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Why is asparagine concentration so important?

Asparagine is one of the 20 amino acids that are used by all living things on Earth to make proteins. However, asparagine accumulates in its free (soluble, non-protein) form in wheat grain at much higher concentrations than are required just to make proteins. This can be exacerbated by other factors, such as the lack of sufficient sulphur in the soil. It is important because free asparagine can be converted to a toxic contaminant called acrylamide when flour prepared from the grain is used to make food. Just about all food products made from wheat flour are affected, including bread, especially after toasting. Acrylamide is regarded as ‘probably cancer-causing’, and has other toxic effects at high doses (more information is available from the European Food Safety Authority Panel on Contaminants in the Food Chain: Scientific opinion on acrylamide in food.  EFSA Journal, 13, 410, 2015).

If we could reduce the free asparagine concentration in wheat grain it would lower the exposure of consumers to acrylamide from their diet. It would also make it easier for food businesses to comply with regulations on the amount of acrylamide that is present in their products.

What’s the difference between GM and gene-editing?

In the case of GM, extra DNA is introduced into the plant, adding one or a small number of additional genes to a plant’s complement of several tens of thousands of genes. In the case of gene-editing (GE), the technique allows for small changes to be made to a precisely targeted gene that is already present in the plant.

Why are these plants both GE and GM?

The gene editing of these plants was undertaken using a technique called CRISPR. This technique requires some genes to be introduced into the plant by GM, one to make a nuclease (a protein that will do the editing) and one to make what is called a guide RNA molecule (gRNA) that interacts with the nuclease and targets it to the chosen gene. The plants also had a marker gene in them that made them selectable in the lab using a herbicide. Now that the editing has been done these additional genes can be removed to produce GE but non-GM plants. This is done by self-pollinating the plants through several generations and identifying individuals that have lost the additional genes. The plants to be used at the start of the trial all have one or two of the additional genes removed but retain at least one, so for now are GM. The ability to screen more plants in the field trial will enable us to identify completely non-GM individuals and by the third year of the trial we expect the plants to be entirely GE but not GM.

What gene has been edited?

Asparagine is made by enzymes called asparagine synthetases. Wheat has five genes that make these enzymes, called ASN1, ASN2, ASN3.1, ASN3.2 and ASN4. We have studied this small gene family and discovered that ASN2 is the most active in the grain and is not active anywhere else in the plant, and this is the gene that we have knocked out using CRISPR. Our theory is that the low activity of the other ASN genes in the grain will provide enough asparagine to make proteins but that free asparagine will not accumulate in the way that it does when ASN2 is working. The rest of the plant should not be affected. Studies on the plants growing in a glasshouse have shown this to be the case, but the real test is how the plants fare in the field, and whether asparagine concentrations continue to be low in grain produced under field conditions.

Is GE subject to the same regulations as GM?

In the EU, GE has been classified as a form of GM since July 2018. In many other parts of the world, regulators and scientists consider GE not to be a form of genetic modification and are regulating GE and GM crops differently. The UK has now left the EU, of course, but regulations have been carried over for now. Under these rules, our plants would have to be treated like GM plants for the duration of the project. However, DEFRA held a public consultation on gene editing earlier this year and the outcome of that should be made public in the summer. This may lead to changes in how the UK regulates GE plants.

Could asparagine levels be lowered by other methods?

We have been working on genetic and agronomic approaches to reducing the asparagine concentration of wheat, rye and potato since 2004, and our work has informed the development of regulations on acrylamide in food in the EU, and helped food businesses and their supply chain to comply with those regulations. However, the reduction in asparagine concentrations in the grain of our CRISPR wheat goes way beyond anything we have achieved by other methods. We do have plants in which the ASN2 gene has been mutated using the much older technique of treating seeds with a chemical mutagen. This technique creates mutations at random positions in the plant’s genome, in contrast to the targeted changes induced by CRISPR, but it has been used in plant breeding since the 1950s and plants made in that way are not subject to the same regulations as GM/GE plants. Those plants are behind the GE plants in development and we do not know yet if they are low in asparagine, but some will be grown alongside the GE plants in the field trial to compare them.

Why are these experiments necessary?

We have already studied our low asparagine wheat plants in our laboratories and glasshouses and published the data showing that the asparagine concentration in the grain is very low. The results of those experiments have been published (Raffan et al., Plant Biotechnology Journal, 2021, Doi: 10.1111/pbi.13573). We now need to test the plants in the field, to find out if the low asparagine trait is maintained under field conditions, and assess how the plants perform in the field in other ways, such as their grain yield and protein content. The greater numbers of plants grown in the field trial will also allow us to identify plants that have lost the added genes i.e. plants that are GE but not GM.

How important is this research?

This is very much a research project, but it may lead in the future to the development of commercial wheat varieties with very low concentrations of asparagine in the grain. Flour made from such grain would have greatly reduced potential for acrylamide formation during baking. This would have obvious consumer safety benefits and would make it easier for food businesses to comply with regulations on the presence of acrylamide in their products. The project is also the first field trial of GE wheat in the UK and Europe, and one of the first in the public domain anywhere, so it will be a landmark event for the development of crop gene editing.

What is the nature of the genetic modifications you have made?

We originally made three genetic modifications to the plants. Two of these were to enable the CRISPR process to work, with one introducing a gene that makes the Cas9 nuclease, which is the protein that does the editing, the other a gene that makes the guide RNA (gRNA) molecule. The gRNA is designed to guide the Cas9 nuclease to the target gene, in this case the ASN2 gene. The third modification introduced a Bar gene, which allows the plants to tolerate phosphinothrycin-type herbicides. This enabled us to identify plants that had been successfully genetically modified in the lab.

