CRISPR/dCas-based modulation of bacterial IS-transposition

In this work, a plasmid-based system is applied to inhibit the transposition of bacterial insertion sequences (IS). Using multiple guide RNAs, inactivated Cas9 was directed to simultaneously bind to the left end of IS1, IS5, IS3 and IS150 in Escherichia coli, in vivo. As a result, the transcription of IS1, and IS5 was successfully silenced, in certain cases by two orders of magnitude. The transposition rate of all four targeted elements nevertheless dropped to negligible levels, as verified at the cycA and bgl chromosomal loci. A GFP-expressing plasmid, known to be predominantly inactivated by insertion mutations also displayed a significant increase in stability. The transposition-silencing effect was easily transferable between various E. coli strains by plasmid transformation. Our portable system, or other plasmids constructed likewise can serve as useful tools to eliminate insertion mutagenesis or selectively study distinct transposable elements in numerous prokaryotic species. The transcriptome of three samples were analyzed originating from the IS-knockdown strain. Three controls were also analyzed from cells carrying a mock-silencing plasmid.