The final instalment of Technology Networks Explores the CRISPR Revolution is an interview with Dr Alison Van Eenennaam, a livestock geneticist based at the University of California, Davis. The focus of her laboratory's research is the utilization of DNA-based biotechnologies in the production of beef cattle and in agricultural systems.
Dr Van Eenennaam has previously been involved in research that saw the use of gene editing technologies to produce hornless cattle. One of her ongoing projects uses CRISPR-mediated gene knock-in of the SRY gene to trigger a genotypically female cow to develop male characteristics.
Dr Van Eenennaam is a widely-published researcher and has won a variety of awards throughout her career. In our interview, she speaks passionately about the applications of her research and gene editing technologies, in addition to commenting on the future of genetically modified organisms (GMO) and the current regulations surrounding them.
MC: Why is CRISPR gene-editing superior to other gene-editing techniques in this field of research?
Alison Van Eenennaam (AVE): It's really a tool that can be used to introduce useful genetic variation into our food, plant and animal, breeding programs. There's a lot of people looking at using CRISPR, for example, to inactivate proteins or the products of genes that, for some reason or another, make an animal or plant more susceptible to disease. You can take out an exon of a gene, or perhaps knock out the entire gene if it's not essential for other purposes and generate pigs that are resistant to diseases like porcine reproductive and respiratory syndrome (PRRS) virus. Others are working on making pigs resistant to African swine fever, which of course, is a huge issue at the moment as it spreads through China, the biggest pork producer on Earth.
I'm so glad people are doing analogous things and trying to develop fungus resistant strawberries and blight resistant wheat. We lose about 20% of animal production to disease globally, and so obviously, if you can avoid diseases, then you're 20% more efficient all of a sudden, and you don't have to use chemicals to address whatever the disease problem was. You don't need to use other preventative measures, or treatments like antibiotics in the case of sick animals.
It's kind of a win/win from a sustainability perspective. That's where I see the greatest potential to use the technology as it relates to food production and security.
As for CRISPR being superior, well that question has an embedded assumption that it is. Genome editing tools are all just a fancy pair of scissors. With TALENS and zinc fingers, the disadvantage of those is that you need to develop a separate protein for every target site that you want to try and cut, which is difficult and time expensive. Of course, with CRISPR Cas9, you just need to design a separate guide RNA which is a lot cheaper and easy to design.
Only CRISPR is amenable to targeting multiple guide sites at the same time, and it's not that much more expensive to make four guides perhaps than it would be to make four separate individual proteins in the case of TALENS and zinc fingers. But they also have their own advantages, for example, the overhang generated by FORK1 nuclease that is associated with TALENS and zinc fingers. That overhang has some advantages when it comes to homology directed repair. But there are new Cas proteins that also generate an overhang. So, I think CRISPR in conjunction with new Cas proteins opens up the technology to new target sites.
MC: As an animal science expert, what would you consider to be the most exciting breakthroughs in the application of CRISPR in this field?
AVE: I think if we could develop the African swine fever resistant pig, that would be huge. China produces a billion pigs per year, and I read somewhere that they're going to lose half of their stock. That's a hugely important animal source of food for that country with its one and a half billion population.
It's not a human health issue, as humans can't get African swine fever, but it's a potential food security issue of enormous magnitude, like hard to put into words magnitude. So I think that's would be a breakthrough with the potential to affect billions of people’s existence.
The opportunity we have is to potentially move alleles from one breed to another without bringing along what's called "linkage drag", which is all the unwanted genetics from a less productive breed. It means you can move a useful allele for disease resistance using CRISPR Cas9 in a very targeted way by using a homology repair pathway. This is where the real opportunity lies, if in fact we are ever able to use the technology. Currently it is very limited due to the regulatory constraints.
MC: Previously, you used CRISPR technology to add the SRY gene to bovine cells. The SRY gene can trigger a female to develop male characteristics. Please can you tell us more about:
• The purpose of the research
• These characteristics
• The methods used to conduct the study
• The most exciting study findings?
AVE: This particular gene, SRY, which is the sex determining region on the Y chromosome, is necessary and sufficient to become male. It basically triggers the male differentiation pathway. It's located on the Y chromosome, not surprisingly. There's been documented cases of translocations of the SRY region from the Y chromosome onto the X chromosome in humans, and the result is a phenotypic male. This is someone that appears to be male, but genetically they are XX, and they have the SRY gene transposed onto the X chromosome.
The resulting phenotype does vary, but typically, it's an infertile male, an individual with male characteristics who lacks the other genes required for fertile sperm that are located on the Y chromosome.
This has been seen in humans when an individual may go to the doctor and discover they cannot have children, and genetic analysis reveals this transposition. It's also been seen in horses and other high value species. We've never seen it in cattle, probably because if I have an infertile bull, what happens to him? I don't do a genetic workup because, basically, he goes to market and becomes a hamburger. I think that's probably the reason we haven't seen it.
The purpose of this research is twofold. One is biological interest. Are we able to move the gene from the Y chromosome onto the X chromosome and generate phenotypic males? There are two interests here, one is that in beef cattle production, males are more efficient at converting feed to meat and thus are the preferred gender to have for beef production.
Secondly, if they are infertile, which we hypothesise they would be, it offers an opportunity to safely add other transgenes to these animals. This would be a genetic containment strategy, as of course the bull would be infertile and unable to pass the genetics along. There has been a long-standing concern of "transgenes" getting out into the environment, which doesn't really make a lot of sense when you're talking about cattle, because it's not like we have a lot of wild cattle running around "spreading their seed", but that was one of the rationales that was used in obtaining the grant funding to do the project. It has been a real bear of a project. We were targeting the X chromosome to transfer the SRY gene on to it. Unfortunately, in the bovine genome, the X chromosome is badly annotated; we don't have a very good map of it. We thought we were targeting a region that was "intergenic", meaning between two genes, but when a thorough annotation of the bovine gene assembly came out, we discovered that we'd been working right next to an essential gene, which perhaps explained why we'd been having trouble getting viable cells.
We've since moved to a safe-harbor site called H11 on an autosome chromosome, and this is where we've been targeting SRY. We really want the proof of concept. Ideally we would like to target the X chromosome, but given how poorly mapped that chromosome is, we thought it was safer to go into an area that we know is between genes, where you can insert a gene, without interfering with the viability of the animal.
During the course of this research, the Food and Drug Administration (FDA) made a determination that all “intentional alterations” in genome-edited animals are going to be considered new animal drugs, which dramatically changed the regulatory picture for these animals. We were hoping that because we were moving around an endogenous gene, it wouldn't trigger GMO regulations; but with this 2017 FDA guidance, effectively the animals in the study are all classed as unapproved animal drugs meaning we basically have to incinerate them all, rather than allowing them to enter commerce.
If the animals are going to be considered to be an unapproved animal drug irrespective of the genomic alteration, we decided we may as well include a reporter gene along with the SRY knock-in. So, we inserted a reporter gene known as green fluorescent protein that enables us to see if we have successfully knocked-in SRY into the autosome. Before we do embryo transfers, a fluorescent green embryo confirms that we have a successful SRY knock-in embryo to transfer.
Video taken from SciFri.
MC: In a 2018 interview, when referring to gene editing technologies, you said, "We are being blocked from using these technologies because of the discussion around the crops." Please can you expand on what you meant by this? And, if this still applies, how do you anticipate overcoming this issue?
AVE: I was no doubt referring to the controversy surrounding GMOs and the unsubstantiated safety issues that have been raised, particularly in Europe. It's frustrating. The inability to cultivate GMOs in European agriculture is, to me, "hypocrisy on steroids", given they import large quantities of GM feed for their livestock populations. The GMO controversy has meant that there have been essentially no GM animals brought to market.
Globally, there is one approved food product and that is the fast-growing salmon, which was basically developed around 30 years ago and has only just obtained full regulatory approval. The characteristic of the salmon is that it grows faster, meaning it gets to market weight in half the time. That is not a trait that normally is faced with a high regulatory bar. Fast growth is a trait that is routinely selected for in almost every livestock species. It is hardly a new phenotype. Because of the issues and controversies around GMOs, there has been virtually no investment or public funding available to do genetically engineered animal research.
Genetic engineering, of course, is an older technology, where you randomly introduce a transgene into the genome and "cross your fingers" that it lands in a place in the genome where it does no harm and it is expressed.
When gene editing came along, where you can go in and tweak the DNA and perhaps inactivate an endogenous gene in the genome, or introduce an allele from one breed of cow into another to prevent them from growing horns (which is one of the projects my lab is collaborating on), we thought, "this is great". This was because we're not moving genes from one kingdom to another, so many of the concerns surrounding genetic engineering and transgenes shouldn't be part of the discussion.
There's no rationale for regulating edits that could have been achieved using conventional selection differently to the current regulation of conventional breeding, which is not formally regulated at all. Other than it is illegal to sell unsafe food, irrespective of the breeding method used to produce that food. That was our thought process, and it's the thought process in countries such as Brazil and Argentina, where if you have not introduced a novel DNA sequence, i.e you haven't introduced a transgene, then you're going to be treated as if it's conventional breeding. There will be no special "GMO regulations" for gene edited products that carry no novel DNA sequences.
In 2017 the FDA came out with a draft guidance that says all intentional alterations in animals, irrespective of novelty, if developed using molecular techniques including genome editing, are going to be treated as a new animal drug. Which means you have to go through a multi-generational, multi-million dollar regulatory drug approval package. That effectively makes it cost-prohibitive for the public sector and small companies to have anything to do with commercializing genome edited food animals. It may even make it difficult for animal geneticists working with large animals in America, to do research in this field. Europe has a similar regulatory approach, anything that is edited is going to be regulated as a GMO.
This is problematic. I know how over-regulation can block genetic innovations. I've been around the GMO debate for over 20 years, and it feels a bit like "Groundhog Day". The same arguments are being used regarding genome editing that were used about GMOs. It is the same debate, the same tired talking points, sometimes even including the same individuals doing the talking. Despite there now being over 20 years' worth of data demonstrating that there haven't been any unique safety concerns associated with GMOs. The debate seems immune to objective-evidence and facts.
As a scientist, you can't prove safety. You can only provide data that supports the absence of harm. When someone asks you to “prove the safety of something in the future”, you know that they're asking you to do the impossible. I can't prove that the sun will come up tomorrow, but I can make a pretty good prediction based on past data.
Credit: Photo by Sunnie-Lee Davison on Unsplash.
MC: In your opinion, what does the future of gene editing in animal science look like?
AVE: It depends, if you want to ask optimistic or pessimistic Alison. Optimistic Alison would say that there are tremendously valuable potential uses, like introducing disease resistance, and addressing animal welfare concerns like dehorning cattle. There's a plethora of problems that gene editing offers the opportunity to address. If we're given the ability to use it, I think it will be limited only by the imagination of researchers and research funding.
Pessimistic Alison is maybe a little jaded after 20 years of the GMO debate and says that the same tactics that were used to derail genetic engineering will be successfully employed to derail gene editing in this space, and often by the same groups who have found quite a lucrative niche in creating fear around that technology.
If you fight innovation in animal agriculture and agriculture more generally, and you are successful at demonising a safe and effective technology, such that you actually make it so that farmers can't use it, you've just increased the environmental footprint of agriculture. If I look at Europe, for example, farmers can't plant or cultivate GMOs. So, they can't plant insect protected crops. Despite the incredible decrease in insecticide use that has been associated with insect-protected GMO crops globally. But in Europe bad luck you can’t use this technology, but the insects are still a problem and farmers spray with insecticides. So you've basically blocked your own farmers’ ability to use a safe and effective genetic technology that would have helped them reduce their use of harmful chemicals.
Pessimistic Alison thinks that marketing forces will be at work to demonize gene editing, because they've made so much money off fear mongering around GMOs. I think they're salivating at the idea that they can continue to monetize misinformation in this space.
Dr. Alison Van Eenennaam was speaking with Molly Campbell, Science Writer, Technology Networks.
Catch up on the previous instalment of Technolology Networks Explores the CRISPR Revolution, an interview with Dr Amy Butler, here.