Review of: "Construction and validation of safe Clostridium botulinum Group II surrogate strain producing inactive botulinum neurotoxin type E toxoid"
Year: 2022
Type: peer-review
Abstract: The paper 'Construction and validation of safe Clostridium botulinum Group II surrogate strain producing inactive botulinum neurotoxin type E toxoid' summarizes a collaborative study led by the group of Miia Lindström and five additional PIs (Birgitte Dorner, Martin Dorner, Rongshen Jin, Nigel Minton, and Andreas Rummel) who are established in the Clostridium botulinum (C.botulinum) field.The paper is well-written and describes the use of CRISPR-Cas technology to create a mutated C. botulinum strain.The CRISPR-Cas technology has only recently been adapted to this organisms, in large part thanks to the efforts of Nigel Minton, who developed the CRISPR-Cas plasmids specifically designed for Clostridia.Few available gene editing methods are available for C. botulinum, which is a Tier 1 Select Agent in the US and thus highly regulated, resulting in only few labs working with this organism.In addition, the low GC content, the gram positive cell wall, and the polyphyletic nature of this 'species' complicates genetic manipulations.Utlization of CRISPR-Cas technology to a C. botulinum strain for site directed mutagenesis in specific genes is thus highly significant and novel.The gene that was targeted for mutation in this study was the botulinum neurotoxin E ( bont/E) gene, introducing three single amino acid mutations that have previously been shown to disrupt catalytic activity by preventing zinc binding and transition state stabilization of the toxin's light chain.This resulted in an attenuated C. botulinum strain that has many potential utilities.Additionally, three silent mutations were included in the same gene near the mutation site to add three unique restriction enzyme recognition sites for easy identification of mutants.This is an elegant approach, provided codon usage of the organism is considered when designing the silent mutations, such that translation efficiency is not altered.Describing the strain as safe and the toxin as inactive is a slight overstatement, as the safety of the strain still remains to be determined in future studies and is dependent on its use, and based on the literature this toxin should be described as attenuated not inactive as it has not been isolated and tested at higher concentrations.The authors clearly describe the significance of the created attenuated C. botulinum Ei strain in the introduction and
DOI: 10.32388/1wsn65
Authors: Sabine Pellett, Travis Wentz
Institution: University of Wisconsin–Madison
Language: English
Subfield: Neurology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
OpenAlex ID: W4226399522