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

Shewanella oneidensis MR-1is apromising electroactive microorganism in environmental bioremediation,bioenergy generation, and bioproduct synthesis. Accelerating the extracellularelectron transfer (EET) pathway that enables efficient electron exchangebetween microbes and extracellular substances is critical for improvingits electrochemical properties. However, the potential genomic engineeringstrategies 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 situprotospacer-adjacent motif (PAM)-flexible dual base editing regulatorysystem (iSpider), for precise and high-throughput genomic manipulation.The iSpider enabled simultaneous C-to-T and A-to-G conversions withhigh diversity and efficiency in S. oneidensis. By weakening DNA glycosylase-based repair pathway and tetheringtwo copies of adenosine deaminase, the A-to-G editing efficiency wasobviously improved. As a proof-of-concept study, the iSpider was adaptedto achieve multiplexed base editing for the regulation of the riboflavinbiosynthesis pathway, and the optimized strain showed an approximatelythree-fold increase in riboflavin production. Moreover, the iSpiderwas also applied to evolve the performance of an inner membrane componentCymA implicated in EET, and one beneficial mutant facilitating electrontransfer could be rapidly identified. Taken together, our study demonstratesthat the iSpider allows efficient base editing in a PAM-flexible manner,providing insights into the design of novel genomic tools for Shewanella engineering.