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Updated: Jul 13, 2026
A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer
Published on: July 12, 2017
The ClosTron: a universal gene knock-out system for the genus Clostridium
John T Heap1, Oliver J Pennington, Stephen T Cartman
1Institute of Infection, Immunity and Inflammation, School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
Abstract:
Progress in exploiting clostridial genome information has been severely impeded by a general lack of effective methods for the directed inactivation of specific genes. Those few mutants that have been generated have been almost exclusively derived by single crossover integration of a replication-deficient or defective plasmid by homologous recombination. The mutants created are therefore unstable. Here we have adapted a mutagenesis system based on the mobile group II intron from the ltrB gene of Lactococcus lactis (Ll.ltrB) to function in clostridial hosts. Integrants are readily selected on the basis of acquisition of resistance to erythromycin, and are generated from start to finish in as little as 10 to 14 days. Unlike single crossover plasmid integrants, the mutants are extremely stable. The system has been used to make 6 mutants of Clostridium acetobutylicum and 5 of Clostridium difficile, exceeding the number of published mutants ever generated in these species. Genes have also been inactivated for the first time in Clostridium botulinum and Clostridium sporogenes, suggesting the system will be universally applicable to the genus. The procedure is highly efficient and reproducible, and should revolutionize functional genomic studies in clostridia.
Insights
A new gene inactivation system using a mobile group II intron (Ll.ltrB) enables stable mutant generation in clostridia. This efficient method accelerates functional genomic studies for species like Clostridium acetobutylicum and Clostridium difficile.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Directed gene inactivation is crucial for understanding clostridial genomes but has been hindered by a lack of effective methods.
- Previous methods relied on unstable single crossover plasmid integration, limiting progress in functional genomics.
Purpose of the Study:
- To adapt and validate a mobile group II intron mutagenesis system (Ll.ltrB) for stable gene inactivation in clostridial hosts.
- To establish an efficient and reproducible method for generating clostridial mutants, revolutionizing genomic studies.
Main Methods:
- Adaptation of the Lactococcus lactis Ll.ltrB group II intron system for use in clostridial species.
- Selection of integrants via erythromycin resistance.
- Generation of mutants within a 10-14 day timeframe.
Main Results:
- Successfully generated stable mutants in Clostridium acetobutylicum (6 mutants) and Clostridium difficile (5 mutants).
- Achieved gene inactivation for the first time in Clostridium botulinum and Clostridium sporogenes.
- Demonstrated high efficiency, reproducibility, and stability of the generated mutants compared to previous methods.
Conclusions:
- The Ll.ltrB intron system provides a robust and efficient method for stable gene inactivation in diverse clostridial species.
- This breakthrough is expected to significantly advance functional genomic research across the Clostridium genus.
- The system overcomes previous limitations, enabling rapid and reliable mutant generation.
