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

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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