By Caleb O’Brien | MU Bond Life Sciences Center

 

James Amos-Landgraf, assistant professor of comparative medicine and genetics at the University of Missouri, is helping develop a pig model for colon cancer using CRISPR. //photo by CALEB O'BRIEN/Bond LSC

James Amos-Landgraf, assistant professor of comparative medicine and genetics at the University of Missouri, is helping develop a pig model for colon cancer using CRISPR. //photo by CALEB O’BRIEN/Bond LSC

James Amos-Landgraf needed a pig.

The assistant professor of comparative medicine and genetics at the University of Missouri had joined forces with a startup company developing a tool to detect early colon cancer-causing lesions. They already tried out a rat-sized model, but still needed a full-sized prototype.

Scientists in Europe had an ideal pig model for colon cancer, but importing the animals presented a problem. It would be prohibitively expensive and time consuming, and the method European scientists used to develop the pig took several years and cost a great many Euros, Amos-Landgraf said.

Those obstacles might have been enough to scuttle the project entirely, but CRISPR, a new gene-editing tool discovered in the DNA of a peculiar bacterium, has changed the equation for scientists everywhere.

So when Amos-Landgraf went to the National Swine Resource and Research Center (NSRRC) to ask about importing pigs, they told him, “‘We can just make you the model,’” Amos-Landgraf said. “‘We should be able to do a CRISPR project within a few months.’”

CRISPR is rapidly reshaping the way biologist around the world do their jobs.

At Mizzou, it’s transforming how researchers learn about viruses and mosquitoes, pigs and zebrafish, and the individual genes affecting development, sickness and health. The tool makes research more efficient, cost-effective and vastly more powerful.

Amos-Landgraf knows firsthand just how time-consuming and laborious generating an animal model was pre-CRISPR.

“What was almost a two-year process just to generate an animal now would take us a matter of months,” Amos-Landgraf said. I think the CRISPR revolution is going to be amazing for all of science. I’m totally intrigued by everything that’s going on with this.”

Borrowing a bacterial relic

CRISPR rolls off the tongue far more readily than its unabbreviated equivalent: “clustered regularly interspaced short palindromic repeats.” The name refers to a strange pattern scientists at the University of California, Berkeley noticed in the genome of a bacterium that lives in acidic, abandoned mines: groups of palindromic bacterial DNA sequences interspersed with segments of viral DNA.

It turned out that the genetic snippets were relics of the bacteria’s prior run-ins with viral invaders, like genetic mug shots on a most-wanted list.

Viruses are tiny packages of genetic material that hijack cells, such as bacteria, in order to reproduce. And when a virus enters a bacterial cell, the host compares the virus’ genetic material to the snapshots preserved in the bacteria’s own DNA. If they match, the bacteria dispatches a bounty-hunter protein called Cas9, which tracks down the virus and slices its DNA in half at the very spot that matched the virus’ genetic fingerprint.

If an unfamiliar virus attacks and the bacterium survives, it will incorporate a segment of the invader’s DNA into its own, adding a new battle scar to its DNA and a new miscreant to the most-wanted list.

When the researchers studying the bacterial immune system figured out how it worked, they realized the process could have implications far beyond the organism’s acidic abode: It could become a powerful, inexpensive, and versatile gene-editing tool.

A ground shift

The journey to better manipulate genes has been a long one.

For decades, scientists relied on various techniques and tricks to tease out the function of genes. The most common tool is forward genetics, where a researcher starts with an interesting characteristic in an organism and then hunts for the gene that caused it. Those characteristics could be traits that occur naturally, such as genetic diseases in purebred dogs or pigmentation in corn kernels, or a scientist could induce defects — essentially altering an organism’s genome by exposing it to a bath of nasty chemicals.

Bond Life Sciences Center Biologist Anand Chandrasekhar and PhD candidate Suman Gurung take a look at some zebrafish they've modified using CRISPR. //photo by CALEB O'BRIEN/Bond LSC

Anand Chandrasekhar, Bond Life Sciences Center biologist and professor in the division of biological sciences, and PhD candidate Suman Gurung take a look at some zebrafish they’ve modified using CRISPR. //photo by CALEB O’BRIEN/Bond LSC

Imagine that an organism is like a car, suggests Anand Chandrasekhar, a Bond Life Sciences Center biologist and professor in the division of biological sciences.

“You take a car that is running nicely and you have some kind of weird mechanic from Hell come in and mess something up — just one thing — and the car doesn’t run. Then you have to figure out why the car doesn’t run by looking carefully for where the defect is.”

Reverse genetics — unsurprisingly — starts on the other end. Researchers pick a gene of interest and try to silence or alter it. If they succeed, then they look for changes in the organism that suggest the altered gene plays a role in the observed characteristic.

This tool shaped how scientists do research and what animals they use in their labs. In fact, model organisms such as mice rose in popularity partly because of how easily reverse genetic techniques like homologous recombination work with them, said Amos-Landgraf. But this approach was time consuming, expensive and didn’t function well on other organisms.

