Construction of recombinant CRISPR plasmid for disruption of virulence pic gene from Shigella flexneri ۲a pathogenic bacterium

The pic gene in Shigella flexneri ۲a is located on the PAI۱ pathogenicity island on the chromosome and has been identified as a unique bacterial virulence factor. This gene leads to the induction of mucin secretion and cleavage, which are involved in causing bloody diarrhea. Antibiotics can be used to treat Shigellosis and reduce the period of bacterial shedding from the host. However, S. flexneri is increasingly developing its antibiotic resistance. One of the applications of the CRISPR system is genome editing in pathogenic bacteria, and therapeutic goals can be achieved by targeting harmful genes, including genes involved in pathogenic mechanisms. The aim of this study was to construct CRISPR recombinant plasmids harboring the gRNAdsDNA segment in order to manipulate virulence pic gene from pathogenic Shigella bacterium using of multiple crisps method. Firstly, gRNAdsDNA primers of the gene of interest were designed using of ChopChop computer software, annealed using suitable temperature and successfully cloned in pCas۹ plasmid with DNA ligase after digestion of vectors by BbsI restriction enzyme. Recombinant plasmids were transformed to host E.coli competent cells via heat-shock experiment. After growing the colonies on solid medium, plasmids were extracted. Then cloning of the gRNAdsDNA was verified using of PCR and DNA sequencing experiments. Construction of editing crispr recombinant plasmid made in this study can lead to disrupt virulence gene of pathogenic microorganisms and help to human health