BOZEMAN – A team of scientists from Montana State University has contributed to research recently published in two leading scientific journals that advances the understanding of how bacteria fend off viruses.
MSU senior Joshua Carter was a lead author of a paper published March 30 in the journal Cell, and MSU research associate MaryClare Rollins was lead author of a paper published April 24, in the journal Proceedings of the National Academy of Sciences, or PNAS.
Carter and Rollins conduct research in the Blake Wiedenheft Lab. Wiedenheft is an assistant professor in MSU’s Department of Microbiology and Immunology in the College of Agriculture and the College of Letters and Science. The Wiedenheft lab focuses on CRISPR research, a biotechnology that has implications in fighting infectious disease and genetic disorders. CRISPR is an acronym for “clustered regularly interspaced short palindromic repeats.”
Wiedenheft explained that CRISPRs function as molecular vaccination cards that snatch short fragments of viral DNA and use these DNA snippets to remember previously encountered viruses. Bacteria use this DNA-based memory to guide a “molecular scalpel” to chop up and destroy invading viral DNA.
“Scientists have ‘borrowed’ these immune systems from bacteria and repurposed them as programmable molecular scalpels that can be used to cut out genetic defects associated with human disease,” Wiedenheft said.
The paper published in Cell, “Structure Reveals Mechanisms of Viral Suppressors that Intercept a CRISPR RNA-Guided Surveillance Complex,” provides a snapshot of the battle between CRISPRs and their viral suppressors, called anti-CRISPRs.
“CRISPR-mediated immune systems rely on ‘ biological machines’ that find and destroy viruses, but viruses evolve their own machines, called anti-CRISPRs, that subvert or intercept these CRISPR-guided machines and essentially block immune function,” said Carter, an Honors College student from Watertown, South Dakota who is majoring in mechanical engineering in the College of Engineering and microbiology in the College of Letters and Science and the College of Agriculture. Carter is also minoring in biochemistry in the College of Letters and Science, is the recipient of a Goldwater Scholarship and a Rhodes Scholarship and has received funding from Montana INBRE.
Carter likened this battle to a molecular arms race, in which bacterial CRISPRs and viral anti-CRISPRs continuously evolve defense and counter-defense systems.
“This paper is exciting because we’re looking at this co-evolutionary ‘arms race’ at the molecular level,” Carter said. “We have a snapshot of these two organisms going back and forth and this tug-of-war to see who wins.”
By flash-freezing purified CRISPR-anti-CRISPR samples, the scientists were able to use powerful electron microscopes to image the samples at magnifications that allowed them to determine the location of every atom in these biological machines. This technique provided a molecular blueprint of the biological machines and helps explain the mechanisms that viral anti-CRISPR proteins use to block the bacterial immune system.
“No one has ever seen what these machines look like and really understood how they function at the molecular level, and that’s what these structures allow us to do,” Carter said.
In 2014, Wiedenheft led an MSU research team that provided the first molecular blueprint of a multi-protein CRISPR machine. The paper was featured in the Aug. 7 issue of the journal Science.
Carter explained that these anti-CRISPR proteins may have applications in tweaking or augmenting CRISPR machines that are being used to cure genetic diseases.
“These viral anti-CRISPRs could possibly be used as ‘off’ switches to more precisely control and modulate CRISPRs, expanding our toolset of what we can do with these CRISPR-guided machines and how well we can control them, which is really important when we think about using them for human health,” Carter said.
The PNAS paper, “Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity,” explains how the nuclease – the DNA scalpel -- is activated.
Rollins said she and her colleagues have determined the structure of one of these nucleases.
She said that most of the CRISPR-based genome editing work thus far has made use of Class 2 CRISPR systems, which are simpler CRISPR systems that consist of a single large protein called Cas9.
“This new paper reports on a more complex, multi-protein machine from a Class 1 system,” Rollins said “There may be advantages in some cases to using a Class 1 system for genome editing, but to do that we need to understand how the nuclease is being regulated. These new findings shed light on these processes.”
Rollins said this research has applications in the potential elimination of genetic diseases, such as muscular dystrophy and cystic fibrosis. Already, there are billions of dollars in commercial investments in CRISPR applications worldwide, as CRISPR systems have been moved into human cells and programmed to cut out and repair defective DNA in human cells, she added.
“CRISPR systems have become a really important biotechnological tool,” Rollins said. “The more we understand about the mechanism, the better and more specifically we can repurpose them for medical and industrial applications.”
With this research published, the Wiedenheft team will continue to research how detection of a DNA target leads to a molecular switch that activates the nuclease.
“We anticipate that a mechanical understanding of how these biological machines function will lead to new applications for genome engineering,” Wiedenheft said. “Transformative new innovations in industry and medicine come from unexpected places, and CRISPRs are great examples of how academic research adds value to our economy and to society.”
Other MSU co-authors of the two papers include research associates Sarah Golden and Royce Wilkinson and undergraduate student Connor Hoffmann, an MSU Presidential Scholar from Boise, Idaho, majoring in chemical engineering in MSU’s College of Engineering. Collaborators Alan Davidson and Karin Maxwell from the University of Toronto contributed to the work published in Cell, while scientist Gabriel Lander and Saikat Chowdury from The Scripps Research Institute were involved in both studies.
Research in the Wiedenheft lab is supported by the National Institutes of Health, the National Science Foundation EPSCoR, the M.J. Murdock Charitable Trust, Montana University System Research Initiative, The Gordon and Betty Moore Foundation and the Montana Agricultural Experimental Station.
Research reported in this publication was also supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM103474. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Blake Wiedenheft, firstname.lastname@example.org or (406) 994-5009