CRISPR/Cas13X-assisted programmable and multiplexed translation regulation for controlled biosynthesis
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
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.
Date Issued
2025-01-13
Date Acceptance
2024-12-20
Citation
Nucleic Acids Research, 2025, 53 (1)
ISSN
0305-1048
Publisher
Oxford University Press (OUP)
Start Page
gkae1293
Journal / Book Title
Nucleic Acids Research
Volume
53
Issue
1
Copyright Statement
© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.
License URL
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/39777467
PII: 7944734
Subjects
CRISPR-Cas Systems
Bacillus subtilis
Protein Biosynthesis
CRISPR-Associated Proteins
Bacterial Proteins
Bioreactors
Gene Expression Regulation, Bacterial
RNA Stability
RNA, Messenger
RNA, Bacterial
Biosynthetic Pathways
Riboflavin
Trisaccharides
Metabolic Engineering
Publication Status
Published
Coverage Spatial
England
Article Number
gkae1293
Date Publish Online
2025-01-07