Xu Xianhao, Lv Xueqin, Liu Yanfeng, Li Jianghua, Du Guocheng, Chen Jian, Ledesma-Amaro Rodrigo, Liu Long
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, No. 1800, Lihu Avenue, Binhu District, Wuxi 214122, China.
Science Center for Future Foods, Ministry of Education, Jiangnan University, No. 1800, Lihu Avenue, Binhu District, Wuxi 214122, China.
Nucleic Acids Res. 2025 Jan 7;53(1). doi: 10.1093/nar/gkae1293.
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.
开发高效的基因调控工具对于优化微生物细胞工厂至关重要，但大多数现有工具仅在转录水平上调节基因表达。翻译水平的调控提供了更快的动态响应，然而开发一种可编程、高效且多重的翻译调控工具仍然是一项挑战。在此，我们基于hfCas13X开发了可在枯草芽孢杆菌中调节基因翻译的CRISPRi和CRISPRa系统。首先，我们构建了基于催化失活的hfCas13X（dhfCas13X）来调节基因翻译的CRISPRi系统。其次，我们设计了独特的mRNA-crRNA对来构建DiCRISPRa（降解抑制型CRISPRa）和TsCRISPRa（翻译起始型CRISPRa）系统，它们可通过增强mRNA稳定性或起始mRNA翻译来激活下游基因翻译。此外，我们发现将dhfCas13X与RNA结合伴侣BHfq融合可显著提高DiCRISPRa和TsCRISPRa系统的激活效率（43.2倍）。最后，我们证明构建的CRISPR系统可用于优化两种与生物技术相关的化合物（核黄素和2'-岩藻糖基乳糖）的代谢网络，使其滴度分别提高3倍和1.2倍。此处开发的CRISPRa和CRISPRi系统为翻译水平的基因表达调控提供了新工具，并为CRISPRa系统的构建提供了新思路。
