PROJECT DESCRIPTION

On the forefront of modern medical research is the search to end the proliferation of superbugs, or harmful antibiotic-resistant pathogens. Most likely due to the over-administration of antibiotics, infectious bacteria have developed resistance to traditional treatments, which is a growing danger to public health. A 2013 report from the Centers for Disease Control and Prevention states that at least 2 million people in the United States alone develop an infection from antibiotic-resistant bacteria. And each year, 23,000 Americans die from these infections ¹.

Currently, the only way to treat antibiotic-resistant infections is by administering other antibiotics—oftentimes more aggressive, expensive ones—to which the strain hasn’t developed resistance. If bacteria develop resistance to every type of antibiotic we have, then there’s simply no antimicrobial agent we can prescribe to fight the infection. New antibiotics can be developed, but the R&D process is markedly slower than the evolution of pathogenic bacteria, and if we’re going to fight pan-resistant infections, we need a solution soon.

Is it possible to combat antibiotic-resistant pathogens some other way?

Scientists have discovered a way to utilize CRISPR-Cas9, a system that allows direct modifications to an organism’s genome, to cut genes coding for antibiotic resistance in pathogens ². If these genes are located in their genomes, they die immediately; if these genes are located in a plasmid (a circular piece of DNA separate from the genome), the harmful bacteria are left defenseless against existing antibiotic treatments.

However, an effective way to deliver the CRISPR system to pathogens in infected individuals has yet to be discovered. Each of the methods previously investigated suffers from important drawbacks. Bacteriophages are too large; infected tissues are not permeable to most phages ³. Hydrodynamic injection causes temporary cardiac dysfunction, increased blood pressure across the liver, and significant expansion of the liver when tested on rats ⁴. Lipid-mediated transfection has never been shown to work for bacteria ⁵.

What do CRISPR Capsules have to do with it?

Northwestern iGEM’s CRISPR Capsules proposes solving the problem of delivery by packaging CRISPR-Cas9 in bacterial outer membrane vesicles (OMVs), which are secreted from the membranes of gram-negative bacteria. Bacteria use these vesicles for many applications, including inoculating other bacteria with DNA and delivering proteins to competing strains ⁶.

NU iGEM has worked on identifying a protein translocation mechanism capable of transporting the Cas9 protein to the periplasm (the space between the inner and outer membranes) of E. coli. During OMV formation, proteins in this space become encapsulated in the vesicles. By harnessing the properties of a strain of E. coli that has been genetically modified to hyper-produce OMVs, large quantities of vesicles containing Cas9 can be secreted from the cell body.

Further developments in genetically modifying the membrane proteins of E. coli to lower toxicity need to be characterized before Cas9-encapsulating OMVs can be delivered to pathogens in an infected patient. However, it has been shown that E. coli can be genetically modified to become devoid of a toxin that is responsible for septic shock in humans ^7,8. Because of such innovations, we foresee CRISPR Capsules being delivered topically, orally, or as a suppository, among a broad range of other clinical delivery possibilities.