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.