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Published On September 12, 2016

BASIC RESEARCH

Made to Measure

New gene-editing techniques let researchers create precisely the laboratory animals they need—but at what ethical cost?

Looking through a microscope, Youngnam Jin—a postdoctoral fellow in Randy Peterson’s lab at Massachusetts General Hospital’s Cardiovascular Research Center—arranges one-celled zebrafish embryos, each about 10 minutes old, into rows of 20, spinning them with a micro spatula to turn their nuclei toward the ultrathin glass needle of a micro-injection device. Jin moves efficiently down the line, puncturing the cell wall of each embryo and injecting the nucleus with a tiny drop of a molecular soup that will change the embryo’s genetic makeup. 

Someone like Jin, with good technique, can do 200 to 300 shots at a time, racing to finish before the cells start to divide. In zebrafish, that gives you about 10 minutes. After injection, the embryos are put back into a cell culture dish, where they divide and develop; after 30 hours or so, the hatched larvae look recognizably fishy under a microscope. Adult fish move into a sort of high-rise fish city, with hundreds of clear Plexiglas tanks stacked, barcoded and labeled with the mutation of the fish inside. 

This is CRISPR-Cas9 at work, as it is now used in countless research labs around the world. That injected substance, which contains a “guide and scissors” system for replacing precisely targeted pieces of genetic code, is the hottest biotechnology tool to come along in decades. CRISPR-Cas9 (CRISPR for short) lets scientists (or even amateurs with basic skills and equipment) open up the genome of any living thing to find, cut out and replace one or many genes with unprecedented precision and speed. 

Researchers at the University of California, Berkeley and the Broad Institute of Harvard and MIT separately published fundamental CRISPR methods in 2012 and early 2013. Since then, the number of published papers referring to CRISPR has ballooned from some 300 in 2013 to more than 1,400 in 2015. Scientists around the world have used and refined the system to tweak the DNA of a wide variety of organisms. A handful of biotechnology startups are vying to bring CRISPR-based therapies to market, hoping to cure cystic fibrosis and certain forms of blindness, among other conditions, by getting rid of the bad genes that cause them. CRISPR could also make it possible—practically, if not ethically—to create “designer babies,” gene-edited to a parent’s taste before being implanted in the womb. 

The frontier of using CRISPR on human subjects is likely to be filled with both promise and controversy. But the technology is already changing human health on another front, by allowing researchers to create genetically modified animals that can aid in understanding human diseases. CRISPR not only allows researchers to create these animals faster and more precisely than before, but also to move beyond a relatively small menagerie of standard model species—fruit flies, mice, zebrafish—to create a Noah’s ark of mutant dogs, pigs, primates and more. Unlike earlier, less flexible tools, CRISPR can work in just about any species. 

These animals could lead to unprecedented insights into the mechanisms of disease, and unexpected new treatments. Or, critics say, CRISPR could simply increase the number and variety of animals sacrificed in the name of science, with little to show for it. A 2006 study in the Journal of the American Medical Association that analyzed frequently cited animal studies in top scientific journals found that the results of only about a third of those trials accurately predicted what would happen to people. With odds like that, does it make sense to use CRISPR to create countless new varieties of nonhuman species?

Before CRISPR—an acronym for clustered regularly interspaced short palindromic repeats—there were already about 2,000 strains of mutant zebrafish. But most had been developed using a time-consuming process—one that exposed the fish to chemicals that may generate random genetic mutations, identified those that happened to have a mutation of interest, and then generated mutant strains through traditional breeding. “You kind of had to take what you got,” says Peterson. Two newer gene-editing methods—zinc finger nucleases, used since the late 1990s, and TALENs, first described in 2011—allowed more precise modifications, he says, “but there was a real art and skill required, and only a handful of labs could do those.” 

CRISPR was different. In 2013, Peterson and his colleagues Joanna Yeh and Keith Joung were first to use the new technology to engineer a new strain of animal—a zebra-fish missing the GSK3ß gene, which encodes an enzyme involved in energy metabolism and the development of cell and body structures as an embryo grows. “After we saw the first CRISPR papers in 2012, Woong Hwang, a technician in our lab, canceled his vacation plans and did most of the work over Christmas break,” says Peterson. “It took about two months total—it blew me away. It was so fast.” 

Today, ready-made proteins and synthetic guide RNAs—programmed to seek out a specific location in an organism’s genome for the Cas9 enzyme to cut—are available from online suppliers. Lab techs and students can now routinely whip up designer fish on their own. By using multiple RNA guides, researchers are able to “knock out”—that is, effectively delete or turn off—zebrafish genes with close to 90% efficiency vs. about 5% efficiency using earlier methods. That means that 9 out of 10 embryos that get the CRISPR injection will carry the desired change in their DNA. Establishing a stable strain of fish that consistently passes on its mutations to offspring might take three months. Before CRISPR, thousands of strains of mutant “knockout” mice had been produced using earlier biotechnology methods. But CRISPR’s speed, efficiency and ability to target multiple genes simultaneously are making it the new standard for knockouts in mice, zebrafish and many other species. Knock-ins, which require inserting a new gene, have been more challenging, but researchers expect those to become routine as well. 

