Efficient and Precise Genome Editing in Shewanella with Recombineering and CRISPR/Cas9-Mediated Counter-Selection

Dissimilatory metal-reducing bacteria, particularly those from the genus Shewanella, are of importance for bioremediation of metal contaminated sites and sustainable energy production. However, studies on this species have suffered from a lack of effective genetic tools for precise and high throughput genome manipulation. Here we report the development of a highly efficient system based on single-stranded DNA oligonucleotide recombineering coupled with CRISPR/Cas9-mediated counter-selection. Our system uses two plasmids: a sgRNA targeting vector and an editing vector, the latter harboring both Cas9 and the phage recombinase W3 Beta. Following the experimental analysis of Cas9 activity, we demonstrate the ability of this system to efficiently and precisely engineer different Shewanella strains with an average efficiency of >90% among total transformed cells, compared to ≤5% by recombineering alone, and regardless of the gene modified. We also show that different genetic changes can be introduced: mismatches, deletions, and small insertions. Surprisingly, we found that use of CRISPR/Cas9 alone allows selection of recombinase-independent S. oneidensis mutations, albeit at lower efficiency and frequency. With synthesized single-stranded DNA as substrates for homologous recombination and Cas9 as a counter-selectable marker, this new system provides a rapid, scalable, versatile, and scarless tool that will accelerate progress in Shewanella genomic engineering.