Anaerobes in animal disease Marker-less deletion of cctA gene of Clostridium chauvoei Sanjay Kumar Gupta a , Sameer Dixit a , Saroj K. Dangi a , Gurpreet Kaur a , Mohmad Mashooq a , Kumaragurubaran Karthik a , Mihir Sarkar b , Sonalika Mahajan c , Viswas Konasagara Nagaleekar a, * a Division of Bacteriology and Mycology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, 243122, India b Division of Physiology and Climatology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, 243122, India c Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, 243122, India article info Article history: Received 5 December 2018 Received in revised form 18 October 2019 Accepted 7 November 2019 Available online 8 November 2019 Handling Editor: TG Nagaraja Keywords: Clostridium chauvoei cctA CRISPR-Cas9 Genome editing abstract Clostridium chauvoei causes blackleg disease in domestic animals, especially cattle and sheep. The pathogen produces several toxins including CctA e a hemolysin and protective antigen. Molecular pathogenesis of the disease is poorly understood, possibly due to lack of genetic manipulation tools for C. chauvoei. In the present study, we report the marker-less deletion of cctA gene using the CRISPR-Cas9 system. The C. chauvoei cctA deletion mutant had negligible hemolytic and significantly reduced cyto- toxic activities. To the best of our knowledge, this is the first report of genetic manipulation of C. chauvoei. The method we used in this study can be applied for genetic manipulation of C. chauvoei to better un- derstand the pathogenesis and genetics of the pathogen. © 2019 Elsevier Ltd. All rights reserved. 1. Introduction Clostridium chauvoei, an anaerobic, Gram-positive bacillus, causes blackleg disease in domestic animals, mainly cattle and sheep [1]. The pathogen has also been isolated from human cases leading to fulminant gas gangrene [2,3]. Despite the seriousness of the disease and the high mortality rate, there is lack of under- standing of the role of virulence factors and the molecular patho- genesis of the disease. This is likely because of lack of availability of methods to genetically manipulate C. chauvoei. For most of the Clostridium species, several genetic manipula- tion methods including transposon-based mutagenesis, ClosTron based insertional inactivation, allele-coupled exchange, and CRISPR-Cas9 methods are available [reviewed in Ref. [4]]. Recently, whole genome sequences of C. chauvoei type strain ATCC 10092 [5] and other strains [6] have been published. However, to the best of our knowledge, so far, no report on genetic modification of C. chauvoei is available. Clustered regularly interspersed short palindromic repeats (CRISPR)-CRISPR associated (Cas) system is an immune mechanism in prokaryotes, which cleaves foreign DNA from phages or plasmids [7]. Among the different types, type II CRISPR-Cas system from Streptococcus pyogenes is the most commonly used genome editing tool. In type II CRISPR-Cas system, Cas9 nuclease, guided by chimeric guide RNA (gRNA), selectively targets specific sequence and causes double strand breakage of DNA [8]. This system selects the bacterial cells, in which the DNA repair occurs by homologous recombination [9]. Owing to its high efficiency, the CRISPR-Cas system has been widely used for genome editing in Clostridia [10e13]. C. chauvoei produces several virulence factors, including siali- dase, hyaluronidase, flagellin and C. chauvoei toxin A (CctA) [14e18]. Among these, CctA is a well-characterized hemolysin and protective antigen [14]. Because a hemolytic assay can readily quantify the phenotypic trait of CctA, we chose this gene to opti- mize the procedures for genetic manipulation of C. chauvoei. In this study, we demonstrated the CRISPR-Cas based genome editing of C. chauvoei. We deleted the cctA gene and characterized and compared the features of mutants to wild-type (WT) C. chauvoei. * Corresponding author. E-mail address: kn.viswas@icar.gov.in (V.K. Nagaleekar). Contents lists available at ScienceDirect Anaerobe journal homepage: www.elsevier.com/locate/anaerobe https://doi.org/10.1016/j.anaerobe.2019.102116 1075-9964/© 2019 Elsevier Ltd. All rights reserved. Anaerobe 61 (2020) 102116