Development And Application Of CRISPRi,sRNAs And CRISPRi-sRNAs In Shewanella Oneidensis MR-1

Exoelectrogens use extracellular electron transfer(EET)pathways to exchange electrons and energy with environments.Shewanella oneidensis MR-1,a well-established exoelectrogen with fully sequenced and annotated genome,is one of the most studied model microorganisms capable of EET.EET of S.oneidensis MR-1 underlies many microbial electro-catalysis processes,including microbial fuel cells,microbial electrolysis cells,and microbial electrosynthesis.Unfortunately,it remains hard to control its EET efficiency owing to the lack of efficient genome regulation tools to regulate genes expression levels in S.oneidensis MR-1.CRISPRi and sRNAs are two genome regulation technologies and have been used in a variety of microbes.CRISPRi regulates gene expression by inhibiting the transcription of the target gene,while the sRNAs technique regulates gene expression by inhibiting the translation of the mRNA transcribed by the target gene.In this study,firstly,we systematically established a transcriptional regulation technology,CRISPRi,in S.oneidensis MR-1 using green fluorescent protein(GFP)as a reporter.We then used this CRISPRi technology to repress the expression levels of target genes,individually and coherently,in the EET pathways and the genes affecting the formation of electroactive biofilms of S.oneidensis,which in turn enabled efficient regulation of EET rate.We further established a translational regulation technology,the Hfq-dependent small regulatory RNAs(sRNAs),in S.oneidensis through repressing the GFP reporter and mtrA.To achieve a coordinated transcriptional and translational genome regulation,the CRISPRi and Hfq-dependent sRNAs systems were incorporated into a single plasmid harbored in recombinant S.oneidensis,which enabled an even higher repression efficiency of mtrA gene in the EET pathways than the CRISPRi and Hfq-dependent sRNAs,respectively,as exhibited by the reduced electricity output.This CRISPRi-sRNAs synergistic genome regulation technology would be a toolbox in enabling regulation of EET and metabolisms of microorganisms.