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Microbial Magic

The Potential of CRISPR-Cas Gene Editing Technology

Five years ago, the term CRISPR-Cas was familiar to only a handful of microbiologists. Today, thousands of scientists around the world are using this novel gene editing technology to advance research in basic science, medicine, agriculture, and industry.

The CRISPR-Cas technology is simpler, quicker, more reliable and less expensive than other gene editing methods, and it has brought forth new prospects for curing genetic diseases like cystic fibrosis, muscular dystrophy, and sickle cell anemia.

The explosive growth of CRISPR-Cas technology has also spawned several new biotechnology companies—as well as a patent dispute with billions of dollars at stake. With two separate research teams claiming ownership of the gene editing technology, it will be up to the U.S. Patent and Trademark Office to decide who invented CRISPR-Cas first: a team at the University of California, Berkeley, led by Jennifer Doudna or scientists at the Broad Institute (associated with the Massachusetts Institute of Technology and Harvard University) led by Feng Zhang.

No matter who wins the patent—and probable Nobel Prize—it is clear that the CRISPR-Cas process did not simply appear out of thin air. In fact, it was adapted from existing functions many bacteria use to survive. While microbes have evolved these functions over millions of years, today we humans exploit them in laboratory and kitchen alike for our own purposes.

The story of CRISPR-Cas begins in the kitchen, where both chemists and cooks use microbes in starter cultures to produce wine, beer, bread, vinegar, sauerkraut, pickles, soy sauce, cheese, and yogurt. In cheesemaking, for example, as the microbial culture grows in milk it converts the sugar lactose into lactic acid, thereby ensuring the correct level of acidity, helping to curdle the milk, and suppressing the growth of harmful microbes that might spoil the cheese. As the cheese ripens, the microbial culture gives it a balanced aroma, taste, and texture (it is also responsible for the “holes” in Swiss cheese). 

But a culture can be easily disrupted by viruses called bacteriophages (or phages) that infect bacteria. These viruses are, unfortunately, ubiquitous and can interfere with the production of foods that rely on a starter culture. 

When a bacteriophage infects a bacterial cell, it injects its genetic material (usually DNA) into the cell and forces the cell to use viral genes instead of its own genes to produce more viruses, which go on to infect more bacterial cells. If bacteriophages get into a batch of yogurt in a dairy plant, they may kill bacteria in the starter culture, causing the resulting yogurt to be runny or grainy, or otherwise unacceptable.

To counter this problem, food ingredient companies like Danisco (now owned by DuPont) spend a lot of time and money on ways to make the bacteria in starter cultures resistant to phage infection. In the early 2000s, a group of Danisco scientists in Madison, Wisconsin, together with colleagues in Dangé-Saint-Romain, France, were looking at an unusual genetic sequence called CRISPR in bacteria as a way to identify particular strains.

First described by Spanish researcher Francisco Mojica in the early 1990s, CRISPR (short for clustered regularly interspaced short palindromic repeats) consists of a series of identical 29-base repeats interspersed with unique 32-base sequences called spacers along a strand of DNA. At the time of discovery, CRISPR was considered to be a genetic anomaly of no particular value.

Yet when the Danisco team infected Streptococcus thermophilus (an important bacterium for dairy cultures) with phages, they found that the surviving bacterial cells were identical to the parent cells with one difference: there was extra DNA in the CRISPR sequences that matched the viral DNA. The Dansico researchers had discovered a sort of bacterial immune system that could remember dangerous viruses.

Horvath and Barrangou’s team was the first to demonstrate a direct biological relationship between CRISPR and phage resistance. It works like this: When a virus infects a bacterial cell, the cell clips a bit of the viral DNA and stores it as a CRISPR spacer—a sort of viral mug shot. If the cell is ever infected by the same virus, the cell is able to recognize that virus by making an RNA copy of the mug shot and combining it with a Cas (CRISPR-associated) protein. The pair form a DNA-cutting enzyme called a nuclease that recognizes a virus that is returning to the scene of the crime and cuts the invader’s DNA using the Cas protein, thereby stopping the phage infection. It also turned out that subsequent generations of bacteria keep the mug shot in their CRISPR spacer and their resistant properties, which meant that the Dansico group had discovered a way to alter an entire bacterial germline. 

