CRISPR/Cas13X-assisted programmable and multiplexed translation regulation for controlled biosynthesis

Developing efficient gene regulation tools is essential for optimizing microbial cell factories, but most existing tools only modulate gene expression at the transcriptional level. Regulation at the translational level provides a faster dynamic response, whereas developing a programmable, efficient and multiplexed translational regulation tool remains a challenge. Here, we have developed CRISPRi and CRISPRa systems based on hfCas13X that can regulate gene translation in Bacillus subtilis. First, we constructed a CRISPRi system to regulate gene translation based on catalytically deactivated hfCas13X (dhfCas13X). Second, we designed unique mRNA-crRNA pairs to construct DiCRISPRa (degradation-inhibited CRISPRa) and TsCRISPRa (translation-started CRISPRa) systems, which can activate downstream gene translation by enhancing mRNA stability or initiating mRNA translation. In addition, we found that fusing dhfCas13X with the RNA-binding chaperone BHfq significantly improved the activation efficiency of the DiCRISPRa and TsCRISPRa systems (43.2-fold). Finally, we demonstrated that the constructed CRISPR systems could be used to optimize the metabolic networks of two biotechnologically relevant compounds, riboflavin and 2'-fucosyllactose, increasing their titers by 3- and 1.2-fold, respectively. The CRISPRa and CRISPRi systems developed here provide new tools for the regulation of gene expression at the translation level and offer new ideas for the construction of CRISPRa systems.