Development of a Conjugation-Based Genome Editing Tool in Shewanella Oneidensis MR-1 Using CRISPR/Cas9 and Recombineering

Shewanella oneidensis MR-1 is a facultative anaerobic bacterium capable of respiring a vast number of electron acceptors including insoluble toxic metals, radionuclides, and carbon electrodes. The metabolic pathways and mechanisms by which extracellular electron transfer occurs in MR-1, however, are still poorly understood. Considering the potential application of this organism towards bioremediation of contaminated aquatic and subsurface environments as well as microbial fuel cells, it is important to have efficient genome-editing tools that allow us to easily engineer this organism for better overall understanding of its biological capabilities. While MR-1 is amenable to targeted genomic mutagenesis techniques, current technologies are laborious, time-consuming, and must be conducted individually rather than collectively.

Recently, a technology utilizing a high-throughput approach coupling massive oligomer synthesis to CRISPR/Cas9 and λ-Red systems, named CREATE (CRISPR-enabled trackable genome engineering) has been demonstrated to be a powerful tool for highly efficient and precise targeted genomic editing in Escherichia coli. Introduction of CREATE into E. coli, however, requires the use of electroporation to deliver the system into the cell. While Shewanella and other environmentally relevant microbes are recalcitrant to electroporation, conjugation is a commonly used technique allowing for plasmid transfer and uptake.

Here, we show our progress towards the development of a conjugation-based CREATE system for use in MR-1. Conjugation-based CREATE utilizes the Streptococcus pyogenes type II CRISPR/Cas9 system as well as phage recombineering as previously demonstrated in E. coli. We have optimized existing MR-1 conjugation protocols for the purpose of introducing a CREATE library and provide evidence of efficient CRISPR/Cas9 function in MR-1 as a selection. Additionally, we are developing a minimal inducible system consisting of Cas9 and recombineering proteins in one plasmid for ease portability and adaptability across microbial species.

Our work primarily aims to expand the metabolic engineering toolbox of the environmentally relevant organism MR-1, allowing for expansion of bioremediation technologies. Additionally, this developed conjugation-based CREATE system can be broadly applied to other microbes that are intractable via electroporation and non-recombinogenic, while simultaneously overcoming the dependence upon suicide vectors and/or random mutagenesis approaches.