The CRISPR animal kingdom

 

 

 

 

 

Early one February morning, researchers harvest six eggs from a female rhesus macaque—one of 4000 monkeys chirping and clucking in a massive outdoor complex of metal cages here at the Yunnan Key Laboratory of Primate Biomedical Research. On today’s agenda at the busy facility, outside Kunming in southwest China: making monkey embryos with a gene mutated so that when the animals are born 5 months later, they will age unusually fast. The researchers first move the eggs to a laboratory bathed in red light to protect the fragile cells. Using high-powered microscopes, they examine the freshly gathered eggs and prepare to inject a single rhesus sperm into each one. If all goes well, the team will introduce the genome editor CRISPR before the resulting embryo begins to grow—early enough for the mutation for aging to show up in all cells of any offspring.

But as often happens when eggs are retrieved, all does not go well. Only one egg in this morning’s batch is mature enough to fertilize. “We were a little unlucky today,” says Niu Yuyu, who with facility director Ji Weizhi runs the gene-editing research. The group can afford a little bad luck, though. Through a combination of patience, ingenuity, and enormous animal resources, the team has already used CRISPR to create an astonishing range of genome-edited monkeys to serve as models for studying human diseases.

Ji, Niu, and colleagues were the first to harness CRISPR in monkeys, as they reported in 2014, and they remain leaders in the field. They’ve built on that success, exploiting CRISPR’s speed and precision to create monkey models of muscular dystrophy, autism, and cancer. In a tie with developmental biologist Yang Hui and co-workers at Shanghai’s Institute of Neuroscience (a branch of the Chinese Academy of Sciences, CAS), they were first to use CRISPR in monkeys to introduce, or knock in, a gene—a particularly difficult feat that the two teams reported in back-to-back papers in 2018 in Cell Research.

The team also collaborated with He Jiankui, well before the Chinese biophysicist created the first CRISPR-edited human babies. Almost 2 years ago, in a proof of principle for He’s infamous experiment, they knocked out in monkey embryos the gene for the immune cell protein CCR5, a mutation that makes humans resistant to infection with the most commonly transmitted variant of HIV. That work leaves them uneasy today. “We had no idea he was going to do this in a human being,” Niu says, stressing that their study had yet to evaluate how the editing affected the monkeys when news of the edited babies broke. “It’s unbelievable.”

China now has at least four groups of CRISPR researchers doing gene editing with large colonies of monkeys. “The most startling part of what is coming out of China is seeing how they have just a brute-force approach,” says reproductive biologist Jon Hennebold at the Oregon National Primate Research Center in Hillsboro. “The level of animal support they have to do those experiments is really astounding.”

It’s not just monkeys. China’s researchers have racked up a long list of CRISPR firsts in dogs, mice, rats, pigs, and rabbits. That research promises higher quality meats, disease-resistant livestock, and new medical treatments and organs for human transplantation. So far, many of the animals are simply proofs of concept. Despite the multitude of CRISPR-altered monkeys, for example, Chinese teams have published “very little follow-up in terms of characterizing what these mutations mean from a [disease] model or a treatment perspective,” Hennebold says.

But few people doubt that China will persist with its animal-editing binge. “This is a country and a culture that really values science and technology,” says Jennifer Doudna of the University of California, Berkeley, who helped develop CRISPR into an editing tool. “Their government has put very serious money into it, and they’re walking the walk.”

 

TO GET A FEEL FOR one of China’s advantages, ask Hennebold—one of the few U.S. researchers using CRISPR to develop monkey models of disease—how many edited monkey embryos he has transferred. “You’re going to laugh,” he says. “Maybe 10.” In contrast, Ji and Niu routinely implant embryos with the same CRISPR edit into 50 to 100 surrogate females. Another group—also at the Institute of Neuroscience—recently combined cloning and CRISPR to create 325 monkey embryos with the same mutation, one that disrupts circadian rhythms in people and is connected to sleep disorders, diabetes, and cancers. After implanting the embryos into 65 surrogate females, the team produced five genetically identical monkeys with the disorder.

In addition to having access to large colonies of monkeys and other species, animal researchers in China face less public scrutiny than counterparts in the United States and Europe. Ji, who says his primate facility follows international ethical standards for animal care and use, notes that the Chinese public has long supported monkey research to help human health. “Our religion or our culture is different from that of the Western world,” he says. Yet he also recognizes that opinions in China are evolving. Before long, he says, “We’ll have the same situation as the Western world, and people will start to argue about why we’re using a monkey to do an experiment because the monkey is too smart, like human beings.”

