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

Abstract

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.

Development of Whole Genome‐Scale Base Editing Toolbox to Promote Efficiency of Extracellular Electron Transfer in Shewanella oneidensis MR‐1

“…The CRISPR/dCas9‐AID can target CGA (Arg), CAG (Gln), and CAA (Gln) in the coding strand and CCA in the noncoding strand of a gene to create TGA, TAG, and TAA stop codons by converting the C to T. [ 15c ] Compared to the CRISPR/Cas9‐based gene knockout approach in S. oneidensis MR‐1, [ 11a ] which only needs to have available PAM sites on the target fragment, base editing is significantly more dependent on adjustable editing window and available PAM. It is obvious that CRISPR/dCas9‐AID based gene deactivation strategy have lower genome‐scale coverage.…”

“…Many gene expression and regulation toolbox have been developed in S. oneidensis MR‐1, including plasmid expression toolkit [ 10 ] and CRISPR (clustered regularly interspaced short palindromic repeat)‐mediated genome editing and regulation approaches. [ 11 ] A CRISPR‐mediated base editing system (pCBEso) was recently developed in S. oneidensis MR‐1, [ 12 ] in which C to T conversion in a 6‐nt editing window could be achieved by the fusion protein of nCas9 (D10A) and cytidine deaminase rAPOBEC1. Base editing could achieve gene deactivation via mutating the CAG, CAA, CGA, TGG codons into premature stop codons (TAA, TAG, and TGA).…”

Evidence for quinol oxidase activity of ImoA, a novel NapC/NirT family protein from the neutrophilic Fe(II) oxidizing bacterium Sideroxydans lithotrophicus ES-1

“…pBBR1MCS-2 was a gift from Kenneth Peterson (Addgene plasmid # 85168 ; http://n2t.net/addgene:85168 ; RRID:Addgene_85168). The resulting plasmids were then transformed into Shewanella using electroporation (56). In these constructs, the ribosome binding site (RBS) present at the pBAD2020/D-TOPO vector was also included and the reverse primer contained a stop codon to guarantee that a native protein (i.e.…”

Rapid and highly efficient genomic engineering with a novel iEditing device for programming versatile extracellular electron transfer of electroactive bacteria

“…Because of their great importance, many molecular tools have been developed to facilitate the engineering of EAB for enhanced EET output. For example, a set of expression vectors (Cao et al ., 2019) and the trimethylamine N‐oxide inducible promoter (West et al ., 2017) have been developed to facilitate the construction of gene circuits; the CRISPR‐ddAsCpf1 has been designed for gene interference (Li et al ., 2020); and the CRISPR‐based ssDNA recombination (Corts et al ., 2019) has been developed to introduce small‐scale genomic modifications. However, the importance of creating an efficient genomic engineering method for rapid construction of genome‐edited strains, which are applicable in sophisticated environmental settings, has been neglected.…”