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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要哺乳动物胃肠道是复杂而多样的微生物的家园在代谢，宿主免疫和中枢神经系统功能中发挥关键作用。尽管增长了对平衡微生物组对人类健康和行为的重要性以及广泛范围营养不良，我们在体内研究和修改复杂微生物群落的能力可能导致的疾病仍然受到严格限制。测序工作可以详尽地对细菌的多样性和丰度分类，但仅提供观察信息；小鼠的gnotobiotic研究可以严格控制定殖，但无法代表自然的宿主 - 微生物组相互作用；基因工程可用于操纵精选微生物中的特定基因或途径，但不在天然环境中。解决这些缺点，我们建议开发一种创新的平台技术，用于精确微生物组工程这将首次在体内实现基因和物种特异性的编辑。我们的方法围绕两个我们的实验室中最近取得的突破：一种使用CRISPR-生成精确DNA插入的方法转座子系统（集成技术）和一种动员肠内基因有效载荷的方法使用宽大的范围偶联向量（魔术技术）。通过结合和扩展这些工具，我们将开发可编程的，自动驾驶的元素，这些元素在保留精美的核苷酸 - 靶基因组的水平特异性。我们的初步数据提供了有力的证据来证实我们的提案的基础，并证明了可行性。在最近的一项合作工作中，我们开发了集成kilobase尺度细菌基因组通过系统地评估一系列指南RNA和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AIM 1中，我们将识别自主和增殖功能的多活性整合变体，并开发一个全面的指南RNA设计算法，该算法包含经验脱离目标数据和大型元基因组组装信息。在AIM 2中，我们将结合魔术与集成以实现移动传输和在复杂的体外社区以及小鼠模型中有针对性的整合。最后，在AIM 3中，我们将将我们的工具应用于体内功能丧失和功能丧失研究：1）输送胆汁盐水解酶基因在鼠类肠道中，研究它们对微生物组组成和宿主代谢的相应影响， 2）在克雷伯氏菌肺炎模型中灭活多药抗性基因。总体而言，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，灵活地应用于任何基础研究和最终治疗应用的复杂细菌社区。