Developing a PAM-Flexible CRISPR-Mediated Dual-Deaminase Base Editor to Regulate Extracellular Electron Transport in Shewanella oneidensis

Shewanella oneidensis MR-1 is a promising electroactive microorganism in environmental bioremediation, bioenergy generation, and bioproduct synthesis. Accelerating the extracellular electron transfer (EET) pathway that enables efficient electron exchange between microbes and extracellular substances is critical for improving its electrochemical properties. However, the potential genomic engineering strategies for enhancing EET capabilities are still limited. Here, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-mediated dual-deaminase base editing system, named in situ protospacer-adjacent motif (PAM)-flexible dual base editing regulatory system (iSpider), for precise and high-throughput genomic manipulation. The iSpider enabled simultaneous C-to-T and A-to-G conversions with high diversity and efficiency in S. oneidensis. By weakening DNA glycosylase-based repair pathway and tethering two copies of adenosine deaminase, the A-to-G editing efficiency was obviously improved. As a proof-of-concept study, the iSpider was adapted to achieve multiplexed base editing for the regulation of the riboflavin biosynthesis pathway, and the optimized strain showed an approximately three-fold increase in riboflavin production. Moreover, the iSpider was also applied to evolve the performance of an inner membrane component CymA implicated in EET, and one beneficial mutant facilitating electron transfer could be rapidly identified. Taken together, our study demonstrates that the iSpider allows efficient base editing in a PAM-flexible manner, providing insights into the design of novel genomic tools for Shewanella engineering.