And then there’s money: massive—if hard to quantify—government investment in both new facilities and ambitious research projects. As a result, China has become the center of the CRISPR animal kingdom, attracting top researchers. “In the States, it is harder to do monkey studies, so I moved back,” says Yang, who did a postdoc in the United States and now is part of China’s Thousand Talents Program, which aims to reverse the brain drain.

 

 

 

One major Chinese effort to edit animals traces its origins to 1998, when developmental biologist Lai Liangxue left China to join a lab at the University of Missouri in Columbia run by Randall Prather, a reproductive physiologist studying pigs. Prather, Lai, and co-workers set out to genetically engineer a pig whose organs could be transplanted into humans without rejection. Human immune systems won’t tolerate cross-species, or “xeno,” transplants because many pig genes code for incompatible proteins, and the Prather group wanted to cripple production of one protein that triggers particularly strong antibody responses. Four years later, the team reported in Science (8 February 2002, p. 1089) that it had engineered the world’s first knockout pig.

“It took us so much time and so much money,” Lai says, because the team had to rely on an imprecise and inefficient combination of transgenic and cloning technologies. The cumbersome knockout process required inserting a bacterial gene that would land randomly in the genome, which risked accidentally turning on a cancer gene or causing other problems. Over the next 10 years, Lai says, the team and others created only a few more genetically modified pigs.

In 2007, Lai returned to China and set up labs in Guangzhou and at Jilin University in the northeastern city of Changchun. By 2013, when other groups first showed CRISPR could alter animals, his team had adopted earlier genome-editing tools dubbed zinc fingers and transcription activatorlike effector nuclease (TALEN). With zinc fingers, Lai’s team could edit a pig embryo in 2 to 3 months, and TALEN dropped the timeline to a month. “But with CRISPR, in just 1 week you can get everything done,” Lai says. Everyone in his lab—nearly 30 people—began to use CRISPR to edit rabbits, dogs, and pigs.

To date, they have successfully made 40 different genetic modifications in pigs with CRISPR. “He’s doing great stuff,” Prather says. “I’ve trained all my competition over there, and I’ve trained them well.”

 

OVER THE PAST DECADE, most of Lai’s work has focused on creating pig models of human disease, ultimately to test new medicines. At his pig facility near Jiangmen, about a 2-hour drive from his lab, dozens of swine with edited genomes fill three barns. Lai has engineered some to age prematurely or develop neurodegenerative afflictions that mimic diseases such as Alzheimer’s, Parkinson’s, amyotrophic lateral sclerosis (Lou Gehrig’s disease), and Huntington. Lai’s group has also used CRISPR to knock in a humanized gene for albumin, a blood product given in cases of traumatic shock or liver failure. Lai hopes the protein, purified from the blood of the pigs, will enter human trials next year.

Across the country at CAS’s Institute of Zoology in Beijing, geneticist Zhao Jianguo—another Prather alumnus—uses CRISPR to engineer improved pigs for the pork industry. He and colleagues 2 years ago endowed their pigs with a gene for uncoupling protein 1, which is found in most mammals and helps them form heat-producing brown fat. Swine, however, have lost the protein, and newborn pigs often die from hypothermia. Pigs with the gene restored by CRISPR stay warmer in cold environments, Zhao’s group reported in 2017. The pigs also have about 5% less white fat, which makes for leaner meat. Colleagues on the same campus, led by Wang Haoyi and Zhou Qi, used CRISPR to make a genetic change that speeds growth in pigs, meaning farms could produce meat faster.

The Chinese groups and others are also turning to CRISPR to thwart several diseases that often devastate the pig industry. A gene added with CRISPR made pigs resistant to classical swine fever, Lai reported late last year, together with Ouyang Hongsheng and colleagues at Jilin University. Months earlier, Prather’s team had reported creating pigs altered to resist the industry’s costliest infection, porcine reproductive and respiratory syndrome virus.

When those advances will reach farmers in China is not clear, Zhao says. “The big barrier for Chinese companies is they don’t want to put too much money into genetic modification for pig breeding because right now we don’t have a timeline about how long it will take to get the genetically altered pig into the market,” he says. China has not yet decided whether or how it will regulate CRISPR-modified food, casting a pall of uncertainty over the efforts (see p. 422). “It is moving, but the progress is very slow.”

Both the U.S. Food and Drug Administration (FDA) and China’s Ministry of Agriculture now regulate CRISPR-modified pigs like old-school genetically modified organisms, which are severely restricted in both countries. That “just doesn’t make sense to me,” Prather says, who notes that the genetic changes introduced by CRISPR are often indistinguishable from natural mutations. Genus, a U.K.-based company, is counting on China to reconsider its current policy. Genus licensed the technology to commercialize Prather’s virus-protected pigs and in May formed a collaboration with a Chinese company to bring them to market there—a process the firm says could take several years.