The next step forward were Zinc-Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). Both act like guided missiles to strike at a gene of interest, targeting a specific region of genetic material and breaking both strands of the organism’s DNA at that spot. Once the DNA is broken, the cell’s natural repair mechanism intervenes and stitches the gene back together.

However, the process is prone to errors — mutations — that can alter or silence a gene. ZFNs and TALENs reliably worked in a broader array of species.

CRISPRs represent the next advancement in this process and is far faster than previous techniques.

“Let’s say if you had 15 or 20 genes that you wanted to study: You could design a CRISPR reagent for each one of them in a couple of afternoons, whereas in the ‘olden days’ (three or four years ago) with TALENs that could have taken you months,” Chandrasekhar said. “And if you were using ZFNS… you would not even imagine doing it, because you would have been crazy.”

Elizabeth Bryda, a professor of veterinary pathobiology at MU who heads the Rat Resource and Research Center, stands by a Genetic Analyzer machine. //photo by CALEB O'BRIEN/Bond LSC

Elizabeth Bryda, a professor of veterinary pathobiology at MU who heads the Rat Resource and Research Center, stands by a Genetic Analyzer machine. //photo by CALEB O’BRIEN/Bond LSC

At MU’s NIH-funded Rat Resource and Research Center (RRRC), scientists think CRISPR will help break dependence on default model organisms. The RRRC is the only center of its kind in the US and one of two in the world, serving as a repository and distribution center for rats that model human diseases.

“We’re always preaching, use the species that’s most appropriate for the question you’re asking,” said Elizabeth Bryda, a professor of veterinary pathobiology at MU who heads the RRRC. “If you’re studying human disease, use the species that best recapitulates that disease. I think CRISPR will give people the flexibility to really work in the species they want to be working in.”

For example, the center is using CRISPR to develop rat models of human inflammatory bowel diseases, such as Crohn’s disease. “All of those barriers to making rat models are no longer issues,” Bryda said, “CRISPR is easy and finally allows us to manipulate rats in ways we haven’t before.”

That’s good news for the RRRC: “I do think we’re going to see a huge increase in the number of rat models,” Bryda said, “which would increase our inventory.”

Seeing through the zebrafish

Zebrafish are another model organism that might become even more important thanks to CRISPR.

Originally found in the rice paddies and streams of India and Myanmar, the minnow-like fish is an important model organism. They’re easy to care for, produce abundant offspring and — because their embryos are transparent — make great tools for studying development.

Chandrasekhar uses zebrafish to study cranial motor neurons, the neurons that connect to, and control, muscles in the head. His lab is especially interested in the way those cranial motor neurons are deployed during development: how the neurons know where to go and to which muscles they should link.

“CRISPR is a really big boon for research, because now even small labs can test tens of genes over a short period of time for their effect on a particular biological process,” Chandrasekhar said. “That’s how we use it: We study the process of cell migration within a nervous system, and we want to study a whole slew of associated genes.”

Researchers have identified hundreds of new and potentially important genes using advance genomics, but the old techniques of reverse genetics were too slow and tedious to keep up with the new discoveries.

“CRISPR has removed the bottleneck,” Chandrasekhar said. “We can rapidly go through and, hopefully, find new genes and new signaling pathways that might be playing a very specific role for the migration and the biological process that we study.”

But finding a new gene is just the beginning, Chandrasekhar said.

“We have one student who is testing five genes, and if even one or two of those genes turn out to be important, that will then be sufficient for the lab to continue working on them for two or three years.”

Although scientists primarily use CRISPR as Chandrasekhar does, to silence genes in model organisms, new genes can also be introduced.

Through a process called homologous directed repair, scientists select a location where they want to introduce a gene and design a CRISPR to target that region.

Daniel Davis, a PhD candidate and lab manager for Assistant Professor of Veterinary Pathobiology Catherine Hagan, is developing a technique to screen potential antidepressant drugs by leveraging CRISPR technology and the advantages of the zebrafish. //photo by CALEB O'BRIEN/Bond LSC

Daniel Davis, a PhD candidate and lab manager for Assistant Professor of Veterinary Pathobiology Catherine Hagan, is developing a technique to screen potential antidepressant drugs by leveraging CRISPR technology and the advantages of the zebrafish. //photo by CALEB O’BRIEN/Bond LSC

Daniel Davis, a PhD candidate and lab manager for Assistant Professor of Veterinary Pathobiology Catherine Hagan, is developing a technique to screen potential antidepressant drugs by leveraging CRISPR technology and the advantages of the zebrafish.

When a zebrafish is stressed, it produces a neurotoxic compound, but when the fish is calm, it produces a different compound, one that is neuro-protective. The difference depends on which key enzyme the fish produces — in a stressful situation, the fish produces more of the enzyme that leads to neurotoxicity.

Davis is using CRISPR to try to link different fluorescent proteins genes to each branch of this stress pathway: If the fish produces more of the stressful compound, it will also produce a red fluorescent protein. If the other pathway is taken, the fish will assemble green fluorescent protein.

