Genetically engineered plasmid with CRISPR-Cas9 to remove Antibiotic Resistance Gene
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Genetically engineered plasmid with CRISPR-Cas9 to remove Antibiotic Resistance Gene

Scientists have now engineered a plasmid to remove an antibiotic resistance gene from the Enterococcus faecalis bacterium which is an accomplishment that could lead to new methods for combating antibiotic resistance.

In vitro, and in mouse models, the genetically engineered plasmid removed the antibiotic resistance gene from Enterococcus faecalis. In mouse models, the plasmid reduced the abundance of the antibiotic resistance gene threefold.

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Breck A. Duerkop, Ph.D., Assistant Professor of Immunology and Microbiology, University of Colorado School of Medicine, Anschutz Medical Center, Aurora, said that their concerns with organisms that cause hospital-acquired infections which are resistant to many antibiotic therapies motivated this research. He is the co-senior author of the research study published this week in Antimicrobial Agents and Chemotherapy Journal of the American Society for Microbiology.

Enterococcus faecalis is part of the normal, benign intestinal flora, but when antibiotics kill off beneficial intestinal flora, Enterococcus faecalis can become pathogenic. It can also acquire single drug or multidrug resistance. Antibiotic-resistant E. faecalis infections are a major problem in hospitals.

The mechanism that is used to remove antibiotic resistance genes is the specialized protein called CRISPR-Cas9. This can make cuts just about anywhere in DNA.

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Along with the CRISPR-Cas9 system, RNA sequences homologous to DNA within the antibiotic resistance gene have been added to the engineered plasmid. These RNAs guide the CRISPR-Cas9 system to make the cuts in the right places of the gene.

Previous studies in animal models by co-senior investigator Kelli L. Palmer, Ph.D., found that CRISPR-Cas9 could prevent intestinal Enterococcus faecalis from acquiring resistance genes. Dr. Palmer is Fellow, Cecil H. and Ida Green Chair in Systems Biology Science, Associate Professor of Biological Sciences, University of Texas, Dallas.

The delivery vehicle for the genetically engineered plasmid is a particular strain of Enterococcus faecalis, which conjugates with Enterococcus faecalis of various different strains. Conjugation is the process where the bacteria come together to transfer genetic materials from one to the other via their direct cell to cell contact.

Dr. Duerkop said that Enterococcus faecalis strains were used to deliver these plasmids to drug-resistant strains of Enterococcus faecalis which are immune to acquiring drug-resistant traits carried by the target cells. The genetically engineered plasmid can significantly reduce the occurrence of antibiotic resistance in the target bacterial population rendering it more susceptible to antibiotics. The team envisions that this type of system could be used to re-sensitize antibiotic-resistant Enterococcus faecalis to antibiotics, he added.

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Nonetheless, he cautioned that it remained possible that Enterococcus faecalis could still circumvent the engineered plasmids. Some bacteria have anti-CRISPR systems which can block CRISPR-Cas9 protein function, and some bacterias have systems that can degrade foreign DNA. Further studies in the future will need to be done to address such an issue as Enterococcus faecalis avoiding the targeting system and also under what conditions this may happen, said Dr. Duerkop.


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Ria Roy completed her Post Grad degree at the Visvesvaraya Technological University. She has a great grounding in the skills, including technical, analytical and research skills. She is a motivated life science professional with experience of working in famous research institutes