Today commercial producers can take advantage of the natural CRISPR-Cas system to provide dairy cultures that are already “immunized” by exposure to common bacteriophages and contain the desired CRISPR sequences to make them resistant. 

“It was so simple,” says Dennis Romero, who was part of that Danisco team and is now principal senior scientist in the Department of Culture Development at DuPont Nutrition & Health in Madison. “We had the bacteria, exposed them to phage, and looked at what survived.”

It was an elegant solution to a pervasive problem and all the genetic modification was done by the bacteria themselves, as they likely have been doing since before humans existed. As Romero put it, “Nature finds the simplest and most efficient system.”

In 2011, Jennifer Doudna (University of California, Berkeley) and Emmanuelle Charpentier (Umeå University, Sweden) began studying how the CRISPR sequences and the Cas protein worked together. What they discovered would change the biotechnology field forever.

The two researchers wondered if the method bacteria had evolved to cut phage DNA in specific spots could be adapted to other cells, even other species. Doudna and Charpentier tinkered with the system and, as they reported in 2012, it was indeed possible to cut DNA anywhere by changing the sequence of the guide RNA that associates with the DNA-cutting Cas protein.

It turned out that Cas was a programmable nuclease that could add, inactivate, or modify genes much more easily, cheaply, and quickly than anything being used in labs at the time. To use a computer analogy, the Cas protein is like the hardware. To edit a new gene with CRISPR, all that was required was new software, a short RNA sequence. Previous systems (using techniques called zinc fingers and TALENS) required an entirely new protein for each new genetic target, which would be analogous to building a different computer for each gene. [See infographic here.]

Within months, researchers at the Broad Institute had used CRISPR-Cas to edit genes in cultured cells from mice and humans. Researchers at the University of Wisconsin–Madison were quick to see the potential of the technique. A collaborative effort that included the laboratories of Melissa Harrison (assistant professor of Biomolecular Chemistry), Kate O’Connor-Giles (assistant professor of Genetics), and Jill Wildonger (assistant professor of Biochemistry) resulted in the first published account of CRISPR-modified fruit flies. Before long, genes were being edited in frogs, mice, fish, roundworms, pigs, corn, rice, and tobacco.

“It just became a flood,” says Romero. Work could now be done in weeks instead of years, with hundreds of dollars instead of hundreds of thousands of dollars.

CRISPR-edited cells and organisms can be used to answer fundamental questions about how genes and cells work, providing insight into the ways cells regulate their growth, interact with each other, or sense what’s going on around them. At UW–Madison, modified fruit flies are being used to study the development of embryos and the nervous system.

The technique has the potential to make current animal models for human disease much more precise. For example, if epidemiological evidence links a particular genetic change with a disease, that genetic change can be introduced into an experimental animal in the same place as the normal gene. If the animal model mimics the human disease, it can be studied to see how the disease progresses and find potential targets for drugs. 

Around the world, CRISPR is being used to study cardiovascular disease, diabetes, AIDS, cancer, and a host of other diseases, but its usefulness is not limited to the medical sciences. Edited genes are producing virus-resistant pigs, non-allergenic peanuts, drought-tolerant crops, and malaria-resistant mosquitoes. Trees are being developed that would be easier to convert into biofuels. Using CRISPR-Cas technology, researchers can also tweak cells to produce more of a desired product, such as alcohol, insulin, or antibiotics. 

Investigators continue to make technical advances in the CRISPR-Cas field. Huge computer databases allow them to design the most precise guide RNAs, while high throughput systems are able to test thousands of genes at a time. New Cas proteins are being discovered, and others are being engineered to improve their accuracy or efficiency.

“It certainly is an exciting time at UW–Madison to be the ‘CRISPR guy’,” says C. Dustin Rubinstein. After a postdoctoral stint in the O’Connor-Giles fruit fly lab, Rubinstein was hired as the director of the Translational Genomics Facility, which, along with the Transgenic Animal Facility, makes up the genome-editing core of the UW–Madison Biotechnology Center. The facility connects investigators with the tools and expertise they need to use CRISPR-Cas for their particular research efforts.

