Spontaneous mutagenesis of synthetic genetic constructs by mobile genetic elements
frequently results in the rapid loss of engineered functions. Previous efforts to
minimize such mutations required the exceedingly time-consuming manipulation of bacterial
chromosomes and the complete removal of insertional sequences (ISes). To this aim,
we developed a single plasmid-based system (pCRIS) that applies CRISPR-interference
to inhibit the transposition of bacterial ISes. pCRIS expresses multiple guide RNAs
to direct inactivated Cas9 (dCas9) to simultaneously silence IS1, IS3, IS5 and IS150
at up to 38 chromosomal loci in Escherichia coli, in vivo. As a result, the transposition
rate of all four targeted ISes dropped to negligible levels at both chromosomal and
episomal targets. Most notably, pCRIS, while requiring only a single plasmid delivery
performed within a single day, provided a reduction of IS-mobility comparable to that
seen in genome-scale chromosome engineering projects. The fitness cost of multiple
IS-knockdown, detectable in flask-and-shaker systems was readily outweighed by the
less frequent inactivation of the transgene, as observed in green fluorescent protein
(GFP)-overexpression experiments. In addition, global transcriptomics analysis revealed
only minute alterations in the expression of untargeted genes. Finally, the transposition-silencing
effect of pCRIS was easily transferable across multiple E. coli strains. The plasticity
and robustness of our IS-silencing system make it a promising tool to stabilize bacterial
genomes for synthetic biology and industrial biotechnology applications.
