CRISPR-interference-based modulation of mobile genetic elements in bacteria

Abstract

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.

A single plasmid based CRISPR interference in Synechocystis 6803 – A proof of concept

“…The resulting plasmid, pCRISPathBrick has been characterized elsewhere to be able to suppress multiple genes in E . coli successfully [9, 10]. These plasmids contain the p15A origin of replication and are therefore unable to replicate outside E .…”

“…The same system was also used to improve E . coli strain genome stability by silencing multiple insertion elements [10].…”

The future of self-selecting and stable fermentations

“…Improved mitigation strategies employ CRISPR-based abolishment and genetic conjugation to make hybrid genomes from IS-less genomic regions in related strains [59]. Lately, also strategies using CRISPRi to silence the transposase genes driving the IS have been shown for E. coli strains and Acinetobacter baylyi [25,44]. Silencing strategies are fast and easy to test, yet actual deployment of CRISPRi during production is likely a major burden in itself [13] with risk for quick mutation and indirect metabolic costs to biosynthetic production.…”

Systematic genome engineering approaches to investigate mutational effects and evolutionary processes

“…Szybalski's first notes on synthetic biology, the ability to rationally engineer biological systems has led to a plethora of novelties which have transformed both biotechnology and basic research. Briefly, biological engineering has paved the way for unprecedented advancements, including the production of antimalarial artemisinin in baker's yeast [4], the de novo design of enzymes with novel catalytic activities [5,6], as well as the manufacturing of artificial protein and DNA shells to specifically deliver payloads [7,8], and the construction of programmable genetic circuits and memories within living bacteria, yeasts, and mammalian cells [9,10].…”