RiboCas: A Universal CRISPR-Based Editing Tool for Clostridium

Cañadas, Inés C.; Groothuis, Daphne; Zygouropoulou, Maria; Rodrigues, Raquel; Minton, Nigel P.

Abstract

Members of the genus Clostridium represent a diverse assemblage of species exhibiting both medical and industrial importance. Deriving both a greater understanding of their biology, while at the same time enhancing their exploitable properties, requires effective genome editing tools. Here, we demonstrate the first implementation in the genus of theophylline-dependent, synthetic riboswitches exhibiting a full set of dynamic ranges, also suitable for applications where tight control of gene expression is required. Their utility was highlighted by generating a novel riboswitch-based editing tool—RiboCas—that overcomes the main obstacles associated with CRISPR/Cas9 systems, including low transformation efficiencies and excessive Cas9 toxicity. The universal nature of the tool was established by obtaining chromosomal modifications in C. pasteurianum, C. difficile, and C. sporogenes, as well as by carrying out the first reported example of CRISPR-targeted gene disruption in C. botulinum. The high efficiency (100% mutant generation) and ease of application of RiboCas make it suitable for use in a diverse range of microorganisms.

Citation

Cañadas, I. C., Groothuis, D., Zygouropoulou, M., Rodrigues, R., & Minton, N. P. (2019). RiboCas: A Universal CRISPR-Based Editing Tool for Clostridium. ACS Synthetic Biology, 8(6), 1379-1390. https://doi.org/10.1021/acssynbio.9b00075

• Journal Article Type: Article
• Acceptance Date: May 26, 2019
• Online Publication Date: Jun 7, 2019
• Publication Date: Jun 7, 2019
• Deposit Date: Jun 11, 2019
• Publicly Available Date: Jun 25, 2019
• Journal: ACS Synthetic Biology
• Electronic ISSN: 2161-5063
• Publisher: American Chemical Society
• Peer Reviewed: Peer Reviewed
• Volume: 8
• Issue: 6
• Pages: 1379-1390
• DOI: https://doi.org/10.1021/acssynbio.9b00075
• Public URL: https://nottingham-repository.worktribe.com/output/2171704
• Publisher URL: https://pubs.acs.org/doi/10.1021/acssynbio.9b00075
• Contract Date: Jun 11, 2019