CRISPR-based genome editing has already been successfully applied to sickle cell disease and additional companies are working on developing their CRISPR-based therapeutics. It seems only natural that the technology would make its way to animals. Now a team of scientists at Genus, a British animal genetics company with research facilities in Wisconsin and Tennessee, have developed a new generation of CRISPR-edited pigs that are resistant to porcine reproductive and respiratory syndrome (PRRS) virus, a disease that has had a widespread impact on porcine populations around the world for decades.
Details of exactly how the pigs were edited are published in a new report published in The CRISPR Journal titled, “Generation of a Commercial-Scale Founder Population of Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs Using CRISPR-Cas.”
The Genus team describes a scaled gene editing program that introduced “a single modified CD163 allele into four genetically diverse, elite porcine lines.” The work produced healthy pigs that “resisted PRRS virus infections as determined by macrophage and animal challenges.” Edited pigs showed “no signs of infection or viral replication in lung and lymph node tissue when challenged with PRRSV.” The Genus team believes this is potentially the first integration of CRISPR gene editing into a livestock breeding program and could completely eliminate a major infectious disease in swine.
“This is a milestone study illustrating the potential of CRISPR-based technologies for commercial livestock breeding,” said Rodolphe Barrangou, PhD, professor of food, bioprocessing, and nutrition sciences at North Carolina State University and editor-in-chief of The CRISPR Journal. “A commercially-relevant proof of concept that genome editing can be used to generate healthy PRRS-resistant pigs that are otherwise indistinguishable from the parent population sets the stage for deploying that approach for other diseases and traits of commercial interest.”
“They were able to generate in a couple of generations a founder population of breeding boars (10–15 per line) and gilts to serve as a gene edited nucleus herd for ultimate commercial pork production and sale using classical breeding,” said Alison Van Eenennaam, PhD, an extension specialist in animal genomics and biotechnology in the department of animal science at the University of California, Davis. “There are more sophisticated approaches to guarantee the edited allele is in a homozygous state and absent off-target indels in all animals produced, e.g., using edited porcine embryonic stem cells, but at the end of the day the approach they used did the job.”
Genus works on improving the genetics of livestock to ensure healthy, robust pigs and cattle for agricultural production, Elena Rice, PhD, Genus’ CSO and head of research and development and a co-author on the paper told GEN. “Livestock have a lot of diseases, and there are several that [are] just really devastating for farmers,” Rice said. Until the recent emergence of African swine flu, PRRS infections topped that list.
Murky beginnings
Also known as blue-ear pig disease, the first PRRS cases in the United States were reported in 1987. While the exact origins of the virus are murky, its impact is all too clear. The disease is now found in pig production facilities around the world, accounting for the death of as much as 20% of livestock produced annually. Its impact is environmental, psychological, and personal—not to mention economically devastating for the swine industry. By some estimates, annual losses in the United States exceed $600 million.
Multiple vaccines have been developed to stop the spread of PRRS, but with limited effectiveness, according to Rice. As explained in the paper, the PRRS virus has “a high rate of mutation due to an error-prone viral RNA-dependent RNA polymerase and a significant rate of genetic recombination.”
Vaccines are typically administered when the pigs are already showing symptoms, but by then it is often too late to save them. Even if the pigs survive the infection, their immune systems are usually too weak to combat secondary infections like pneumonia that can develop. Just a few infected pigs are enough to decimate an entire herd. Attempts to breed PRRS-resistant pig populations have also not worked out well to date.
Alongside breeding efforts, scientists in academia have also studied the genetic basis for PRRS infections. It turns out that several genes are involved in viral infection, including CD163, which encodes the entry receptor for the virus. In pigs, this particular protein is expressed on the surface of macrophages and monocytes and mediates inflammation among other functions.
