Endless possibilities

What is CRISPR?

ECRISPR, which stands for “clustered regularly interspaced short palindromic repeats,” was originally discovered as a defense mechanism by bacteria to protect against viruses. The DNA repeats are interspersed with small segments called “spacers,” which bacteria take from viruses and use as a “memory” to recognize and stop viruses that attack again. A portion of the CRISPR section is then copied and processed into CRISPR RNA (crRNA). The strand of crRNA guides the Cas protein, or “CRISPR associated” protein, to cut and deactivate the virus’ DNA.
The technology today known as CRISPR-Cas is a toolbox of enzymes adapted from that “bacterial immunity” system and was first developed in 2012. Scientists use CRISPR to precisely cut DNA and then generate a desired change that is incorporated when the DNA repairs itself. Using this tool, possibilities are endless.

Engineering research is creating CRISPR breakthroughs

Few technological developments have created as much of a buzz as CRISPR, the gene editing tool that, like scissors, can cut DNA, making it much easier to create edits that can cure severe genetic and infectious diseases, or improve crop resistance to disease and drought.

In just a few years, CRISPR has revolutionized biological sciences and opened avenues across engineering, biomanufacturing and medicine that might have never been possible — or at least not as easy.

At the College of Engineering, faculty members in the Department of Chemical and Biomolecular Engineering (CBE) are growing a rich portfolio of CRISPR-focused technologies by introducing novel CRISPR-Cas reagents, using CRISPR techniques to engineer new organisms and developing new bioprocesses to mass manufacture CRISPR products in anticipation of a slew of clinical approvals from the U.S. Food and Drug Administration.

“I think it is so unique of NC State to be the home of developers, manufacturers and users of CRISPR,” said Stefano Menegatti, assistant professor in CBE. “These three groups of scientists must listen to each other and work with each other to ensure the success of the CRISPR revolutions.”

Developing, using and manufacturing CRISPR at NC State

Rodolphe Barrangou, the Todd R. Klaenhammer Distinguished Professor in the Department of Food, Bioprocessing and Nutrition Sciences within the College of Agricultural and Life Sciences, leads the CRISPR Lab at NC State. He was one of the first to show how bacteria incorporated new spacers into their CRISPR regions after viral attacks in a seminal 2007 paper, and has remained one of the field’s leading researchers.

“(NC State) actually used to be quite ahead of the curve, in covering the whole CRISPR spectrum,” he said. “Others have caught on since then. And it’s been democratized enough that other universities can aspire to catch up, is a nice way to put it.”

As a land grant university, NC State is well-positioned to turn CRISPR discoveries into usable technology.

“That’s where engineering programs come in, so we can take some of that science and turn that into tech. CRISPR, the immune system to CRISPR, the genetic tech,” said Barrangou. “And then if you are applications-minded, if you are real world, Grand Challenges, humanity-minded, like we are, some of what you should do is apply that technology and deploy it toward addressing real problems.”

Within CBE, Menegatti and fellow assistant professor Nathan Crook, along with associate professor Chase Beisel, are working on developing scalable tools that will increase the availability of CRISPR reagents — such as the CRISPR-Cas proteins and the guide RNA — that are needed to implement CRISPR technologies.

Crook’s research focuses on using CRISPR to engineer microbes to improve human and environmental health. For example, microbes in our gut can be engineered to degrade a toxin. In the ocean, they can be made to break down microplastics. Within roots, engineered microbial bacteria can help plants grow faster.

“CRISPR is a great tool because it’s basically scissors that you can guide to the genome in a very specific way,” Crook said. “Before CRISPR, that ability was very hard to come by. CRISPR, if you design it right, will cut in exactly one place and nowhere else.”

On the biological side, Beisel’s research group is continuing to examine how bacteria use CRISPR to fight off attacks and how their findings can be translated to better CRISPR technologies. Many bacteria have native CRISPR systems, and while Cas9 is the protein most commonly associated with gene editing, it’s one of many that can cut DNA, Beisel explained.

“My group has been trying to find other ones that exist in nature, and have functions that we could use for different technology purposes,” he said.

As CRISPR tools become more widely understood and utilized by research groups, industries, and — eventually — clinics, the technology to produce them must keep up. That’s where the biomanufacturing side comes in, which both Crook and Beisel agreed is strong at NC State.

Menegatti and his team are developing technology that will make it easier to scale up the production of CRISPR tools to further advance science and address real-world problems.

His lab develops “affinity adsorbents,” namely filter-like materials functionalized with synthetic binders known as “ligands,” to purify biological therapeutics from complex sources. In his recent project related to CRISPR, he developed an affinity adsorbent to selectively purify CRISPR-Cas9 from bacterial cell culture fluids.

“To our knowledge, this is the first adsorbent ever made for the scalable purification of CRISPR Cas proteins,” he said.

This process is just one of a series of ligands Menegatti is developing to make the purification of biomolecular pharmaceuticals cheaper and faster.

“Our goal is to develop today the affinity technology that will serve the pharmaceutical industry of tomorrow, by providing it with a toolbox that will accelerate, simplify and reduce the cost of the bioseparation segment of the biopharmaceutical manufacturing pipeline,” he said. “This way, more drugs with higher efficacy and safety will become available to more people.”

The big picture

Taken all together, CRISPR is a complex and exciting technology, with so much untapped potential. With support from the Office of Research Commercialization, NC State and COE faculty members have launched new companies from their research, including Locus Biosciences, co-founded by Barrangou and Beisel, and LigaTrap, co-founded by Menegatti.

The toolboxes and frameworks are there — with more in development — for future breakthroughs that include improved pest management and crop disease resistance that ensure global food supply keeps up with demand, as well as better treatments of diseases.

“What excites me is that on one hand, we’re studying this really quirky, fascinating defense system that’s in bacteria,” said Beisel. “But at every turn, there can be a new discovery that becomes an entirely new technology, but also technology that can have a very real and immediate impact on society.”


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