; Technology; Therapeutic; Time; Transposase; Variant; Vibrio cholerae; Vision; Work; bacterial community; base; bile acid metabolism; bile salts; carbapenemase; data integration; design; dysbiosis; exhaustion; experimental study; flexibility; gain of function; genetic approach; genetic manipulation; genetic payload; genome-wide; global health; gut microbiome; homologous recombination; host microbiome; in vivo; innovation; insight; interest; loss of function; metabolic engineering; metagenome; microbial community; microbiome; microbiome composition; microbiota; microorganism; mouse model; next generation; novel therapeutic intervention; novel therapeutics; pathogen; pathogenic bacteria; prevent; programs; repaired; resistance gene; reverse genetics; screening; site-specific integration; synthetic biology; tool; transmission process; vector

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.

项目概要 哺乳动物胃肠道是复杂多样的微生物的家园，这些微生物 在新陈代谢、宿主免疫和中枢神经系统功能中发挥着至关重要的作用。尽管不断增长 认识到平衡的微生物组对人类健康和行为的重要性，以及广泛的 生态失调可能导致的疾病，我们研究和修改体内复杂微生物群落的能力 仍然受到严重限制。测序工作可以详尽地记录细菌多样性和丰度，但是 仅提供观察信息；对小鼠进行的限生研究可以严格控制定植，但是 无法代表自然的宿主-微生物组相互作用；基因工程可以用来操纵 特定微生物中的特定基因或途径，但不在天然环境中。为了解决这些 缺点，我们建议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 生成精确 DNA 插入的方法- 转座子系统（整合技术），以及在肠道内调动遗传有效负载的方法 使用广泛宿主范围的接合载体（MAGIC 技术）。通过组合和扩展这些工具，我们 将开发可编程的、自动驱动的元件，在保留精致的核苷酸的同时广泛传播 目标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来证实我们提案的基础并证明 可行性。在最近的一项合作中，我们开发了用于千碱基级细菌基因组的 INTEGRATE 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设计，以及 在多种临床和工业相关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过度活跃的 INTEGRATE 变体，并开发 综合指导RNA设计算法，结合了经验脱靶数据和大型宏基因组 装配信息。在目标2中，我们将MAGIC与INTEGRATE相结合，以实现移动传输和 在复杂的体外群落以及小鼠模型中进行有针对性的整合。最后，在目标 3 中，我们将 应用我们的工具进行体内功能获得和功能丧失研究：1) 传递胆汁盐水解酶基因 在小鼠肠道中并研究它们对微生物组组成和宿主代谢的相应影响， 2) 灭活肺炎克雷伯菌疾病模型中的多药耐药基因。总的来说，我们的 研究将推进强大的新合成生物学工具，这些工具可以广泛、灵活地应用于任何领域 感兴趣的复杂细菌群落，用于基础研究和最终的治疗应用。