Zebrafish have several built-in advantages for this work. Their genome has been fully sequenced, and at least 70% of human genes have a zebrafish equivalent. Smaller, cheaper and easier to breed than mice, zebrafish in their juvenile phase can actually live in the tiny amount of liquid—ranging from tens of nanoliters to several milliliters—that fits in the wells of a standard 96-well plate used to screen drug compounds. That makes it relatively simple to gauge the effectiveness of large numbers of drug candidates in treating a condition caused by a particular mutation. 

In cardiac research, Peterson has used mutant zebrafish to understand how a dozen or so drugs work. But his group and others are now also using zebrafish—which have spinal cords, central nervous systems and a range of well-defined behaviors—in neuroscience research, to study neurodevelopmental disorders such as autism and schizophrenia. 

Many genes are implicated in those diseases in humans, but it’s often hard to tell which ones actually cause the disease and which are merely associated with it somehow. Using CRISPR, a researcher can take a collection of several human genes and knock out the corresponding genes in zebrafish one by one. Then, by running each altered fish through a battery of established behavioral tests—measuring such factors as social interactions, addictive tendencies, sleep and fear response—researchers can see which mutations correspond most strongly with actual behavioral changes. Finally, fish with those mutations could then be screened to identify potential drug treatments. 

One postdoc in Peterson’s lab, for example, is using zebrafish to study opioid addiction, trying to identify genes that heighten drug-seeking behavior and thus could be useful targets for helping human addicts. Another lab member is studying the role the sigma-1 gene plays in regulating fear response, testing how normal zebrafish react to a stimulus—the flash of a strobe light—and comparing that with the response of sigma-1 knockouts. 

Beyond expanding researchers’ ability to study problems in traditional animal models, CRISPR is also facilitating work in animals that couldn’t be modified by earlier gene-editing technologies. “CRISPR opens up the menagerie of organisms,” says Keith Joung, associate chief of pathology for research at MGH. 

Itamar Harel, a postdoc at Stanford University, for example, is using CRISPR to study aging and its role in a range of complex human diseases, using the African turquoise killifish. This fish, native to Zimbabwe, is the shortest-lived vertebrate that can be housed in captivity, with a natural life span of just four to six months under optimal lab conditions. Since the late 1960s, it has been used to gauge the impact of specific changes—a shift in diet or receiving a particular drug—on life span. 

What researchers couldn’t do, however, was to create specific disease-causing mutations in killifish, and watch them unfold in fast-forward over the course of the fish’s short life. Researchers who studied aging could on the one hand use short-lived invertebrates, such as yeast, worms and flies, whose genomes have been sequenced, for which there are both lots of available mutant strains as well as established techniques for gene editing. Or they could use vertebrates such as mice or zebrafish, which have more of the complexity of human organs and systems but, Harel jokes, “have a life span similar to that of an average postdoc.” 

Thanks to two recent breakthroughs—the discovery of CRISPR and the mapping of the killifish genome (produced by Harel’s colleagues at Stanford and published in December 2015)—Harel earlier this year was able to create strains of killifish with mutations that give rise to a wide range of diseases associated with tissue degeneration in humans. It took just two to three months to produce stable fish lines, and he was able to observe age-related problems in fish as young as two months, as compared with six to eight months in zebrafish and much longer in mice. That lets researchers systematically evaluate the roles that specific gene variants might play. “The aging process itself is the main risk factor in cardiac disease, cancer, Alzheimer’s and other major causes of death for humans,” says Harel. “If we understand the biology behind aging, we may be able to understand what all of these diseases have in common.” 

Meanwhile, CRISPR also makes it easier to edit the genomes of relatively large animals, whose metabolism, physiology and anatomy are closer to humans than previous models have been. The go-to model for studying Duchenne muscular dystrophy (DMD) has been so-called mdx mice, a mutant strain discovered in the early 1980s. Like humans with the disease, these mice have a mutation that reduces production of the muscle-building protein dystrophin. But the mice don’t display many of the symptoms of DMD that humans have, notes Dongsheng Duan, a professor of molecular microbiology and immunology at the University of Missouri’s School of Medicine. 

Dogs are considered a much more realistic animal model, and several breeds have naturally occurring dystrophin-gene mutations. Researchers have established several experimental colonies of these natural mutants, but before CRISPR they had never used genetic engineering to create DMD mutant dogs. In the fall of 2015, however, Chinese researchers showed off CRISPR-engineered beagles with a kind of DMD in reverse—they have a mutation in the myostatin gene that makes them hypermuscular. The scientists, from Nanjing University, say they also intend to create dog models of muscular dystrophy, Parkinson’s and other human diseases. 

Thanks to CRISPR, in the past couple of  years alone, the list of genetically modified firsts has grown steadily: Mutant rabbits, sheep, pigs and other animals are being used to study everything from congenital cataracts to ALS, Parkinson’s disease and hearing loss. “Whether those turn out to be perfect models for a disease or not, the ability to make targeted mutations in whole animal models will provide much richer and more complex information to study,” says Joung. 

