Herding Innovation in Livestock

ABOVE: Alison Van Eenennaam uses genome-editing technology in cattle to address animal welfare and sustainability challenges. University of California, Davis

When Alison Van Eenennaam, an animal geneticist and cooperative extension specialist at the University of California (UC), Davis, grew up in metropolitan Melbourne, Australia, she never expected to establish her career in a city across the Pacific Ocean. Her trajectory towards genetic engineering, a career path that did not exist during her childhood, came as an even bigger surprise.

During her undergraduate studies at the University of Melbourne, Van Eenennaam focused on agricultural science. In her senior year, she met geneticist Juan Fernando Medrano at UC Davis during an exchange program, which led to her first foray into animal genetics. Van Eenennaam went back to Australia for a few years before taking the leap to emigrate to California. She eventually joined Medrano’s group to pursue a master’s degree in animal science and a doctoral degree in genetics.

In 1994, she worked as a research scientist at Calgene, focusing on using genomic techniques to modify plants. During this time, she witnessed Calgene’s commercialization of the first genetically engineered plant, the Flavr Savr tomato. However, animal agriculture and extension remained her passions. 

One of my favorite parts of working with Alison is that right off the bat, she took the time to really try and understand what my interests were both from a research and future career standpoint. 

 —Maci Mueller, Kansas State University

In 2002, UC Davis began a search for someone to fill a role at the intersection of animal biotechnologists, livestock producers, and the public to explain these technologies and combat the misinformation around genetically modified organisms (GMO). Van Eenennaam, a perfect fit, jumped at the opportunity.

Van Eenennaam’s role since then has encompassed a blend of research and science communication. With her genome editing research, she strives to underscore the technology’s utility for augmenting livestock breeding and its potential for improving animal models for biomedical applications. 

Dehorning Cattle

A vast majority of cows have horns, which pose a risk of injury for fellow cattle and the animal handlers. To achieve hornless or polled cows, farmers apply heat to the horn bud to destroy the growing horn cells. According to Van Eenennaam, this practice is unpleasant for the cows. 

To circumvent the need for dehorning, livestock producers painstakingly conduct selective breeding for polled cows. Angus beef cattle are naturally polled and can be bred with Holstein dairy cows; however, these crosses may inadvertently alter other desirable traits such as milk production. 

Van Eenennaam and other researchers saw a better alternative to this practice: gene editing. The polled allele is dominant to the horned allele. It involves a naturally occurring insertion of approximately 110 base pairs in the promoter region of the polled gene. Recombinant DNA technologies could speed up this process by introducing a specific trait while retaining other desired characteristics in an animal.

In 2016, Van Eenennaam collaborated with a group of researchers from Recombinetics, a biotechnology company that offers gene editing solutions for human health and animal agriculture. Together, the research team used transcription activator-like effector nucleases to produce hornless dairy cattle.¹ Later, they transferred the animals to UC Davis for genetic and phenotypic characterization to ensure the success of the polled genome edit.² In a breeding experiment, none of the offspring developed horns, and the animals were all healthy. There were no unintended phenotypic or genotypic alterations observed.

That’s the real beauty of large animal models. You can do quite meaningful experiments in a larger animal model that you just can’t do in a mouse, and so [this work] is exciting. 

 —Alison Van Eenennaam, University of California, Davis

“Gene editing for polled in cattle is a win-win all around. First and foremost, it benefits the animal in avoiding the dehorning process and the producer by saving time and labor,” remarked Maci Mueller, an animal geneticist at Kansas State University and a former student of Van Eenennaam. 

Mueller’s interest in animal breeding and genetics stemmed from witnessing her family’s Angus cattle operation while growing up and experiencing firsthand how innovations in genetics help cattle producers. When she met Van Eenennaam during a lecture series at the University of Nebraska-Lincoln, they hit it off right away. Mueller conducted her master’s thesis project and doctoral studies under Van Eenennaam’s mentorship. “One of my favorite parts of working with Alison is that right off the bat, she took the time to really try and understand what my interests were both from a research and future career standpoint.”

Although Mueller’s primary interest was in the molecular side of animal genetics, she sought a comprehensive viewpoint of the field by diving into quantitative genetics. In her master’s project, Mueller conducted comparative studies using quantitative genetics tools, such as computer simulation and modeling. She evaluated how gene editing technology could introduce a beneficial trait, specifically polled, into different cattle populations such as dairy cattle in the United States or beef cattle from Northern Australia.^3,4 These scenarios demonstrated that genetic editing for the polled allele appeared more beneficial than conventional selective breeding methods. 

Having experienced the quantitative side of this field, Mueller’s doctoral work then brought her to the laboratory bench; she produced in vitro embryos, performed micromanipulations, and injected gene-editing reagents into bovine zygotes. “The advantage to genome editing is introducing a specific trait without diluting the genetic merit or excellence of the background DNA,” said Van Eenennaam.

SRY Brings All the Boys

Aside from polled cows, Van Eenennaam figured that livestock producers still had a laundry list of desired traits, including sex. The motivation behind skewing the sex ratio toward maleness is efficiency. Beef ranchers would largely benefit as males grow bigger and faster to generate more beef. So, she began the “All Boys” project and used CRISPR-Cas9 technology to alter sex ratios. 

         

The researchers used these technologies to insert the sex-determining region of the Y chromosome (SRY) gene, which is responsible for initiating male development, into bovine embryos. Previous attempts to insert the gene into the X chromosome to generate only male offspring were unsuccessful, so the researchers chose chromosome 17, where genomic edits would not affect the expression of adjacent genes.

