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，特别是胃肠道中的本地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，但我们目前缺乏强有力的、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，甚至通过干扰特定物种或基因并观察其对更广泛社区的影响来调查它们所起的作用。该建议寻求利用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，通过工程改造降解噬菌体基因的细胞能够在噬菌体存在的情况下胜过易感细菌，同时，通过工程改造降解致病基因的可移动遗传元件可以在细菌种群传播时对其进行免疫。由于噬菌体和可移动元件可以高度特异性，这些创新可以控制目标基因和物种的丰度，而不会影响混合培养中不相关的微生物。在K99阶段，我将学习与动物模型和致病菌一起工作，以便在体内测试这些方法的有效性。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的有害微生物，2)利用可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。在获得了新的技能并探索了这些新技术的能力之后，我将在R00阶段应用它们：3)通过消除带有可移动Cas9元件的毒素编码基因，并通过在肠道中定植保护性菌株，来治疗和预防志贺毒素介导的肠道疾病；4)利用稳定的微生物种群，在炎症上皮附近分泌一致水平的抗炎分子，来减少小鼠的炎症。我希望这些创新的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.