Genus’ work builds on the research into the role of CD163. “We had some evidence from university studies that we could edit a single host gene to confer resistance,” Brian Burger, PhD, senior research manager at Genus and first author on The CRISPR Journal paper, told GEN. “The challenge was: how do you go from that proof of concept work to a commercial breeding program?” It seemed like the ideal opportunity to bring CRISPR technology to bear on animal disease.
CRISPR’s efficiency also made it the right fit for editing animals. Finding the right edit for a gene without introducing dangerous off-target effects requires a lot of trial and error. “Ethically and morally, it’s very important [that] we designed the whole process so that we eliminate unnecessary production of animals,” Rice told GEN.
Farm to table?
To edit the genomes, the Genus scientists injected CRISPR-Cas9 editing reagents into the genomes of pig zygotes. Their goal was to make a precise deletion in CD163 that removed a single exon encoding the domain that directly interacts with the virus. Importantly, the edit did not impact CD163’s function in the new population. They also genotyped the edited animals to ensure that the edit was consistent across the animals, there were no unforeseen off-target effects, and there was enough genetic diversity within the potential breeding population. Edited pigs that passed muster were then moved through the breeding process to establish a population of pigs that are PRRS-resistant. The paper also covers details of how the team optimized their editing reagents to ensure that their work would translate to a commercial breeding program, Burger added.
The repercussions of this work could be major. Genus is not the only company working on PRRS by targeting CD163. But Genus certainly hopes to be the first to market with its edited pigs. It has its sights set on getting the edited pigs through the FDA’s regulatory process.
Rice told GEN that the company is also working with regulatory agencies in other countries to seek broader approval as well. “Everything is going well, but it’s just a long process,” she said. ”The FDA is making a lot of effort to create a much better environment for gene editing [in livestock]. We’re learning together as we go through the process.”
Barrangou noted that “regulatory approval will be critical” and that “there are already established Ag-relevant frameworks for crops, notably in the U.S. and very recently in the EU that set the stage and precedence.” Furthermore, “the enthusiasm related to last week’s EU vote on the use of new genomic techniques and plant breeding technologies in Ag” makes the publication of this study “very timely and encouraging,” he added.
Van Eenennaam has some reservations about the approval process in the United States. “The United States FDA is alone in the world in regulating ‘intentional’ genetic alterations including single base pair deletions as an animal drug, and requiring a ‘new animal drug’ approval for commercialization. This expensive regulatory path is basically a non-starter for smaller companies,” she told GEN in an email.
Furthermore, “Until the pigs are approved, they are all considered unsalable new animal drugs and therefore cannot enter commerce or the food supply,” she added. “That means all of the 435 edited pigs produced in the paper in the development and testing process need to be incinerated, composted, or buried. Such a multiyear endeavor requires very deep pockets.” And the road to approval will likely be quite long. She pointed to the multi-year approval timeframe that was required for the genetically engineered AquAdvantage salmon as an example of just how long the process can be.
But regulatory approval is just the first hurdle. Genus scientists hope their pigs will be widely disseminated in the livestock industry to stop losses due to PRRS. Building PRRS-resistant piglets requires two gene-edited parents. Genus plans to sell aliquots of semen from gene-edited males to breeders who could use it to begin breeding programs that ultimately produce PRRS-resistant pigs after a few generations.
If all goes well, the public could be faced with the choice of eating pork from gene-edited animals. Historically, the conversation around the production and consumption of genetically modified foods has been very polarizing. But the public response in this case may not follow historical trends given the central reason for the gene editing in the first place. Early consumer research conducted by Genus indicates that consumers are more open to CRISPR-edited foods when there is a good reason for the genetic modification. Given the impact PRRS has had on the livestock industry, there is a clear benefit here, Rice said.
Genus’ headquarters are in the U.K. but its business divisions are based in the U.S. In addition to pigs, Genus also has a bovine division focused on using genetics to boost beef and dairy production. Genus’ PIC division—focused on the pigs and pork production—is headquartered in Hendersonville, TN, while the headquarters for its bovine division are located in Madison, WI.
