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September 22, 2014

CRISPR Snips Away Antibiotic Resistance

This scanning electron micrograph shows numerous clumps of an all-too-familiar antibiotic-resistant bacterium—MRSA, or methicillin-resistant Staphylococcus aureus. [Janice Haney Carr/Centers for Disease Control and Prevention]

  • A gene editing system bacteria use to shield themselves from viruses has been used by MIT scientists as a sword to vanquish antibiotic resistance. Thus far, the scientists have used the gene editing system, called CRISPR, to improve survival in moth larvae infected with a harmful form of Escherichia coli. Next, they intend to explore its efficacy in mice. Ultimately, the scientists, led by MIT’s Timothy Lu, M.D., Ph.D., hope that a more fully developed version of their CRISPR approach will treat infections or remove unwanted bacteria in human patients.

    The CRISPR system devised by Dr. Lu’s team meddles with bacterial genes that confer antibiotic resistance. For example, in a newly published study, the system was used to target NDM-1 and SHV-18. NDM-1 is an enzyme that confers resistance to carbapenems and other beta-lactam antibiotics. SHV-18, a mutation in the bacterial chromosome, helps bacteria resist quinolone antibiotics. It is also a virulence factor in enterohemorrhagic E. coli.

    The CRISPR system, the researchers found, was even capable of providing a kind of bonus: It selectively removed specific bacteria from diverse bacterial communities. To achieve this feat, the researchers engineered RNA-guided nucleases (RGNs) against different genetic signatures, enabling simultaneous targeting of a variety of virulence factors and resistance genes. This approach, the researchers suggested, raises the possibility of “microbiome editing”—and not just in antimicrobial applications.

    The RGNs used by Dr. Lu’s group were described in the September 21 issue of Nature Biotechnology, in an article entitled “Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases.” Guide RNAs are key components of any CRISPR system because they work with Cas9, a DNA-snipping enzyme. Once Cas9 binds with a short guide RNA, it is primed to recognize to specific base sequences.

    In the current study, the RGNs targeting specific DNA sequences were designed to serve as narrow-spectrum antimicrobials. They were delivered to microbial populations using bacteriophage or bacteria carrying plasmids transmissible by conjugation. After they were delivered, the RGNs significantly improved survival in a Galleria mellonella infection model, the authors reported.

    RGNs, the authors explained, constitute a class of highly discriminatory, customizable antimicrobials that enact selective pressure at the DNA level to reduce the prevalence of undesired genes, minimize off-target effects, and enable programmable remodeling of microbiota.

    “Owing to the modularity and simplicity of CRISPR-Cas engineering, libraries of multiplexed RGNs could be rapidly constructed to simultaneously target a plethora of antibiotic resistance and virulence determinants and to modulate the composition of complex microbial communities,” they wrote. “The addition of facile, sequence-informed, rational design to a field that has been dominated by time- and cost-intensive screening for broad-spectrum, small molecule antibiotics has the potential to reinvigorate the pipeline for new antimicrobials.”

    Dr. Lu’s group indicates that it is exploring multiple means of curbing antibiotic resistance. Besides the CRISPR approach reported in the Nature Biotechnology study, the MIT-based team is developing a strategy called combinatorial genetics en masse (CombiGEM).

    CombiGEM was detailed August 11 in the Proceedings of the National Academy of Sciences, in an article entitled “Enhanced killing of antibiotic-resistant bacteria enabled by massively parallel combinatorial genetics.” This article described how CombiGEM could be used to search for genetic combinations that sensitize bacteria to different antibiotics.

    “We created ∼34,000 pairwise combinations of E. coli transcription factor (TF) overexpression constructs,” wrote the authors. “Using Illumina sequencing, we identified diverse perturbations in antibiotic-resistance phenotypes against carbapenem-resistant Enterobacteriaceae. Specifically, we found multiple TF combinations that potentiated antibiotic killing by up to 106-fold and delivered these combinations via phagemids to increase the killing of highly drug-resistant E. coli harboring [NDM-1].”

    “This platform allows you to discover the combinations that are really interesting, but it doesn’t necessarily tell you why they work well,” Dr. Lu said. “This is a high-throughput technology for uncovering genetic combinations that look really interesting, and then you have to go downstream and figure out the mechanisms.”

    Whether using CRISPR to knock down genes that confer antibiotic resistance, or identifying gene combinations that work together to enhance antibiotic resistance, Dr. Lu’s group hopes to contribute to the urgent search for new treatments against drug-resistant bacterial infections. “This is a pretty crucial moment when there are fewer and fewer new antibiotics available, but more and more antibiotic resistance evolving,” Dr. Lu explained. “We’ve been interested in finding new ways to combat antibiotic resistance, and these papers offer two different strategies for doing that.”

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