Leveraging cell‐free gene expression for RNA‐based biopesticides production: mechanisms and applications

As RNA-based biopesticides have been drawing increasing attention globally, lack of efficient production becomes a major bottleneck. Cell Free gene Expression (CFE), which has been widely used for protein synthesis, has emerged as a promising and transformative approach to achieve cost-effective yet high-quality RNA production. This review analyzed the key processes and core components of CFE technology based on the protein synthesis protocol, and made adaptation as well as suggestions for RNA synthesis. Drawing from successful examples from both industry and the lab, the authors proposed a streamlined cell free RNA synthesis (CFRS) workflow. This review also illustrated the advantages and bottlenecks through comparison with traditional RNA synthesis methods such as chemical synthesis, in vitro transcription (IVT) and microbial fermentation. It pointed out that preparing high quality cell lysates and ensuring RNA stability are the two critical technical aspects for successful CFRS, while commercialization of RNA-based biopesticides still face regulatory hurdles.