“If you take some fish, subject them to a stressor and test a variety of potential therapeutics on them, you could visualize the fluorescent proteins to see which therapeutics are more protective,” Davis said.

Altering the host to understand the virus

 Other models present special challenges. In mosquitoes, for instance, it’s hard to knock out genes from its genome using traditional methods.

“The problem is that in mosquitoes such as Aedes aegypti, ‘traditional’ knockouts never really worked, so people tried out new techniques such as ZFNs and TALENs,” said Alexander Franz, assistant professor of veterinary pathobiology at MU. But the other techniques had flaws, too: they were expensive, complicated to assemble and often posed issues of efficiency and specificity.

Franz studies arthropod-borne viruses (arboviruses), specifically dengue virus and chikungunya virus. The life cycle of an arbovirus requires its circulation between arthropods, such as mosquitoes, and vertebrate hosts, such as humans. Because vaccines exist for only a few mosquito-borne viruses — yellow fever and Japanese encephalitis, for example — people usually rely on conventional and often ineffective environmental controls to thwart disease: bed nets, the elimination of breeding areas, insecticides.

Alexander Franz (far right), an assistant professor of veterinary pathobiology at MU who demonstrated that CRISPR is effective in mosquitoes, stands with his lab. From left to right: Velmurugan Balaraman, Asher Kantor, Hannah Gerlt, and Shengzhang Dong. //Photo by Alexander Franz

Alexander Franz (far right), an assistant professor of veterinary pathobiology at MU who demonstrated that CRISPR is effective in mosquitoes, stands with his lab. From left to right: Velmurugan Balaraman, Asher Kantor, Hannah Gerlt, and Shengzhang Dong. //Photo courtesy of Alexander Franz

Franz is pursuing a different avenue for protection that uses genetic manipulations to interrupt the transmission cycle of a virus in the mosquito.

“If you can stop the virus from taking hold in the mosquito, you can block transmission of the virus to its vertebrate host,” he said. “But to do so, you need an effective way to manipulate the mosquito’s genome.”

This is where CRISPR comes into play. “When people started reporting using the CRISPR system for genome editing in Drosophila or zebrafish, we immediately had the idea to try it out in mosquitoes.” Working with two postdocs, Franz demonstrated for the first time that the CRISPR system was capable of disrupting genes in mosquitoes.

To do so, he started with a line of transgenic mosquitoes that had already been modified to produce red and blue fluorescent proteins in their eyes. The lab designed a CRISPR to silence the gene responsible for the blue fluorescent protein. After trying a few different methods, they found a technique that turned off the target gene when they injected the CRISPR into mosquito embryos.

Because it is a very powerful and easy-to-handle genome editing technique, CRISPR has been recently utilized and further developed by other groups studying mosquito-pathogen interactions.

Other MU researchers focus on the viral interaction with human host cells.

Marc Johnson, associate professor of molecular microbiology and immunology at the Bond Life Sciences Center, studies the way a virus puts itself together inside a host cell and fights off the cell’s defenses. //photo by CALEB O'BRIEN/Bond LSC

Marc Johnson, associate professor of molecular microbiology and immunology at the Bond Life Sciences Center, studies the way a virus puts itself together inside a host cell and fights off the cell’s defenses. //photo by CALEB O’BRIEN/Bond LSC

Marc Johnson, associate professor of molecular microbiology and immunology at the Bond Life Sciences Center, studies the way a virus puts itself together inside a host cell and fights off the cell’s defenses.

“We don’t know all the cellular genes, cellular machinery and cellular pathways that viruses are harnessing,” Johnson said. “The best way to say that a virus requires a particular gene would be to knock it out of the cell and see if the virus can still replicate.”

“CRISPR is a real ground shift in how we can do science,” he said. “Things that took 6 months to a year to make one gene before, now we can do half-a-dozen in a week.”

The technique has altered the rate at which Johnson’s research proceeds and expanded the scope of his lab’s work. “It’s allowed me to take a step back and think about the whole genome, as opposed to being totally focused on this one thing or that one thing,” Johnson said. “I’d never really taken a step back to think about the whole genome — every gene, where are they and what families. It’s changed my outlook on the cell, the way I can think about it.”

The CRISPR era

 Amos-Landgraf and the researchers at NSRRC are still in the process of validating their pig model: developing primers to identify the mutation and creating the CRISPRs themselves. Once everything is ready, they’ll test out the lesion-detecting colonoscope, and if all goes well, move into human trials — far faster and more economically than would have been possible a few years ago.

But Amos-Landgraf is tantalized by the possibilities the technology offers beyond increased speed and reduced costs: To be able to tease apart not just a single gene in a pathway, but maybe think about knocking out or altering all the genes in a pathway and looking at combinations of those pathways… You can start thinking about multiple gene knockouts, multiple gene manipulations all within the same experiment,” he said.

“And that is not only cost saving, but it becomes a really powerful tool when you want to interrogate biology. We’ve entered a new era of genetics and genomics.”