Targeted Gene inactivation in Salmonella typhi byCRISPR/Cas۹

Background and Aim : Targeted gene inactivation (TGI) is a widely used technique for the studyof genes’ functions. There are many different methods for TGI, however, most of them are socomplicated and time-consuming. New promising genetic engineering tools are developing for thispurpose. In the present study, for the first time we inactivated a virulence gene from SalmonellaTyphi (S. Typhi), located in the bacterial chromosome using CRISPR/Cas۹ system.Methods : For this aim, pCas۹plasmid containing Cas۹ enzyme and required proteins for HDRrecombination was prepared and transferred to S. Typhi by electroporation. On the other hand, aspecific guide RNA (gRNA) was designed using CRISPOR online tool. Synthetic gRNA wascloned into pTargetF plasmid. Also, a DNA fragment (HDR) was designed to incorporate into thebacterial chromosome following the cleavage of the bacterial genome by Cas۹ enzyme. pTargetFcontaining gRNA and HDR were co-transferred to S. Typhi containing pcas۹ plasmid. Thetransformed bacteria were screened for recombination using PCR, restriction digestion andsequencing.Results : The results of PCR, restriction digestion and sequencing showed the successfulrecombination of S. Typhi, in which the gidA gene is disrupted. At last, foreign plasmids werecured by adding IPTG and growing in ۳۷ ˚C.Conclusion : In the present study we developed a rapid and specific method for targeted geneinactivation in a bacterial species, S. Typhi. This procedure can be exploited for disruption of otherSalmonella’s genes as well as other bacteria.

کلیدواژه ها:

Targeted gene inactivation (TGI) ، CRISPR/Cas system ، Salmonella Typhi ، gidAgene ، Live attenuated vaccines