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External guide sequences (EGSs) have successfully been used to inhibit expression of target genes at the post-transcriptional level in both prokaryotes and eukaryotes. We previously reported that EGS accessible and cleavable sites in the target RNAs can rapidly be identified by screening random EGS (rEGS) libraries. Here the method of screening rEGS libraries and a partial RNase T1 digestion assay were used to identify sites accessible to EGSs in the mRNA of a global virulence regulator MglB from  Francisella tularensis , a Gram-negative pathogenic bacterium. Specific EGSs were subsequently designed and their activities in terms of the cleavage of mglB mRNA by RNase P were tested  in vitro  and  in vivo . EGS73, EGS148, and EGS155 in both stem and M1 EGS constructs induced mglB mRNA cleavage  in vitro . Expression of stem EGS73 and EGS155 in  Escherichia coli  resulted in significant reduction of the mglB mRNA level coded for the  F. tularensis mglB  gene inserted in those cells.

Inhibition of Expression in Escherichia coli of a Virulence Regulator MglB of Francisella tularensis Using External Guide Sequence Technology