Sculpting the Enteric Microbiota with CRISPR-Cas Systems

描述（由申请人提供）：人类健康与生活在皮肤、口腔，特别是胃肠道中的固有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社区的组成提供了一个窗口，但我们目前缺乏强有力和有针对性的干预方法。除了少数例外，这种技术的缺乏使我们无法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的破坏，甚至通过干扰它们并观察对更广泛社区的影响来调查特定物种或基因所扮演的角色。 该提案旨在利用CRISPR-Cas获得性免疫系统来控制微生物群中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，经过基因工程改造，能够降解噬菌体基因的细胞能够在有细菌存在的情况下战胜易感细菌，而且经过基因工程改造，能够降解致病基因的移动的遗传元件能够在细菌种群传播时免疫它们。由于可移动和移动的元素可以是高度特异性的，这些创新可以控制目标基因和物种的丰度，而不会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，以测试这些方法在体内的有效性。具体来说，我将寻求1）在小鼠肠道中用保护性菌株稳定地取代潜在的有害微生物，2）使用移动的遗传元件将其有效载荷复制到宿主基因组中，通过天然微生物群传播Cas9介导的免疫力。在获得了新技能并探索了这些新技术的能力之后，我将在R 00阶段将其应用于3）通过用移动的Cas9元件消除毒素编码基因并通过用保护性菌株定殖肠道来治疗和预防滋贺毒素介导的肠道疾病，和4）使用稳定的微生物群体减少小鼠中的炎症，所述微生物群体被工程化以分泌一致水平的抗-炎症分子紧密靠近发炎的上皮。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。

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.