Type I-F CRISPR-PAIR platform for multi-mode regulation to boost extracellular electron transfer in Shewanella oneidensis

Bio-electrochemical systems are based on extracellular electron transfer (EET), whose efficiency relates to the expression level of numerous genes. However, the lack of multi-functional tools for gene activation and repression hampers the enhancement of EET in electroactive microorganisms (EAMs). We thus develop a type I-F CRISPR/PaeCascade-RpoD-mediated activation and inhibition regulation (CRISPR-PAIR) platform in the model EAM, Shewanella oneidensis MR-1. Gene activation is achieved (3.8-fold) through fusing activator RpoD (σ(70)) to Cas7 when targeting the prioritized loci upstream of the transcription start site. Gene inhibition almost has no position preference when targeting the open reading frame, which makes the design of crRNAs easy and flexible. Then CRISPR-PAIR platform is applied to up-/down-regulate the expression of six endogenous genes, resulting in the improved EET efficiency. Moreover, simultaneous gene activation and inhibition are achieved in S. oneidensis MR-1. CRISPR-PAIR platform offers a programmable methodology for dual regulation, facilitating in-depth EET studies in Shewanella spp.