CRISPR-Cas9 gene drives to fight antimicrobial resistance

抗菌药物耐药性（AMR）是我们这个时代最大的威胁之一，预计到2050年，每年将导致1000万人死亡，总成本达100万亿美元。最近，一种革命性的技术被开发出来，称为CRISPR-Cas9，它可以用来从微生物群落中根除AMR。不过，这项技术只在实验室条件下进行了测试，还没有准备好在真实的世界中使用。我提出的研究将通过首次测试CRISPR-Cas9是否可以从猪肠道分离的复杂微生物群落中根除AMR，将这项技术的发展提升到一个新的水平。猪肠道微生物群落是一个高度相关的研究系统，因为数十年来动物工业中持续过度使用抗生素以促进动物生长和限制疾病，导致动物肠道中的AMR水平非常高，对人类和动物健康造成严重风险。由此产生的AMR进入环境是由于施用动物粪便用于土壤施肥，这被认为也有助于AMR在人类群体中的出现。在我的研究中，我将首先开发遗传工具，这将有助于通过微生物群落传播CRISPR-Cas9。我将监测CRISPR-Cas9的传播和相关的AMR下降。至关重要的是，我还将仔细监测其后果：往往在没有考虑生态和进化风险的情况下应用有希望的病虫害控制策略，有时会对生态系统的功能产生破坏性影响，并导致抗药性的出现。我预见了与实施这项新技术相关的两个科学挑战。首先，去除AMR基因可能会导致功能冗余的其他AMR基因的不必要或意外增加。第二，CRISPR-Cas9靶向的AMR基因可能会进化为对靶向产生抗性，这可能会破坏该技术。为了应对这些挑战，我将监测去除AMR基因后猪肠道微生物群落的变化，以及CRISPR-Cas9的抗性是否会进化。这将涉及在长期（12个月）实验中将CRISPR-Cas9引入猪肠群落，并在短期实验（2个月）中将CRISPR-Cas9引入更简单的衍生群落。对于这两种类型的实验，我将研究发生的生态和进化变化。这些实验将在封闭的实验室环境中进行，以避免CRISPR-Cas9意外释放到环境中。至关重要的是，这些实验的数据将输入一个数学模型，以生成一个理论框架，使我能够预测其他微生物群落的生态和进化后果。这项研究将在埃克塞特大学进行，埃克塞特大学是研究微生物群落生态学、AMR和CRISPR-Cas9进化的卓越中心之一。我将被嵌入在一个高度协作的研究环境中，与相关学科的主要专家共享实验室空间。BBSRC未来领袖奖学金将使我能够开始建立自己的研究小组，并成为一个完全独立的研究人员。我将利用这个奖学金作为跳板，以吸引更多的资金来扩大我的研究计划的范围，并追求与这个项目相吻合的新的研究路线。AMR现在被联合国大会认为是我们社会面临的最紧迫的问题之一，也是BBSRC研究资金的关键战略优先事项。发现新的策略来最大限度地减少人类和动物的AMR负担将是真正具有开创性的。测试CRISPR-Cas9是否可以从复杂的微生物群落中消除AMR，并了解AMR去除的后果，将是推动这一突破的重要一步。

Antimicrobial resistance (AMR) is one of the greatest threats of our time causing a predicted 10 million human deaths per year by 2050 with a total cost of $100 trillion. Recently, a revolutionary technology has been developed, known as CRISPR-Cas9, which can be used to eradicate AMR from microbial communities. However, this technology has only been tested under laboratory conditions, and is not yet ready for use in the real world. My proposed research will take the development of this technology to the next level by testing for the first time whether CRISPR-Cas9 can eradicate AMR from a complex microbial community, isolated from the pig gut. The pig gut microbial community is a highly relevant study system, since decades of consistent overuse of antibiotics in animal industries to boost animal growth and limit disease has selected for very high levels of AMR in the animal gut, causing severe risks for human and animal health. The resulting AMR enters the environment due to the application of animal manure for soil fertilization, which is thought to also contribute to AMR emergence in human populations.In my research I will first develop genetic tools that will help to spread CRISPR-Cas9 through a microbial community. I will monitor the spread of CRISPR-Cas9 and the associated AMR decline. Crucially, I will also carefully monitor the consequences: too often have promising pest and disease control strategies been applied without considering the ecological and evolutionary risks, sometimes leaving a devastating impact on ecosystem functioning and causing resistance to emerge. I foresee two scientific challenges associated with implementing this new technology. First, removal of an AMR gene may cause unwanted or unanticipated increases in other AMR genes that are functionally redundant. Second, the AMR gene that is targeted by CRISPR-Cas9 may evolve to become resistant to targeting, which could undermine the technology.To address these challenges, I will monitor the changes in a pig gut microbial community caused by the removal of AMR genes, and whether resistance to CRISPR-Cas9 will evolve. This will involve introducing CRISPR-Cas9 to the pig gut community in a long-term (12 months) experiment, and in parallel introducing CRISPR-Cas9 to simpler derived communities in short-term experiments (2 months). For both types of experiments, I will then examine the ecological and evolutionary changes that occur. These experiments will take place in a contained laboratory environment, to avoid unintended release of the CRISPR-Cas9 into the environment. Crucially, the data from these experiments will feed into a mathematical model to generate a theoretical framework that allows me to predict these ecological and evolutionary consequences in other microbial communities. This research will be carried out at the University of Exeter, one of the centers of excellence for studying microbial community ecology, evolution of AMR and CRISPR-Cas9. I will be embedded in a highly collaborative research environment, sharing laboratory space with key experts from the relevant disciplines. The BBSRC Future Leader Fellowship will allow me to start building my own research group, and to become a fully independent researcher. I will use this fellowship as a springboard to attract further funding to expand the scope of my research program, and to pursue novel lines of research that dovetail from this project.AMR is now recognized by the UN General Assembly as one of the most urgent problems that our society is facing, and a key strategic priority for BBSRC research funding. Discovering new strategies to minimize the burden of AMR in both humans and animals would be truly groundbreaking. Testing whether CRISPR-Cas9 can eradicate AMR from a complex microbial community and understanding the consequences of AMR removal will be a major step forward to push such a breakthrough.