Researchers work with Rubinstein to design guide RNA that will produce the most accurate editing and to obtain and purify the materials they will need for their purposes. If the guide RNA is to be used to create an animal model, it is handed off to the staff of the Transgenic Animal Facility to be microinjected into animal embryos at the single-cell stage.

Animal models are crucial to biomedical research, but they are not perfect models for human disease (after all, they aren’t human). Human stem cell research can help bridge that gap. Krishanu Saha, Assistant Professor of Biomedical Engineering at UW–Madison, works with human induced pluripotent stem (iPS) cells. Unlike embryonic stem cells, iPS cells are body cells—such as skin or blood cells—that are reprogrammed so that they can be coaxed into becoming almost any kind of cell. They can be grown in culture to become microtissues or organoids, which are collections of cultured human cells that emulate the structure and functions of specific tissues or organs.

Saha uses CRISPR-Cas to induce genetic defects, producing “diseases in a dish” on which he can test drugs and study disease processes in very controlled conditions. He and researchers at the Waisman Center use these methods to study neurological disorders, such as ALS (amyotrophic lateral sclerosis), fragile X syndrome, Rett syndrome, and Down syndrome, as well as stroke and diseases of the retina.

Saha, Rubinstein, Harrison, O’Connor-Giles, and Wildonger, along with faculty, staff, and students across campus participate in a collaborative effort called Genome Editing and Engineering at Wisconsin. GEEwisc allows them to compare notes, troubleshoot, and work together, bridging basic and applied genetic research.

The rapid rise of CRISPR in research labs has been “a big boon to the local [Madison] economy,” says Rubinstein. Biotechnology companies see that there is new revenue to be generated keeping laboratories supplied with the reagents they need.

As the use of CRISPR-Cas technology expands in academia and industry, there will be increased need for workers skilled in these new techniques. Madison College has this covered. Madison College is the first and only two-year college in the nation to prepare students to be professionals in the area of human embryonic stem cell technology. Opened in 2013, their 2,700-square-foot Advanced Cell Culture Education Suite supports stem cell biotechnology education and hosts courses and program offerings in cell culturing, protein purification and molecular biology.

Under the guidance of project director Thomas Tubon and with funding from the National Science Foundation, the Human Stem Cell Technologies Education Initiative at Madison College is developing curricula to give students hands-on experience with stem cells and CRISPR-Cas gene editing. These “cutting-edge curricula,” says Tubon, “will be more feasible and affordable to bring into the classroom” and will be integrated into biotechnology programs across the country. The initiative also cultivates interest in biotechnology in younger students by offering opportunities for grades K–12 to work with stem cells and CRISPR. 

The simplicity, power, and low cost of CRISPR-Cas technology, while holding the potential for important discoveries and therapies, also raises some difficult questions. Could so called “biohackers” edit genes in their garages? What happens if CRISPR-ized organisms are released into the environment? And what about changes to the human germline—changes that can be inherited?

A report last year that a group of scientists in China had used CRISPR to make changes in human embryos created controversy and widespread discussion about the ethical repercussions of such a powerful tool, hinting at the possibility of eugenics or “designer babies.” There were also questions about its safety and potential ecological impact. Similar concerns were raised when genetic engineering first became possible and a meeting was held to address these issues at Asilomar Beach, California, in 1975.

With a nod to the Asilomar Beach meeting, the International Summit on Human Gene Editing brought experts from many disciplines to Washington DC in December 2015. Four UW–Madison faculty—Krishanu Saha, Pilar Ossorio (professor of Law and Bioethics), Alta Charo (professor of Law and Bioethics), and Dietram Scheufele (professor of Life Sciences Communication)—attended the summit. [Editor’s note: Both Charo and Scheufele are recognized as Wisconsin Academy Fellows for their public service and achievement in respective fields.] The summit’s organizing committee produced a set of professional guidelines, proposed a moratorium on inheritable changes in the human germline, and encouraged continued discussions as the technology evolves. 

For a biotechnology tool that is less than five years old, CRISPR-Cas has made a huge impact in basic and applied research in medicine, agriculture, and industry. The journal Science named CRISPR-Cas its 2015 “Breakthrough of the Year.” We will have to wait to see if, as some have predicted, it will become the Breakthrough of the Century. 

Contributors

Jacqueline Houtman is a freelance biomedical science writer and editor based in Madison.

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