CRISPR/dCas9-RpoD-Mediated Simultaneous Transcriptional Activation and Repression in Shewanella oneidensis MR-1.

Extracellular electron transfer (EET) of electroactive microorganisms (EAMs) is the dominating factor for versatile applications of bio-electrochemical systems. Shewanella oneidensis MR-1 is one of the model EAMs for the study of EET, which is associated with a variety of cellular activities. However, due to the lack of a transcriptional activation tool, regulation of multiple genes is labor-intensive and time-consuming, which hampers the advancement of improving the EET efficiency in S. oneidensis. In this study, we developed an easily operated and multifunctional regulatory tool, that is, a simultaneous clustered regularly interspaced short palindromic repeats (CRISPR)-mediated transcriptional activation (CRISPRa) and interference (CRISPRi) system, for application in S. oneidensis. First, a large number of activators were screened, and RpoD (σ70) was determined as the optimal activator. Second, the effective activation range was identified to be 190-216 base upstream of the transcriptional start site. Third, up- and downregulation was achieved in concert by two orthogonal single guide RNAs targeting different positions. The activation of the cell division gene (minCDE) and repression of the cytotoxic gene (SO_3166) were concurrently implemented, increasing the power density by 2.5-fold and enhancing the degradation rate of azo dyes by 2.9-fold. The simultaneous CRISPRa and CRISPRi system enables simultaneous multiplex genetic regulation, offering the potential to further advance studies of the EET mechanism and application in S. oneidensis.

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

Shewanella oneidensis MR-1, as a model electroactive microorganism (EAM) for extracellular electron transfer (EET) study, plays a key role in advancing practical applications of bio-electrochemical systems (BES). Efficient genome-level manipulation tools are vital to promote EET efficiency; thus, a powerful and rapid base editing toolbox in S. oneidensis MR-1 is developed. Firstly a CRISPR/dCas9-AID base editor that shows a relatively narrow editing window restricted to the "-20 to -16" range upstream of the protospacer adjacent motif (PAM) is constructed. Cas9 is also confined by its native PAM requirement, NGG. Then to expand the editable scope, the sgRNA and the Cas-protein to broaden the editing window to "-22 to -9" upstream of the PAM are engineered, and the PAM field to NNN is opened up. Consequently, the coverage of the editable gene is expanded from 89% to nearly 100% in S. oneidensis MR-1. This whole genome-scale cytidine deaminase-based base editing toolbox (WGcBE) is applied to regulate the cell length and the biofilm morphology, which enhances the EET efficiency by 6.7-fold. WGcBE enables an efficient deactivation of genes with full genome coverage, which would contribute to the in-depth and multi-faceted EET study in Shewanella.