By making it vastly easier to create mutant strains of a growing variety of species, CRISPR is likely to increase the use of animals in research. That would amplify a trend that began about 15 years ago when knockout mice first became common. It may, however, also divert funding and attention away from promising nonanimal alternatives for testing disease processes and drug treatments. Organs-on-chips, for example, re-create organ systems in tiny detail and can be used to test drugs and understand how the body works. “There had been a decline in the use of animals, and genetic modification technologies turned that around,” says Thomas Hartung, director of the Center for Alternatives to Animal Testing at Johns Hopkins Bloomberg School of Public Health in Baltimore. 

Hartung acknowledges that CRISPR animal models may somewhat ease the ethical and logistical burdens on laboratories. Because the technology to create them is more efficient, fewer animals may now be required. And the ability to make incremental genetic changes, such as adding “reporting” genes that glow fluorescent, for example, might let researchers measure changes noninvasively in animals rather than using painful probes or euthanizing them. 

Also, Hartung says, there is a clear advantage in using the higher species that CRISPR can manipulate. “Many of the past’s meaningless results came from using the cheapest, fastest animals available: mice,” he says. 

Yet there are no guarantees that experiments involving even closely related species will predict what will happen in people. For example, a recent trial of a new medication for mood disorders, tested on chimpanzees, was later linked to one human death and irreversible brain damage in three other people. 

Yet researchers are rising to the task of making this research more predictive. The task is especially delicate with primates, who share many physiological and cognitive similarities with humans. Guoping Feng, a professor of neuroscience at MIT and investigator at the McGovern Institute for Brain Research at MIT., is collaborating with colleagues in the United States and China to develop less superficial—and more useful—nonhuman primate models. Feng shares the critics’ sober assessment that, at least in neuroscience, animal models have failed to deliver breakthrough treatments. Despite the availability of knockout mice and other animals, most current drug treatments were discovered serendipitously decades ago and are often unspecific and ineffective. Promising drug candidates in animals have consistently failed to translate to humans. Development of treatments for diseases of the central nervous system, Feng says, “has essentially stalled.” 

Still, Feng believes that animals are crucial for understanding disorders of the brain in particular. “Cancer is difficult, but we have a harder problem to deal with,” he says. “With autism and schizophrenia, you can’t just get tissue to study. We need to understand how billions of neurons in different parts of the brain talk to each other, and how malfunctions in neural connections get expressed as behavioral abnormalities.” 

This requires observing these mechanisms—circuits—in animals that have brain circuits similar to those in humans. Primates are the logical candidate. “The mouse is a great model for lots of things, but the human brain is different from the mouse brain,” Feng says. Nonhuman primates (NHPs)—like humans, but unlike mice—have a well-developed prefrontal cortex, a brain region responsible for higher mental functions that shows significant defects in many human neurocognitive conditions.

Feng and colleagues Robert Desimone, director of the McGovern Institute, and MIT professor Feng Zhang, a co-inventor of the CRISPR technology (pending U.S. Patent and Trademark Office investigation and ruling), are working with Chinese researchers to develop CRISPR-modified macaques—a monkey found in many Old World environments—to model human schizophrenia and autism. Unlike earlier animal models of central nervous system disorders that have emphasized trying to re-create behaviors that “look like” the diseases in humans, Feng aims to model the dysfunctional brain circuitry that underlies the behaviors. “Human behavior is different from any other species,” Feng says. But he believes that similar brain-signaling patterns underlie behaviors in animals with similar brain structures. 

Feng’s group is focusing first on conditions with the strongest human genetic data, such as Huntington’s disease, in which a single gene causes a disorder, and using advanced neuroimaging technology and other tools to understand how the mutation causes brain circuits to misfire. In the future, they hope to explore more genetically complex disorders.

There are still major technical and biological issues to overcome—including the long gestation period of primates—as well as care and ethical issues. “If we can study something in mice or any other model, we will not use primates,” Feng says. “The most important thing is thinking how best to use the resources of the primate models and sharing data so researchers don’t repeat the same kind of work.” 

Will all of this effort and sacrifice, human and animal, be worth it? Will this use of higher mammals, modeled and modified through the marvels of CRISPR, lead to effective therapies for some of our most intractable diseases? It will likely take another 10 to 15 years for potential new treatments to make their way into human trials. But encouraging tests in primates could dramatically increase confidence—and investment—among drugmakers. “I think this can make a direct and huge impact on future patients,” Feng says. “Many of us are very optimistic.”  

Dossier

“Zebrafish as Tools for Drug Discovery,” by Calum MacRae et al., Nature Reviews Drug Discovery, October 2015. This review paper covers the history and future role of zebrafish in drug discovery, and the key role that genetic modification will play.

“Generation of Gene-Target Dogs Using CRISPR/Cas9 System,” by Qingjian Zou et al., Journal of Molecular Cell Biology, October 2015. Chinese researchers describe using CRISPR to change the myostatin gene in dogs, which results in hypermusculature.

VIDEO: “How Does CRISPR-Cas9 Gene Editing Work?” CRISPR/Cas9 co-discoverer Jennifer Doudna explains the process.