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Published on: September 8, 2012
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An Attenuated CRISPR-Cas System in Enterococcus faecalis Permits DNA Acquisition.
Karthik Hullahalli1, Marinelle Rodrigues1, Uyen Thy Nguyen1
1Department of Biological Sciences, The University of Texas at Dallas, Richardson, Texas, USA.
Mbio
|May 3, 2018
Summary
Enterococcus faecalis exhibits CRISPR tolerance, allowing transient survival despite CRISPR-Cas targeting of its chromosome. This study uncovers the mechanisms behind this phenomenon and develops a novel genome-editing tool for this opportunistic pathogen.
Area of Science:
- Microbiology
- Bacterial Genetics
- Genome Defense Systems
Background:
- Antibiotic resistance is a major public health threat, with horizontal gene transfer (HGT) accelerating its spread.
- Enterococcus faecalis, an opportunistic pathogen, readily disseminates antibiotic resistance via conjugative plasmids.
- Multidrug-resistant E. faecalis strains often lack CRISPR-Cas systems, which normally defend against foreign DNA.
Purpose of the Study:
- To investigate the phenomenon of "CRISPR tolerance" in Enterococcus faecalis, where mobile genetic elements targeted by CRISPR-Cas systems can be transiently maintained.
- To understand the molecular basis for CRISPR tolerance in E. faecalis.
- To develop a robust CRISPR-assisted genome-editing platform for E. faecalis.
Main Methods:
- Investigated CRISPR tolerance by introducing self-targeting constructs into E. faecalis.
- Analyzed gene expression, focusing on DNA repair and prophage induction during CRISPR targeting.
- Quantified cas9 expression levels and their impact on cell viability.
- Developed and validated a CRISPR-based genome-editing scheme.
Main Results:
- E. faecalis can tolerate chromosomal cleavage by CRISPR-Cas9 at a fitness cost, with no upregulation of DNA repair genes but strong induction of integrated prophages.
- Low cas9 expression was identified as a key factor contributing to transient nonlethality.
- A novel, robust CRISPR-assisted genome-editing platform for E. faecalis was successfully developed with minimal off-target effects.
Conclusions:
- Enterococcus faecalis exhibits "CRISPR tolerance," a mechanism facilitating DNA acquisition by attenuating its CRISPR-Cas genome defense system.
- This tolerance allows for the transient maintenance of targeted mobile genetic elements, potentially aiding in adaptation.
- The developed CRISPR editing platform simplifies genetic modifications in E. faecalis, offering new avenues for research and antimicrobial development.
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