Construction of recombinant CRISPR plasmid for disruption of virulence SetA۱ gene from Shigella flexneri ۲a pathogenic bacterium

Shigellosis, infection by shigella bacterium, is estimated to cause more than ۸۰ million cases of hospitalizations and ۷۰۰ ۰۰۰ deaths worldwide annually. Shigella isolates are resistance to antibiotics from ۵۰% to ۸۳%. Chromosomal genes, set۱A and set۱B, encode the Shigella enterotoxin ۱ (ShET-۱), and are among the factors associated with the watery phase of diarrhea. CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR associated (Cas) proteins) systems, originating from the bacterial adaptive immune system, have been engineered as genome editing tools for a variety of organisms. The aim of this study was to construct an applicable recombinant crispr plasmid for mutagenesis of setA۱ gene in shigella pathogen. Forward and reverse oligonucleotides were designed to produce the ۲۰ bps guide molecules by computer chopchop software. In addition, Watson and crick primers were also designed to verify cloning of the segment of interest in crispr plasmids. Annealing of single nucleotides was carried out with DNA ligase buffer. CRISPR plasmids were digested with BBsI restriction enzyme. Ligation of guide segment and digested vector was performed using of DNA ligase. Transformation of recombinant plasmids was done using of heat shock method on Escherichia coli DH α competent cells. After colony formation, in order to cloning verification, PCR experiment was done using of extracted recombinant plasmids as template DNA. Finally, PCR analysis showed the successful cloning of guide molecule in vector. Production of such editing recombinant crispr vector can help us to disrupt virulence gene in bacterial pathogens.