A versatile one-step CRISPR-Cas9 based approach to plasmid-curing

Abstract

BackgroundPlasmids are widely used and essential tools in molecular biology. However, plasmids often impose a metabolic burden and are only temporarily useful for genetic engineering, bio-sensing and characterization purposes. While numerous techniques for genetic manipulation exist, a universal tool enabling rapid removal of plasmids from bacterial cells is lacking.ResultsBased on replicon abundance and sequence conservation analysis, we show that the vast majority of bacterial cloning and expression vectors share sequence similarities that allow for broad CRISPR-Cas9 targeting. We have constructed a universal plasmid-curing system (pFREE) and developed a one-step protocol and PCR procedure that allow for identification of plasmid-free clones within 24 h. While the context of the targeted replicons affects efficiency, we obtained curing efficiencies between 40 and 100% for the plasmids most widely used for expression and engineering purposes. By virtue of the CRISPR-Cas9 targeting, our platform is highly expandable and can be applied in a broad host context. We exemplify the wide applicability of our system in Gram-negative bacteria by demonstrating the successful application in both Escherichia coli and the promising cell factory chassis Pseudomonas putida.ConclusionAs a fast and freely available plasmid-curing system, targeting virtually all vectors used for cloning and expression purposes, we believe that pFREE has the potential to eliminate the need for individualized vector suicide solutions in molecular biology. We envision the application of pFREE to be especially useful in methodologies involving multiple plasmids, used sequentially or simultaneously, which are becoming increasingly popular for genome editing or combinatorial pathway engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0748-z) contains supplementary material, which is available to authorized users.

CRISPR‐based curing and analysis of metabolic burden of cryptic plasmids in Escherichia coli Nissle 1917

“…In this work we demonstrated CRISPR-Cas9-assisted curing of two cryptic plasmids in EcN. Curing of the first was straightforward and similar to previous works in which CRISPR-Cas9 has successfully cured plasmids [27,[34][35][36][37], but homology to the EcN genome in the second necessitated the insertion of a selection cassette followed by targeting of that cassette. This latter method is likely more broadly applicable as the degree of homology of a cryptic plasmid to the organisms genome may not be well known without a full genome sequence.…”

“…CRISPR-Cas9 creates RNA-guided dsDNA breaks and has previously been used for programmed curing of plasmids [27,[34][35][36][37]. A schematic of our CRISPR-based curing approach appears in Figure 1A.…”

Potentiation of curing by a broad-host-range self-transmissible vector for displacing resistance plasmids to tackle AMR

“…The fact that our experimental regime did not first eliminate the natural microbiota by streptomycin treatment may play a role in the difference. This may also be encouraging for related use of IncP-1 plasmids such as the recent demonstration of RK2 as a vehicle for targeting plasmids with CRISPR-cas9 [51]. Indeed combining both approaches into a single plasmid might provide an even more powerful strategy for tackling antibiotic resistance in the future.…”

Development of a CRISPR/Cas9n-based tool for metabolic engineering of Pseudomonas putida for ferulic acid-to-polyhydroxyalkanoate bioconversion

“…Fourth, the CRP strategy was also expanded to plasmid-curing application, as a fast, single-step plasmid-curing platform. Traditional methods are often tedious, requiring sequential rounds of growth under stressful or non-selective conditions to promote the appearance of plasmid-free segregants, consequently increasing the chance of accumulating unwanted mutations and are time consuming 49 . In our strategy, the functions of genome editing and plasmid curing are combined in one plasmid.…”