Sculpting the Enteric Microbiota with CRISPR-Cas Systems

使用 CRISPR-Cas 系统塑造肠道微生物群

• 批准号: 8751445
• 负责人: Kevin Esvelt
• 金额: $ 9万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8751445
• 关键词: Acute; Address; Affect; Animal Model; Anti-Inflammatory Agents; Anti-inflammatory; Antibiotics; Antibodies; Bacteria; Bacterial Genes; Bacteriophages; Cells; Characteristics; Chromosomes; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; Development; Diabetes Mellitus; Diet; Digestive System Disorders; Disease; Ecology; Ecosystem; Elements; Engineering; Ensure; Enteral; Enterobacteriaceae; Epithelium; Equilibrium; Escherichia coli; Escherichia coli EHEC; Escherichia coli Infections; Future; Gastrointestinal tract structure; Gene Targeting; Genes; Genome; Grant; Guide RNA; Health; Human; Immune System Diseases; Immune system; Immunity; In Situ; Indigenous; Individual; Infection; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Intervention; Knowledge; Laboratories; Lead; Learning; Life; Link; Malignant Neoplasms; Measures; Mediating; Mentors; Methods; Microbe; Mobile Genetic Elements; Modification; Mus; Mutation; Nutrition Disorders; Obesity; Oral cavity; Pathogenesis; Patients; Phase; Plasmids; Play; Population; Prevalence; Prevention; Probability; Role; Shiga Toxin; Signal Transduction; Skin; Stable Populations; Stress; Supplementation; System; Techniques; Testing; Therapeutic; Toxin; Work; base; cellular engineering; commensal microbes; community living; comparative efficacy; cytokine; deep sequencing; efficacy testing; foodborne infection; improved; in vivo; innovation; metagenomic sequencing; microbial; microbial community; microbial genome; new technology; novel therapeutics; nuclease; pathogen; pathogenic bacteria; prevent; public health relevance; skills

DESCRIPTION (provided by applicant): Human health is intrinsically linked to the health of the indigenous microbial ecosystems living in the skin, the mouth, and especially the gastrointestinal tract. While deep sequencing has provided a window into the composition of these communities, we currently lack robust and targeted methods of intervention. With few exceptions, this dearth of techniques prevents us from restoring unbalanced ecosystems to a healthy state, immunizing the microbiota against further disruption, or even investigating the roles played by particular species or genes by perturbing them and observing the effects on the wider community. This proposal seeks to harness the CRISPR-Cas acquired immune system to control the abundance of specific genes and bacteria within the microbiota for the study and in situ treatment of enteric disease. I have shown that cells engineered to degrade bacteriophage genes are able to outcompete susceptible bacteria in the presence of phages, and also that mobile genetic elements engineered to degrade pathogenic genes can immunize bacterial populations as they spread. Because phages and mobile elements can be highly specific, these innovations can control the abundance of targeted genes and species without affecting unrelated microbes in mixed cultures. During the K99 phase, I will learn to work with animal models and pathogenic bacteria in order to test the efficacy of these methods in vivo. Specifically, I will seek to 1) stably replace potentially harmful microbes with protective strainsin the mouse gut, and 2) spread Cas9-mediated immunity through the native microbiota using mobile genetic elements that copy their payload into the host genome. Having acquired new skills and explored the capabilities of these novel technologies, I will apply them during the R00 phase to 3) treat and prevent Shiga toxin-mediated enteric disease by eliminating the toxin-encoding genes with mobile Cas9 elements and by colonizing the gut with protective strains, and 4) reduce inflammation in mice using stable populations of microbes engineered to secrete consistent levels of anti-inflammatory molecules in close proximity to the inflamed epithelium. I expect these innovative approaches to illuminate the role of the microbiota in human health and establish a new basis for the in situ treatment and prevention of enteric disease.

描述（由申请人提供）：人类健康与生活在皮肤、口腔、尤其是胃肠道中的本土微生物生态系统的健康有着内在的联系。虽然深度测序为了解这些群落的组成提供了一个窗口，但我们目前缺乏稳健且有针对性的干预方法。除了少数例外，技术的缺乏阻碍了我们将不平衡的生态系统恢复到健康状态，使微生物群免受进一步破坏，甚至无法通过扰动特定物种或基因并观察对更广泛群落的影响来研究它们所发挥的作用。 该提案旨在利用 CRISPR-Cas 获得性免疫系统来控制微生物群内特定基因和细菌的丰度，用于肠道疾病的研究和原位治疗。我已经证明，经过改造以降解噬菌体基因的细胞能够在噬菌体存在的情况下击败易感细菌，而且经过改造以降解致病基因的可移动遗传元件可以在细菌群体传播时对它们产生免疫。由于噬菌体和移动元件具有高度特异性，这些创新可以控制目标基因和物种的丰度，而不影响混合培养物中不相关的微生物。 在K99阶段，我将学习使用动物模型和病原菌来测试这些方法在体内的功效。具体来说，我将寻求 1) 用小鼠肠道中的保护性菌株稳定地取代潜在有害的微生物，2) 使用可移动遗传元件将其有效负载复制到宿主基因组中，通过本地微生物群传播 Cas9 介导的免疫力。在掌握了新技能并探索了这些新技术的功能后，我将在 R00 阶段将它们应用到 3) 通过消除带有移动 Cas9 元件的毒素编码基因以及通过用保护性菌株定植肠道来治疗和预防志贺毒素介导的肠道疾病，以及 4) 使用稳定的微生物群来减少小鼠的炎症，这些微生物经过改造可分泌一致水平的抗炎分子 靠近发炎的上皮。我希望这些创新方法能够阐明微生物群在人类健康中的作用，并为肠道疾病的原位治疗和预防奠定新的基础。