Establishing the feasibility of editing the human gut microbiome

建立编辑人类肠道微生物组的可行性

• 批准号: 10222578
• 负责人: Peter James Turnbaugh
• 金额: $ 40.38万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10222578
• 关键词: Actinobacteria class; Address; BCAR1 gene; Bacteria; Bacterial Genes; Bacterial Genome; Bacteriophage Genetics; Bacteriophages; Biochemical; Biological; Biomedical Research; Cardiac; Cell Death; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; Custom; DNA; DNA delivery; Development Plans; Devices; Digoxin; Discipline; Disease; Dissection; Engineering; Exposure to; Family; Foundations; Gastrointestinal tract structure; Gene Combinations; Gene Deletion; Gene Targeting; Genes; Genetic; Genome; Genomic DNA; Genotype; Gnotobiotic; Goals; Growth; Guide RNA; Habitats; Health; Human; Human Microbiome; Immune system; In Situ; In Vitro; Individual; Knowledge; Link; Medicine; Metabolic Pathway; Metabolism; Methods; Modeling; Molecular Genetics; Mus; Pathogenesis; Pharmaceutical Preparations; Physiology; Planets; Plants; Plasmids; Predisposition; Prevalence; Process; Reagent; Regulation; Research; System; Technology; Testing; Therapeutic; Toxin; Validation; Variant; bacterial genetics; base; clinical application; fitness; gene discovery; gut bacteria; gut microbiome; high reward; high risk; host microbiome; human disease; in vivo evaluation; insight; interest; knockout gene; microbial; microbial community; microbial host; microbiome; microbiome research; mutant; novel; nuclease; programs; protein complex; technology development; technology research and development; tool

PROJECT SUMMARY Definitive links between the remarkable inter-individual genotypic variation in the human microbiome and disease are still limited due to the lack of generalizable methods to precisely remove microbial genes from complex microbial communities in situ. Not only would such a technology help transform the microbiome field from a descriptive to a mechanistic discipline, but it would also have immediate clinical applications. The goal of this Focused Technology Research and Development (PAR-19-253) application is to establish a generalizable toolkit to program the endogenous CRISPR-Cas systems in human gut bacteria to knockout genes of interest with unprecedented precision. CRISPR-Cas is a bacterial immune system, composed of RNA-guided nucleases, that protect the cell against bacteriophage and other foreign DNA. Endogenous CRISPR-Cas systems can be programmed to target their own genomic DNA using custom guide RNAs (gRNAs) homologous to a gene of interest. As an initial proof-of-principle we will target a single gut bacterial gene that we discovered is responsible for the inactivation of the cardiac drug digoxin, but if successful this approach could be readily extended to the numerous bacterial species that have now been implicated in host physiology and the predisposition to and treatment of disease. We will pursue the following Specific Aims: (Aim I) the functional validation of a novel CRISPR-Cas system in Eggerthella lenta, a prevalent gut Actinobacterium with multiple links to metabolism and microbial pathogenesis; (Aim II) the isolation and rebooting of bacteriophages that infect human gut bacteria; and (Aim III) the engineering of bacteriophage to program an endogenous gut bacterial CRISPR-Cas system. Our long-term goal is to establish the first modular tools for engineering the gut microbiome to delete one gene, combinations of genes, or even entire metabolic pathways. Importantly, by focusing on endogenous CRISPR-Cas systems, we anticipate that along the way we will uncover basic insights into the diversity, regulation, and function of these systems, with broad implications for our understanding of the complex interactions between bacteriophages, their bacterial hosts, and their shared mammalian habitat. While our early results provide support for feasibility, this high-risk, high- reward technology development plan would have broad implications for the study of microbial communities and host-microbiome interactions while bringing the promise of microbiome-based therapeutics within reach.

项目摘要 人类微生物组中显着的个体基因型变异与 由于缺乏可准确去除微生物基因从 原位复杂的微生物群落。这样的技术不仅有助于改变微生物组领域 从描述性到机械学科，但也将立即使用临床应用。目标 在这项集中的技术研发中（PAR-19-253）的应用是为了建立一个 可概括的工具包，以对人肠道细菌中的内源性CRIS-CAS系统进行编程 具有前所未有的精度的敲除基因。 CRISPR-CAS是一种细菌免疫系统， 由RNA引导的核酸酶组成，可保护细胞免受噬菌体和其他异物DNA的影响。 可以对内源性CRISPR-CAS系统进行编程，以使用自定义指南来针对自己的基因组DNA RNA（grnas）与感兴趣的基因同源。作为初始证明，我们将以一个肠道为目标 我们发现的细菌基因是导致心脏药物地高辛失活的原因，但如果 成功的这种方法很容易扩展到现在已经存在的众多细菌物种 与宿主生理学以及对疾病的倾向和治疗有关。我们将追求以下 具体目的：（目标i）在Eggerthella Lenta中的新型CRISPR-CAS系统的功能验证，这是一个普遍的 肠道细菌具有多种与代谢和微生物发病机理的联系； （AIM II）隔离和 重新启动感染人类肠道细菌的噬菌体； （AIM III）噬菌体的工程 编程内源性肠道细菌CRISPR-CAS系统。我们的长期目标是建立第一个 用于设计肠道微生物组的模块化工具，以删除一个基因，基因的组合，甚至整个基因的组合 代谢途径。重要的是，通过关注内源性CRISPR-CAS系统，我们预计 我们将发现对这些系统的多样性，调节和功能的基本见解，并具有广泛的方式 我们对噬菌体之间复杂相互作用的意义，它们的细菌宿主， 以及他们共同的哺乳动物栖息地。尽管我们的早期结果为可行性提供了支持，但这种高风险，高 奖励技术发展计划将对微生物社区的研究和 宿主 - 微生物组相互作用，同时带来了基于微生物组的疗法的希望。