CRISPRi–sRNA: Transcriptional–Translational Regulation of Extracellular Electron Transfer in Shewanella oneidensis

Abstract

Extracellular electron transfer (EET) in Shewanella oneidensis MR-1, which is one of the most well-studied exoelectrogens, underlies many microbial electrocatalysis processes, including microbial fuel cells, microbial electrolysis cells, and microbial electrosynthesis. However, regulating the efficiency of EET remains challenging due to the lack of efficient genome regulation tools that regulate gene expression levels in S. oneidensis. Here, we systematically established a transcriptional regulation technology, i.e., clustered regularly interspaced short palindromic repeats interference (CRISPRi), in S. oneidensis MR-1 using green fluorescent protein (GFP) as a reporter. We used this CRISPRi technology to repress the expression levels of target genes, individually and in combination, in the EET pathways (e.g., the MtrCAB pathway and genes affecting the formation of electroactive biofilms in S. oneidensis), which in turn enabled the efficient regulation of EET efficiency. We then established a translational regulation technology, i.e., Hfq-dependent small regulatory RNA (sRNA), in S. oneidensis by repressing the GFP reporter and mtrA, which is a critical gene in the EET pathways in S. oneidensis. To achieve coordinated transcriptional and translational regulation at the genomic level, the CRISPRi and Hfq-dependent sRNA systems were incorporated into a single plasmid harbored in a recombinant S. oneidensis strain, which enabled an even higher efficiency of mtrA gene repression in the EET pathways than that achieved by the CRISPRi and Hfq-dependent sRNA system alone, as exhibited by the reduced electricity output. Overall, we developed a combined CRISPRi-sRNA method that enabled the synergistic transcriptional and translational regulation of target genes in S. oneidensis. This technology involving CRISPRi-sRNA transcriptional-translational regulation of gene expression at the genomic level could be applied to other microorganisms.

CRISPR/dCas9‐Mediated Multiplex Gene Repression in Streptomyces

“…As expected, only slight downregulation of actII4 (≈70% of the control) was observed in the strain with pSET‐dCas9‐ actII4‐ Out‐S0 (targeting outside of the gene) at 48 h, but the repression extent was much lower than the strains with sgRNAs targeting the coding region. The results presented above clearly demonstrated that our CRISPRi system could be used for efficient gene repression in S. coelicolor , and further confirmed that sgRNAs targeting the coding regions of target genes on the NT strand are very effective for gene repression as reported in other bacteria …”

“…Many studies showed that sgRNAs targeting the promoter regions are very effective for gene repression irrespective of whether they are targeted to the template (T) or nontemplate strand (NT); however, when sgRNAs are targeted to the coding region, effective repressive effects are only observed on the NT strand . Considering that the promoter regions of most genes in Streptomyces are unknown, in this study, all sgRNAs were designed to target the NT strand of gene coding regions, which were conducted by the software CRISPRy to minimize off‐target effects .…”

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

“…We found that Cas9 must be tightly regulated since high Cas9 expression leads to a loss in cell viability, as shown in other bacteria (Cobb et al 2014;Cui et al 2018;Huang et al 2015;Wang et al 2018). CRISPRi was also recently applied in S. oneidensis for transient control of gene expression with the defective dCas9 protein (Cao et al 2017), however, CRISPRi is not a tool for site-directed mutagenesis (Table 4.2). (Saltikov & Newman 2003;Blomfietd et al 1991;Corts et al 2019), b CFU/µg DNA in E. coli MG1655 (Gray et al 2015;Yang et al 2014), c Measured as repression of GFP expression (Peters et al 2016;Cao et al 2017), d Determined as the number of recombinants among the total surviving cells (this work).…”

Genome-wide targets identification by CRISPRi-Omics for high-titer production of free fatty acids inEscherichia coli

“…Then, we examined the transcriptional repression capability of CRISPRi on gene expression by co-expressing a specific sgRNA. sgRNA- gfp was designed to target on the non-template strand of GFP at a position ∼38 nucleotides downstream of the start codon to expect a high repression efficiency 25 . The plasmid Sg-Hgfp ( Supplementary Fig.…”