The goal of this edit was to investigate whether inheriting SRY would trigger male development in female calves. This would greatly benefit the beef industry with beefier female cattle. After a few years of painstakingly developing the method to insert the gene into a developing embryo and then establishing a pregnancy, the CRISPR calf, Cosmo, was born in the spring of 2020.⁵

Cosmo should produce 75 percent male offspring, including the normal 50 percent male (XY) animals plus the 25 percent of female (XX) animals that inherit the SRY gene. Researchers used an embryo biopsy collected from an XX egg that inherited the SRY gene to generate the next generation; the resulting offspring was born in early 2024. “She was very big, about twice the normal weight potentially due to embryo transfer but had no obvious external genitalia or alterations from conventional females,” said Van Eenennaam. Further investigation is underway to confirm whether the calf inherited the SRY gene and to observe the calf’s growth.

Employing Genetic Shears in Sheep

The scope of Van Eenennaam’s gene-edited farm also extends into biomedical applications. She collaborates with researchers at UC San Francisco using sheep as a model for human research. In humans, a mutation in the bone morphogenetic protein receptor type 2 (BMPR2) gene leads to a high incidence of pulmonary arterial hypertension (PAH) during childhood. While scientists used rats as models for PAH, sheep and lambs are much closer in size to humans, which is an advantage when testing therapeutics and biomedical devices. 

Van Eenennaam’s goal was to create a suitable sheep model and use it for studying this disorder; however, a BMPR2 homozygous knockout is lethal at the embryonic stage.⁶ “To do this, we had to devise an editing strategy that enables one allele to remain in the wild-type sequence and the other allele to be knocked out,” explained Van Eenennaam. Initial attempts were unsuccessful in producing a single cut, so she and her team redesigned the experiment. 

We have some solutions from a genetic toolbox that exactly addresses problems like disease and sustainability to improve efficiency and reduce the environmental footprint of food production. 

 —Alison Van Eenennaam, University of California, Davis

They decided to cut both strands. The first strand was cut and resulted in a base pair deletion. The cut in the second strand targeted a short DNA sequence, the protospacer-adjacent motif site, to initiate homology-directed repair and create a silent mutation that would save the lamb from a lethal edit. A week later, the researchers transferred the edited embryos into a surrogate ewe. As a result of this work, one male and three female lambs were born. 

PAH predominately affects women, and there is a 70 percent penetrance in female animals. The sheep appeared to exhibit symptoms similar to those in human patients, with the female animals being more symptomatic. Unfortunately, roughly a month after birth, two of the female lambs had to be euthanized due to severe symptoms. 

When the male lamb reaches sexual maturity, researchers will use the collected semen to inseminate ewes. The researchers need to produce his offspring to confirm that they will be BMPR2 heterozygous; they expect that 50 percent of his lambs will be wild type and 50 percent will be heterozygous, which gives researchers the two necessary groups for comparison. “That’s the real beauty of large animal models. You can do quite meaningful experiments in a larger animal model that you just can’t do in a mouse, and so [this work] is exciting,” said Van Eenennaam.

Despite her excitement for genetic innovation in larger animal models, Van Eenennaam lamented the slow, uphill battle for genome-edited animal breakthrough into the market. While millions of farmers continue to grow genetically modified crops, the number of approved animals for commerce remains in the single digits in the US. These include AquAdvantage salmon that can grow to full size in half the time as conventional salmon, GalSafe pigs, which lack the alpha-gal sugar on their cell surfaces to make the meat safer for consumption for individuals with allergic reactions to alpha-gal sugar, and cattle bred to have a short, slick haircoat that improves heat tolerance. 

“We have some solutions from a genetic toolbox that exactly addresses problems like disease and sustainability to improve efficiency and reduce the environmental footprint of food production,” said Van Eenennaam. However, she remains cautiously optimistic that more of these technologies will be accepted and enter the market.  

Extension and Education Beyond the Bench

Outside of research, Van Eenennaam actively engages in science communication on a local and global scale. She collaborates with scientists to provide educational resources to the public. Matt Spangler, an animal geneticist at the University of Nebraska-Lincoln, met Van Eenennaam more than a decade ago at the National Beef Cattle Evaluation Consortium. Spangler’s work focuses on the quantitative side of the field of animal genetics, leveraging genetic predictions to advance livestock populations. The difference in their expertise within the genetics domain and background naturally led to collaboration. “One of the things that makes Alison unique is that she does not come from an agricultural background herself. Since she grew up in a metropolitan area, she doesn’t come with the implicit bias of someone who did grow up on an agricultural enterprise,” remarked Spangler. “In a lot of ways, that’s been beneficial to her because she can see things through a different lens.”

Alison is exceptionally unique because she has broadened the audience considerably. Her voice is backed by science, and I think she’s done an exceptional job at making science palatable to the public so that they can digest it and understand it on their terms. 

 —Matt Spangler, University of Nebraska-Lincoln

Van Eenennaam and Spangler often speak to beef cattle producers on how to use various gene editing tools and technologies to advance their enterprises while keeping a keen eye on sustainability. “Alison is exceptionally unique because she has broadened the audience considerably. Her voice is backed by science, and I think she’s done an exceptional job at making science palatable to the public so that they can digest it and understand it on their terms,” said Spangler, who also partnered with Van Eenennaam and other scientists to develop a one-stop shop resource for GMO information, eBeef. This serves as a centralized platform for reliable beef cattle genetics and genomics information, offering expert-recommended resources that has garnered nationwide attention for its educational value.

Despite the slow-moving progress of commercial approval and combating misinformation around genetically edited animals, Van Eenennaam has maintained a positive outlook. Her passion for applying genome-editing technologies to address a myriad of environmental and sustainability issues and sharing this information with others continues to leave a lasting impact.