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

Abstract

Electroactive microorganisms (EAMs) use bidirectional extracellular electron transfer (EET) pathways to exchange electrons with environments, enabling a diverse array of bio-electrochemical systems (BES). [1] These BES include microbial fuel cells (MFC) for electricity production from biomass and organic wastes, [2] electrochemical microbial biosensors for biotoxicity detection, [3] microbial desalination cells for seawater desalination, [4] microbial electrolysis cells for H 2 production, [5] and microbial electrosynthesis (MES) and photoelectrochemical biohybrid systems for the production of chemicals and fuels from CO 2 . [6] The EET efficiency is a dominating factor for the practical applications of BES. [7] Shewanella oneidensis MR-1, as a model EAM with contact-based and electron shuttle-based EET pathways, has been widely used in the EET study. [8] EET is however associated with multiple energy and materials metabolisms and cellular processes, for example, intracellular redox conditions, redox mediators, anaerobic carbon metabolism, and biofilm formation, etc. [9] This complexity makes it urgent to develop powerful genetic manipulation tools to engineer S. oneidensis MR-1 for improving the EET efficiency.Many gene expression and regulation toolbox have been developed in S. oneidensis MR-1, including plasmid expression toolkit [10] and CRISPR (clustered regularly interspaced short palindromic repeat)-mediated genome editing and regulation approaches. [11] A CRISPR-mediated base editing system (pCBEso) was recently developed in S. oneidensis MR-1, [12] in which C to T conversion in a 6-nt editing window could be achieved by the fusion protein of nCas9 (D10A) and cytidine deaminase rAPOBEC1. Base editing could achieve gene deactivation via mutating the CAG, CAA, CGA, TGG codons into premature stop codons (TAA, TAG, and TGA). Compared with the CRISPR-based recombination system that requires multiple components (two plasmids and ssDNA repair template), [11a] the base editing technologies are easier to operate, which avoid introduction of DNA double-strand breaks into the genome. However, to deactivate genes by base editing effectively, the premature stop codon should be introduced as close as possible 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, th...

Genome Editing by CRISPR/Cas12 Recognizing AT-Rich PAMs in Shewanella oneidensis MR-1

“…Various types of CRISPR-Cas systems have been widely employed in gene manipulation in S. oneidensis , ,− of which Cas9 is the most commonly used, but canonically requires a PAM (5′-NGG-3′) sequence to recognize and cleave the target DNA site, which substantially restricts its application. Therefore, in order to extend the application range of the CRISPR-Cas system, we assessed the Cas proteins recognizing different PAMs in S. oneidensis MR-1.…”

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

“…Biofilm-related genes tviB and tviC were selected from the cell surface polysaccharide biosynthesis gene cluster ( Kouzuma et al., 2010 ). It has been reported that base editing-mediated deactivation of these two genes increased the thickness and density of biofilm formed by EAMs on electrode surfaces ( Chen et al., 2022 ), which has a positive effect on both cytochrome-mediated direct EET and shuttle-related indirect EET pathways ( Edel et al., 2019 ). We thus conjectured that interference of tviB and tviC expression might cause thicker and/or denser biofilm, contributing to the improvement of the EET efficiency ( Figure 7 A).…”

“…pPaeCascade was constructed by Genewiz with cas8f (csy1), cas5 (csy2), cas7 (csy3), cas6f (csy4) sequence from pCsy_complex, gift from Prof. Puping Liang ( Chowdhury et al., 2017 ) and crRNA cassette integrated to pCYR011 ( Chen et al., 2022 ). To facilitate rapid construction of crRNA, we introduced Golden Gate assembly strategy ( Engler et al., 2008 ; Fang et al., 2021 ), so the crRNA cassette contained repeat sequences and spacer sequence with BsaI recognition sites synthesized by Genewiz (China).…”

Highly efficient multiplex base editing: One-shot deactivation of eight genes in Shewanella oneidensis MR-1

“…pMBE assembled with multiple gRNAs was removed after editing from S. oneidensis MR-1 as our previous report [ 21 ]. Then PYYDT-C5 (PYYDT- ribADEHC ) plasmid was transformed into the edited strains to construct HRF(3BE) and HRF(8BE) via conjugation with E. coli WM3064.…”

“…It has been demonstrated as a genome-level approach in genetic engineering and gene function identification [ 19 , 20 ]. Despite its conducive features, multiplexed application of base editing in S. oneidensis is still impeded by the low-throughput procedures of the current system [ 21 ]. As far as we know, base editors can edit at most two targets in a round for S. oneidensis , and to edit more than two loci, iterative methods must be used.…”