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Obtaining electroactive microbes capable of efficient extracellular electron transfer is a large undertaking for the scalability of bio-electrochemical systems. Inevitably, researchers need to pursue the co-modification of multiple genes rather than expecting that modification of a single gene would make a significant contribution to improving extracellular electron transfer rates. Base editing has enabled highly-efficient gene deactivation in model electroactive microbe Shewanella oneidensis MR-1. Since multiplexed application of base editing is still limited by its low throughput procedure, we thus here develop a rapid and efficient multiplex base editing system in S. oneidensis. Four approaches to express multiple gRNAs were assessed firstly, and transcription of each gRNA cassette into a monocistronic unit was validated as a more favorable option than transcription of multiple gRNAs into a polycistronic cluster. Then, a smart scheme was designed to deliver one-pot assembly of multiple gRNAs. 3, 5, and 8 genes were deactivated using this system with editing efficiency of 83.3%, 100% and 12.5%, respectively. To offer some nonrepetitive components as alternatives genetic parts of sgRNA cassette, different promoters, handles, and terminators were screened. This multiplex base editing tool was finally adopted to simultaneously deactivate eight genes that were identified as significantly downregulated targets in transcriptome analysis of riboflavin-overproducing strain and control strain. The maximum power density of the multiplex engineered strain HRF(8BE) in microbial fuel cells was 1108.1 mW/m2, which was 21.67 times higher than that of the wild-type strain. This highly efficient multiplexed base editing tool elevates our ability of genome manipulation and combinatorial engineering in Shewanella, and may provide valuable insights in fundamental and applied research of extracellular electron transfer.

Obtaining electroactive microbes capable of efficient extracellular electron transfer is a large undertaking for the scalability of bio-electrochemical systems. Inevitably, researchers need to pursue the co-modification of multiple genes rather than expecting that modification of a single gene would make a significant contribution to improving extracellular electron transfer rates. Base editing has enabled highly-efficient gene deactivation in model electroactive microbe Shewanella oneidensis MR-1. Since multiplexed application of base editing is still limited by its low throughput procedure, we thus here develop a rapid and efficient multiplex base editing system in S. oneidensis . Four approaches to express multiple gRNAs were assessed firstly, and transcription of each gRNA cassette into a monocistronic unit was validated as a more favorable option than transcription of multiple gRNAs into a polycistronic cluster. Then, a smart scheme was designed to deliver one-pot assembly of multiple gRNAs. 3, 5, and 8 genes were deactivated using this system with editing efficiency of 83.3%, 100% and 12.5%, respectively. To offer some nonrepetitive components as alternatives genetic parts of sgRNA cassette, different promoters, handles, and terminators were screened. This multiplex base editing tool was finally adopted to simultaneously deactivate eight genes that were identified as significantly downregulated targets in transcriptome analysis of riboflavin-overproducing strain and control strain. The maximum power density of the multiplex engineered strain HRF(8BE) in microbial fuel cells was 1108.1 mW/m 2 , which was 21.67 times higher than that of the wild-type strain. This highly efficient multiplexed base editing tool elevates our ability of genome manipulation and combinatorial engineering in Shewanella , and may provide valuable insights in fundamental and applied research of extracellular electron transfer., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)