Establishing the feasibility of editing the human gut microbiome

项目总结 人类微生物群中显著的个体间基因变异与 疾病仍然有限，因为缺乏普遍的方法来准确地去除微生物基因 现场复杂的微生物群落。这样的技术不仅有助于改变微生物组领域 从描述性学科到机械性学科，但它也将立即应用于临床。目标是 这项重点技术研究和开发(PAR-19-253)的应用是建立一个 用于编程人类肠道细菌内源性CRISPR-Cas系统的通用工具包 以前所未有的精确度敲除感兴趣的基因。CRISPR-CAS是一种细菌免疫系统， 由RNA引导的核酸酶组成，保护细胞免受噬菌体和其他外来DNA的侵袭。 内源性CRISPR-CAS系统可以被编程为使用定制指南来靶向它们自己的基因组DNA 与目标基因同源的RNA(GRNA)。作为最初的原则证明，我们将针对单个内脏 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 成功的这种方法可以很容易地扩展到许多细菌物种，现在已经 与宿主生理以及疾病的易感性和治疗有关。我们将致力于以下工作 具体目标：(目标I)一个新的CRISPR-Cas系统在兰氏Eggerthella lenta中的功能验证 肠道放线菌与新陈代谢和微生物发病机制有多种联系；(AIM II)分离和 重新启动感染人体肠道细菌的噬菌体；以及(目标III)改造噬菌体以 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以删除一个基因、基因组合甚至整个肠道微生物组的模块化工具 代谢途径。重要的是，通过关注内生CRISPR-CAS系统，我们预计 我们将揭示对这些系统的多样性、监管和功能的基本见解，以及广泛的 对我们理解噬菌体、它们的细菌宿主、 以及它们共同的哺乳动物栖息地。虽然我们的早期结果为可行性提供了支持，但这种高风险、高风险的 奖励技术发展计划将对微生物群落的研究和 宿主-微生物组的相互作用，同时使基于微生物组的治疗的前景变得触手可及。

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.