 

WHEN LAI RETURNED TO CHINA, he wanted to continue the xenotransplantation work he had begun in Prather’s lab but couldn’t get funding. “The Chinese government was not interested,” he says. But over the past few years, attitudes have shifted. The government now strongly backs efforts by Lai and others to create pig organs that could help people with eye problems, diabetes, kidney and liver failure, and heart disease.

China has a critical organ shortage—300,000 people in need and 10,000 organs available—transplant specialist Deng Shaoping of Sichuan Provincial People’s Hospital in Chengdu and his co-authors explained in February in a review article in Xenotransplantation. The shortage, they say, was exacerbated by the government’s 2015 decision to stop harvesting organs from executed criminals. Now, they wrote, “China has the potential to become a world-renowned pig organ center, with the perspective of minimizing or even eliminating the current organ shortage for transplantation.”

International science teams are joining the effort, with some starting commercial ventures in China. One such company is Qihan Biotech in Hangzhou, which collaborates with eGenesis of Cambridge—both co-founded by Harvard University’s George Church, a CRISPR pioneer, and Yang Luhan, who earned her Ph.D. in his lab. They are building on an advance the team reported 2 years ago, when they showed that CRISPR could remove dozens of genetic sequences called endogenous retroviruses from pigs (Science, 22 September 2017, p. 1303).

Those vestiges of ancient infections theoretically could harm humans who receive pig organ transplants, although other researchers downplay the risk. “I don’t think [the sequences are] a very big issue,” Zhao says. “The really big barrier is the immunorejection, not the viruses.” No one knows how many pig genes will ultimately have to be knocked out to prevent human rejection of their organs—estimates range as high as 20—but to date, Lai and co-workers have disabled four in one animal, and at least five other groups in China also are targeting such genes, according to the Xenotransplantationarticle.

As the final step before a human trial, researchers routinely put gene-edited pig tissue or organs into a monkey, widely considered the best testing ground for predicting human responses. Few Chinese investigators have made that crucial step. Lai’s team hopes to transplant a CRISPR-modified pig organ to a monkey next year, which he notes will require a large, skilled team of surgeons and specialists. “It takes so many people to care for the animal, and it’s very, very expensive.”

Peter Cowan, a leading xenotransplantation researcher at St. Vincent’s Hospital in Melbourne, Australia, says China’s recent push to get back into the field, aided by CRISPR, puts them a bit behind the leading groups. “It just takes time for people to catch up and move into the preclinical models and then get enough evidence because no one wants to be the first there and have it turned into a disaster.”

 

 

 

Competition is stiffening. A South Korean group hopes to transplant CRISPR-engineered pig corneas or insulinmaking islet cells into people in clinical trials starting in May 2020. By the end of next year, a U.S. team led by Revivicor in Blacksburg, Virginia, that works with David Cooper of the University of Alabama in Birmingham may test a pig kidney with nine modified genes in people on dialysis. The team is already testing their kidneys in baboons but still needs FDA approval to conduct the human trial. “The timeline is very close now because we’ve got so much encouraging data,” Cooper says.

 

LAI MAY BE A BIT BEHIND in the xenotransplant race, but he’s also competing on more fronts than almost any other CRISPR animal researcher. About 150 kilometers from where his pigs live, a research farm on the forested banks of the Lian’an reservoir houses 2000 beagles used in a variety of scientific experiments. The two most famous are Lai’s creations: a female named Tiangou, the heavenly dog in Chinese legend that eats the sun or the moon, and Hercules, a male whose name more clearly reflects why they have received international attention.

Lai used CRISPR in the two dogs to cripple a gene that normally constrains muscle mass. The beagles are 5 years old now, and their jaws have a hint of pit bull. Their pectoral and thigh muscles bulge, resembling the sculpted humans who compete in bodybuilding contests. But the dogs are sweet, gentle beagles through and through. They hold the distinction of being the world’s first, and to date only, dogs modified with CRISPR.

Although Lai acknowledges that breeders might want to engineer dogs that can jump higher and run faster, he created Tiangou and Hercules in 2015 simply to show that CRISPR works in canines and can be done safely. “This genotype is really easy to observe, so people at the regulatory agency can see it with their own eyes,” Lai says.

The dogs may be just for show. But researchers in China and abroad say the country’s industrial-scale effort to transform animals with CRISPR will soon yield real payoffs for fundamental research, agriculture, and medicine.

Lai, for one, says his university has just invested 60 million yuan (nearly $9 million) in a new, higher-tech facility for both his pig and rabbit research. “Come back next year,” he says. “The whole thing will change.”

 

 

 

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