An experimental evolution approach to studying horizontal transfer of antiviral defences between bacterial species

CRISPR-Cas类似于细菌免疫系统。它允许细菌“记住”外来入侵的DNA（例如来自病毒），最终将它们作为破坏目标。CRISPR-Cas系统特异性识别某些序列的能力导致了该系统作为基因编辑工具的发展，从而彻底改变了分子生物学。尽管如此，相对较少的研究集中在细菌之间转移CRISPR-Cas系统的长期稳定性和可行性上。研究不同物种之间CRISPR-Cas基因序列相似性的研究表明，在自然界中，细菌可能通过水平基因转移（HGT）在物种之间交换这些基因。然而，获得新的CRISPR-Cas系统的进化效应还有待实验研究。目的：本项目的首要目标是研究CRISPR-Cas转录调控在转移到幼稚宿主时如何进化。此外，该项目旨在确定有利于这种水平基因转移事件的选择压力，并使表达获得性CRISPR-Cas系统的细菌在种群中占据主导地位。这项研究将为未来在幼稚细菌物种中生产和维护功能性合成CRISPR-Cas系统提供信息。所产生的结果将模拟水平基因转移事件的潜在的预扩增和进化后果，所述水平基因转移事件被提出为对CRISPR-Cas systems.Objective 1的进化历史做出了贡献：了解在什么环境条件下CRISPR-Cas系统对在缺乏CRISPR-Cas的群体内竞争的细菌产生选择性优势。这将使用铜绿假单胞菌作为已经具有天然CRISPR-Cas系统的模型系统。目的2：将铜绿假单胞菌的I-F型CRISPR-Cas系统的功能性合成版本转移到幼稚宿主-荧光假单胞菌。目的3：了解什么样的选择压力会有利于获得这种CRISPR-Cas系统的荧光假单胞菌，并使其在野生型荧光假单胞菌群体中达到固定。目标4：描述允许荧光假单胞菌维持获得性CRISPR-Cas系统表达的调控变化和补偿突变。以及这些突变如何影响赋予宿主选择优势的新系统

CRISPR-Cas is akin to a bacterial immune system. It allows bacteria to 'remember' foreign invading DNA (e.g. from viruses), ultimately targeting them for destruction. The ability of CRISPR-Cas systems to specifically recognise certain sequences has led to the development of this system as a tool for gene editing, revolutionising molecular biology. Despite this, relatively little research has centred on the long-term stability and viability of transferring CRISPR-Cas systems between bacteria.Studies that have examined the sequence similarities of CRISPR-Cas genes between different species have suggested that in nature bacteria may have exchanged these genes between species by horizontal gene transfer (HGT). However, the evolutionary effects of gaining new CRISPR-Cas systems are yet to be investigated experimentally.Aim: The overarching aim of this project is to investigate how CRISPR-Cas transcription regulation evolves when transferred to a naïve host. In addition, this project aims to identify the selective pressures that favour such horizontal gene transfer events and enable bacteria expressing an acquired CRISPR-Cas system to rise to dominance in a population. This research will inform the future production and maintenance of functional synthetic CRISPR-Cas systems in naïve bacterial species. The results generated will model the potential pre-requisites and evolutionary consequences of the horizontal gene transfer events that are proposed to have contributed to the evolutionary history of CRISPR-Cas systems.Objective 1: To understand under what environmental conditions CRISPR-Cas systems produce a selective advantage to bacteria competing within a population otherwise lacking CRISPR-Cas. This will use P. aeruginosa as a model system that already possess a native CRISPR-Cas system.Objective 2: To transfer a functional synthetic version of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa to a naïve host - Pseudomonas fluorescens. The consequences of this event on the fitness of the host will then be followed.Objective 3: To understand what selection pressures will favour the P. fluorescens that have acquired this CRISPR-Cas system and allow them to reach fixation in a population of wild type P. fluorescens. Objective 4: To describe the regulatory changes and compensatory mutations that allow P. fluorescens to maintain expression of an acquired CRISPR-Cas system. As well as how these mutations influence the new system conferring a selective advantage to the host