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

ABSTRACT:

Background

Plasmids 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.

Results

Based 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 conte

SUBMITTER: Lauritsen I 

PROVIDER: S-EPMC5540278 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature