CRISPR-Cas9 Used to Generate “Surrogate Sire” Livestock that Produce Only Donor Sperm

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One of the "surrogate sire" goats on the WSU Pullman campus [Washington State University]

For the first time, scientists have created pigs, goats, and cattle that could serve as viable “surrogate sires,” male animals that produce sperm carrying only the genetic traits of donor males. The team of U.S. and U.K. researchers used CRISPR-Cas9 gene editing technology to knock out a gene specific to male fertility in animal embryos that would be raised to become the surrogate sires. These male animals were effectively born sterile, but otherwise healthy, and began producing sperm after researchers transplanted stem cells from donor animals into their testes. The sperm produced by the surrogates carried only the genetic material of the donor animals.

The scientists say this approach could be used to help spread desired characteristics in livestock, and improve food production for a growing global population. The technology could also give breeders in remote regions better access to the genetic material of elite animals from other parts of the world, and allow more precision breeding in animals, such as goats, for which techniques such as artificial insemination can be problematic.

The reported achievements are the result of six years of collaborative work by researchers at Washington State University, Utah State University, University of Maryland, and the Roslin Institute at the University of Edinburgh in the U.K. “Genetic improvement of livestock, implemented through application of advanced breeding technologies such as artificial insemination, has been hugely successful in advanced economies,” commented Simon Lillico, PhD, research fellow at the Roslin Institute, who is one of the authors of the team’s published paper, in the Proceedings of the National Academy of Sciences (PNAS). “In our study we used gene editing technology to develop male surrogates that do not produce their own sperm, but can act as incubators for the sperm of other males. This development has potential application for genetic improvement of livestock in low- and middle-income countries, such as those with whom the Centre for Tropical Livestock Genetics and Health works, where small-holder livestock holdings are crucial for food security, nutrition, and income generation.”

“With this technology, we can get better dissemination of desirable traits and improve the efficiency of food production,” added senior author Jon Oatley, PhD, a reproductive biologist at Washington State University’s College of Veterinary Medicine. “This can have a major impact on addressing food insecurity around the world. If we can tackle this genetically, then that means less water, less feed, and fewer antibiotics we have to put into the animals.” Oatley and colleagues reported their work in a paper titled, “Donor-derived spermatogenesis following stem cell transplantation in NANOS2 knockout males.”

Scientists have for decades been searching for a way to create surrogate sires, as a means to overcome the limitations of selective breeding and artificial insemination, both of which require either animal proximity or strict control of their movement, and in many cases, both. While artificial insemination is commonly used in dairy cattle—which are often confined and so their reproductive behavior is relatively easy to control—the procedure is rarely used with beef cattle that need to roam freely to feed. And for pigs, the procedure still requires the animals to be nearby, as pig sperm does not survive freezing well. In goats, artificial insemination is quite challenging and could require a surgical procedure.

The potential to transfer sperm-producing stem cells—­or spermatogonial stem cells (SSCs)—from a donor animal into the testes of a recipient male could have multiple applications, the authors suggested. This approach, known as spermatogonial stem cell transplantation (SSCT), might be used to help improve the genetics of global livestock populations, and also potentially be used to protect endangered species. “This accomplishment could provide an efficacious avenue for improving production characteristics and in turn enhance the capacity to provide food security for the expanding global population,” the investigators wrote. “In addition, SSCT in endangered species has utility for preserving unique genetic lines, thereby contributing to conservation efforts.”

One key requirement for this approach to be feasible, is that the recipient male animals don’t produce any of their own sperm cells, as these would carry the surrogate’s own genetic material. But they should still be otherwise physiologically normal. “ … recipient males must be devoid of endogenous germline but possess normal testicular architecture and somatic cell function capable of supporting allogeneic donor stem cell engraftment and regeneration of spermatogenesis,” the scientists stated.

To generate their surrogate sires, the team used CRISPR-Cas9 gene editing to produce first mice, and then pigs, goats, and cattle, that lacked a gene called NANOS2, which is specific to male fertility. These male NANOS2 knockout (KO) animals grew up sterile but otherwise healthy. “… we show that male mice, pigs, goats, and cattle harboring knockout alleles of the NANOS2 gene generated by CRISPR-Cas9 editing have testes that are germline ablated but otherwise structurally normal,” they wrote.

After being born sterile, animals received stem cells from male donors into their testes—pigs received cells from wild boars, and mice and goats were given cells from different mice and goat species. The animals that received transplants of sperm-producing stem cells from donor males then started producing sperm cells that were derived from the donor’s stem cells. The engraftment was successful, even though the donors and recipients were immunologically incompatible, and the recipients had a fully functional immune system. “In adult pigs and goats, SSCT with allogeneic donor stem cells led to sustained donor-derived spermatogenesis,” the researchers noted.

The technology could be used to improve genetics in beef cattle herds. [Washington State University]
Breeding experiments with the surrogate sire mice confirmed that they could father healthy offspring, which all carried the genes of the donor male animals. Three of the six mice that underwent SSCT before puberty mated to produce a total of 111 donor-derived offspring. Breeding with the larger animals hasn’t yet been carried out, as Oatley’s lab is refining the stem cell transplantation process before taking that next step.

 

Nevertheless, the investigators noted, “Collectively, these advancements represent a major step toward realizing the enormous potential of surrogate sires as a tool for dissemination and regeneration of germplasm in all mammalian species … Moreover, our discovery that inactivation of NANOS2 also leads to germline ablation in male cattle opens the intriguing possibility of one day developing bull “super dads” that then can be harnessed for disseminating desirable genetics in cattle populations around the world.”

The study provides a powerful proof of concept, suggested the Roslin Institute’s Bruce Whitelaw, PhD. “This shows the world that this technology is real. It can be used. We now have to go in and work out how best to use it productively to help feed our growing population.” As the researchers concluded, their results “ … provide compelling support for feasibility of developing the surrogate sire concept in all animals including endangered species and livestock.”

The surrogate sire technology could effectively solve some of the issues of current artificial insemination approaches, and deliver the donor genetic material the natural way, through normal reproduction. Donors and surrogates do not need to be near each other, since either frozen donor sperm or the surrogate animal itself can be shipped to different places. In addition, female NANOS2 knockout animals remain fertile—since the gene only affects male fertility—and so could feasibly be bred to generate sterile males that would be used as surrogate sires.

Jon Oatley. [Washington State University]
The technology has great potential to help food supply in places in the developing world, where livestock farmers still have to rely on selective breeding to improve their stock, said co-author Irina Polejaeva, PhD, a professor at Utah State University. “Goats are the number one source of protein in a lot of developing countries. This technology could allow faster dissemination of specific traits in goats, whether it’s disease resistance, greater heat tolerance, or better meat quality.” The surrogate sires technology could also open up a new option for genetic conservation of endangered species, whose dwindling numbers leave animal communities isolated from each other, limiting their genetic diversity.

 

None of the benefits of surrogate sires could be realized, however, without changes to the current landscape of government regulations. Gene-edited surrogate sires could not be used in the food chain anywhere in the world under current regulations, even though their offspring would not be gene-edited. This is due in part to the misperceptions that gene editing is the same as the controversial gene manipulation, Oatley said. Gene editing involves making changes within a species that could occur naturally. It does not combine DNA from different species.

Acknowledging that there is a lot of work to do outside of the lab, Oatley recently joined the National Task Force on Gene Editing in Livestock, to bring together researchers, industry representatives, bioethicists, and policymakers to find a path forward for the technology. “Even if all science is finished, the speed at which this can be put into action in livestock production anywhere in the world is going to be influenced by societal acceptance and federal policy,” he said. “By working with policymakers and the public, we can help to provide information assuring the public that this science does not carry the risks that other methods do.”

 

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