Eliminating mcr-1-harbouring plasmids in clinical isolates using the CRISPR/Cas9 system
Related Experiment Video
Updated: Jan 23, 2026
Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells
Published on: July 22, 2019
Pengxia Wang1, Dongmei He2, Baiyuan Li1,3
1Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, the South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.
Objectives:
To eliminate mcr-1-harbouring plasmids and MDR plasmids in clinical Escherichia coli isolates.
Methods:
Plasmid pMBLcas9 expressing Cas9 was constructed and used to clone target single-guide RNAs (sgRNAs) for plasmid curing. The recombinant plasmid pMBLcas9-sgRNA was transferred by conjugation into two clinical E. coli isolates. The curing efficiency of different sgRNAs targeting conserved genes was tested. The elimination of targeted plasmids and the generation of transposase-mediated recombination of p14EC033a variants were characterized by PCR and DNA sequencing.
Results:
In this study, four native plasmids in isolate 14EC033 and two native plasmids in isolate 14EC007 were successfully eliminated in a step-by-step manner using pMBLcas9. Moreover, two native plasmids in 14EC007 were simultaneously eliminated by tandemly cloning multiple sgRNAs in pMBLcas9, sensitizing 14EC007 to polymyxin and carbenicillin. In 14EC033 with two mcr-1-harbouring plasmids, IncI2 plasmid p14EC033a and IncX4 plasmid p14EC033b, a single mcr-1 sgRNA mediated the loss of p14EC033b and generated a mutant p14EC033a in which the mcr-1 gene was deleted. An insertion element, IS5, located upstream of mcr-1 in p14EC033a was responsible for transposase-mediated recombination, resulting in mcr-1 gene deletion instead of plasmid curing.
Conclusions:
CRISPR/Cas9 can be used to efficiently sensitize clinical isolates to antibiotics in vitro. For isolates with multiple plasmids, the CRISPR/Cas9 approach can either remove each plasmid in a stepwise manner or simultaneously remove multiple plasmids in one step. Moreover, this approach can be used to delete multiple gene copies by using only one sgRNA. However, caution must be exercised to avoid unwanted recombination events during genetic manipulation.
Insights
CRISPR/Cas9 technology efficiently eliminates antimicrobial resistance plasmids, including mcr-1, from clinical Escherichia coli isolates. This method can remove single or multiple plasmids, and even delete specific genes, offering a novel strategy to combat multidrug resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The emergence of multidrug resistance (MDR) in clinical pathogens, particularly Escherichia coli, poses a significant global health threat.
- Plasmids carrying resistance genes, such as mcr-1 (colistin resistance) and other MDR determinants, are key drivers of antimicrobial resistance.
- Effective strategies are needed to eliminate these resistance plasmids from bacterial populations.
Purpose of the Study:
- To investigate the efficacy of the CRISPR/Cas9 system for eliminating mcr-1-harbouring and MDR plasmids from clinical Escherichia coli isolates.
- To assess the ability of CRISPR/Cas9 to remove single, multiple, or specific genes from plasmids in a stepwise or simultaneous manner.
Main Methods:
- Construction of a plasmid vector (pMBLcas9) for expressing Cas9 and cloning single-guide RNAs (sgRNAs).
- Delivery of the pMBLcas9-sgRNA construct into clinical E. coli isolates via conjugation.
- Testing the efficiency of different sgRNAs targeting conserved plasmid genes for plasmid elimination.
- Characterization of plasmid elimination and potential recombination events using PCR and DNA sequencing.
Main Results:
- Successful stepwise elimination of four native plasmids from E. coli isolate 14EC033 and two from 14EC007 using pMBLcas9.
- Simultaneous elimination of two native plasmids from 14EC007 by tandemly cloning multiple sgRNAs, restoring sensitivity to polymyxin and carbenicillin.
- In isolate 14EC033, a single mcr-1 sgRNA led to the deletion of the mcr-1 gene from one plasmid (p14EC033a) via transposase-mediated recombination, while eliminating another plasmid (p14EC033b).
Conclusions:
- CRISPR/Cas9 is an effective tool for sensitizing clinical isolates to antibiotics by eliminating resistance plasmids in vitro.
- The CRISPR/Cas9 approach allows for flexible plasmid elimination, including stepwise removal of individual plasmids or simultaneous removal of multiple plasmids.
- This technology can also be utilized for targeted gene deletion, but careful consideration of potential unwanted recombination events is necessary.
Related Concept Videos
01:59CRISPR
01:28CRISPR/Cas9 Genome Editing
02:53CRISPR and crRNAs
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
01:28Plasmids
01:17Kinetics of Drug Elimination
Drug clearance depends on the rate of drug elimination and its plasma concentration. Another important parameter is the half-life of a drug, which is the time required for its concentration to decrease by half. In most cases, drug clearance follows first-order...
01:26Enhanced Elimination of Poison
Antidotes serve a crucial role in counteracting the effects of poison by inhibiting enzymes responsible for producing harmful drug metabolites. In some cases, these toxic metabolites can be neutralized by endogenous cosubstrates, which are maintained at specific concentrations to prevent interaction with cellular macromolecules and subsequent cell death.
Renal excretion is the...