Construction of recombinant CRISPR plasmid for disruption of virulence SigA gene from Shigella flexneri a pathogenic bacterium

Shigellosis, infection by shigella, continues to be a main public health problem worldwide, mainly in developing countries where it is endemic.It has been demonstrated that SigA from Shigella pathogen plays a role in the intestinal fluid accumulation associated with S. flexneri infections. Unfortunately, irregular usage of antimicrobial agents has given rise to the increasing resistance of Shigella spp. and the growth of multi-drug resistance against common antibiotics worldwide. CRISPR is a powerful tool for editing genomes, meaning it allows researchers to easily alter DNA sequences and modify gene function. It has many potential applications, including correcting genetic defects, treating and preventing the spread of diseases and etc. the aim of this study was to modify virulence sigA gene from Shigella pathogen by multiple CRICPR systems. Oligonucleotides for forward and reverse were designed to produce a ۲۰ bps gRNAdsDNA as guide molecules. Then produced dsDNA was ligated to CRISPR by DNA ligase after digestion with BBsI restriction enzyme. Recombinant CRISPR plasmids were transformed into E.coli DHα host competent cell via heat shock experiments. After cell culture plasmids were isolated by alkaline lysis method. PCR analysis showed successful cloning of the guide segment for sigA gene on CRISPR editing tool. Construction of such vector is an initial step to modify virulence genes form pathogen microorganisms.

Keywords:

shigella , crispr , siga gene , gene editing