Efficient and Precise Genome Editing in Shewanella with Recombineering and CRISPR/Cas9-mediated Counter-selection

Abstract

Shewanella are invaluable hosts for the discovery and engineering of pathways important for bioremediation of toxic and radioactive metals, to create microbial fuel cells and for understanding extracellular electron transfer. However, studies on this species have suffered from a lack of effective genetic tools for precise and high throughput genome manipulation. Previously, the only reliable method used for introducing DNA into Shewanella spp. at high efficiency was bacterial conjugation, enabling transposon mutagenesis and targeted knockouts using suicide vectors for gene disruptions. In this dissertation, I describe development of simple and efficient genome editing tools for precise and site-directed mutagenesis of Shewanella. First, In Chapter I, I review recent advances in synthetic biology that accelerate the study and engineering of bacterial phenotypes. In chapter II, I show the development of a robust and simple electroporation method in S. oneidensis that allows an efficiency of up to ~108 transformants/µg DNA and which is adaptable to other strains. Using this method for DNA transfer, in chapter III, I characterize a new phage recombinase, W3 Beta from Shewanella sp. W3-18-1 and show its utility for in vivo genome engineering (recombineering) using linear single-stranded DNA oligonucleotides. In my experiments the W3 Beta recombinase gives an efficiency of ~5% recombinants among total viable cells. In addition, I show the functionality of this new system in S. amazonensis, a strain with few genetic studies but of interest given its higher temperature range for growth and wide range of carbon sources utilized. In chapter IV, I demonstrate use of the CRISPR/Cas9 system as a counter-selection to isolate recombinants. When coupled to recombineering, this counter-selection results in an extremely high efficiency of >90% among total surviving cells, regardless of the gene or strain modified. This efficiency allows isolation of several different types of mutations made with recombineering, and even allows identification of rare recombinants that form independently of W3 Beta expression. This is the first effective and simple strategy for recombination with markerless mutations in Shewanella. With synthesized single-stranded DNA as substrates for homologous recombination and CRISPR/Cas9 as a counter-selection, this new system provides a rapid, scalable, versatile and scarless tool that will accelerate progress in Shewanella genomic engineering. Finally, I conclude in Chapter V with an overview of the challenges and future directions of the technologies demonstrated here, discussing possible advancements that could further enhance the study of Shewanella.