Clostridial Genetics: Genetic Manipulation of the Pathogenic Clostridia.

The past 10 years have been revolutionary for clostridial genetics. The rise of next-generation sequencing led to the availability of annotated whole-genome sequences of the important pathogenic clostridia: Clostridium perfringens, Clostridioides (Clostridium) difficile, and Clostridium botulinum, but also Paeniclostridium (Clostridium) sordellii and Clostridium tetani. These sequences were a prerequisite for the development of functional, sophisticated genetic tools for the pathogenic clostridia. A breakthrough came in the early 2000s with the development of TargeTron-based technologies specific for the clostridia, such as ClosTron, an insertional gene inactivation tool. The following years saw a plethora of new technologies being developed, mostly for C. difficile, but also for other members of the genus, including C. perfringens. A range of tools is now available, allowing researchers to precisely delete genes, change single nucleotides in the genome, complement deletions, integrate novel DNA into genomes, or overexpress genes. There are tools for forward genetics, including an inducible transposon mutagenesis system for C. difficile. As the latest addition to the tool kit, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technologies have also been adopted for the construction of single and multiple gene deletions in C. difficile. This article summarizes the key genetic technologies available to manipulate, study, and understand the pathogenic clostridia.