None of these genes is required now that the editing has taken place and they are being removed by self-pollinating the plants and selecting individuals in the next generation that lack them. In our ‘best’ plants, the Cas9 and Bar genes are already gone. The field trial will allow us to grow many more plants than was possible in the glasshouse and complete the process of producing non-GM, GE plants.

What kind of changes has CRISPR made to the target gene?

We identified several different edits to the ASN2 genes in the plants. Most were small deletions (i.e. a small section of DNA was removed, the longest being 173 base pairs long), but there were also some single base pair insertions (i.e. the gene had an extra base pair) and substitutions (where a single base pair had been changed). Bread wheat has three genomes, A, B and D, and some plants only had editing in the A genome ASN2 genes, while others had editing in all of them. Both types will be included in the field trial.

The types of edits that have been induced by CRISPR in our plants can and do occur as natural mutations; indeed, mutations like these occur all the time. The power of the technique is that the editing is targeted to a specific gene.

How have you communicated this research and the possibility of a field trial?

We have been working on this area of research since 2004 and have published many scientific papers on the topic, including a recent paper that described how we had made the plants to be used in the field trial (Raffan et al., Plant Biotechnology Journal, 2021, Doi: 10.1111/pbi.13573). That paper, like most of our publications, is available to download for free, and can be found on-line using the Doi number. The publication of that paper was accompanied by a press release that announced our intention to apply to Defra for a licence to run a field trial of the plants. For more information on our earlier work on the topic, you can look at a review that we published in 2019 (Raffan and Halford, Annals of Applied Biology, 2019, Doi: 10.1111/aab.12536). That too is free to download and can be found on-line using the Doi number.

Will independent experts and the public be consulted?

As part of the statutory process co-ordinated by the Department for Environment, Food & Rural Affairs (Defra), there is a period of public consultation before a decision is made on whether the field trial can go ahead. People can make representations to Defra on any environmental risks that they think the trial might pose. These concerns will be considered by the independent experts of ACRE, the Advisory Committee on Releases to the Environment.

ACRE is a statutory advisory committee appointed under section 124 of the Environmental Protection Act 1990 to provide advice to government regarding the risks to human health and the environment from the release of genetically modified organisms (GMOs).

Why is there a need to carry out this research in the UK?

The UK has world-class plant scientists who should be at the forefront of developing scientific and technological advances that can make agriculture more sustainable while ensuring that the country continues to enjoy a plentiful supply of safe and nutritious food. Plant biotechnology has become an established technique in plant breeding in many parts of the world, and GM crops have been grown commercially since 1994, with large-scale cultivation of GM field crops starting in 1996, a generation ago. GE is a newer technology, but we are already seeing huge investment in its use for crop improvement, particularly in the Americas and Asia. It is important that the UK maintains its expertise in these technologies.

Will the trial be safe?

Yes. Rigorous regulations govern the planting of GM crops in the UK, overseen by Defra and its independent advisory committee, ACRE. There will also be inspections during the trial, carried out by the Genetic Modification Inspectorate, which is part of the UK’s Animal and Plant Health Agency. The ensuing inspection reports are made publicly available.

What is the process of approval for research using GE and GM crops and microbes?

At Rothamsted, as at many universities and research institutes around the world, work is taking place using GM plants and microbes for a variety of projects and to address specific scientific questions. Research is conducted in our laboratories, in our glasshouses and, in some instances, in the fields of our research farm. Where research using GM plants or microbes is undertaken, relevant regulations are followed, and risk assessments carried out. Currently, the same regulations cover the use of GE plants and microbes.

There are several key pieces of legislation specifically concerned with the contained use of such organisms, the most important of which is the Genetically Modified Organisms (Contained Use) Regulations 2000, as amended, covering both human health and environmental aspects of work in laboratories and glasshouses. Rothamsted’s activities comply with the regulations set out in this and other relevant pieces of legislation.  We also follow best practice in risk assessment, as specified by the Compendium of Guidance, which has been put in place by the Health and Safety Executive (HSE), in conjunction with Defra and the Scottish Government.

If a research project requires experimentation using GM or GE crops in the field, as in this case, approval from Defra is required. We must carry out a full risk assessment of the project, which is scrutinised by the institute’s own Biosafety Committee and other experts in-house, and then apply to Defra. In the application, we describe the nature of our experiment, the type of plant material and the specific genetic modifications / edits that we have carried out. We are also required to assess the risks to human health and the environment. The application becomes publicly available on the Defra website and our application is independently assessed by ACRE. As soon as the application is submitted, there are 48 days for public consultation, carried out by Defra; any member of the public can comment or ask any relevant question.

Once the review of the risk assessment has been carried out, ACRE make their recommendation to Defra, which considers it along with any public representations that the department has received. Defra then decides whether to grant a consent to conduct the field trial, and whether to impose specific conditions on it.

What about the risks of cross-pollination with local wild plants or crops?

Wheat is a self-pollinating crop with very low rates of cross-pollination even with other wheat plants. The only wild relatives of wheat commonly found in the UK are in the genera Elymus and Elytrigia (formerly known as Agropyron) and there are no reports of cross-hybridisation between wheat and species of these genera. The two most common inland species are common couch and bearded couch.  Common couch is quite widespread on the Rothamsted estate, whereas bearded couch is confined to woods and hedgerows. Common couch propagates primarily by vegetative reproduction (rhizomes), rather than by sexual reproduction.

Wheat can also be forced using laboratory techniques to cross with a limited number of cultivated cereals, including rye and triticale, but if crosses with these species do occur spontaneously in the field they must be extremely rare events.

Overall, therefore, cross-pollination is extremely unlikely to occur. Nevertheless, the trial site will be surrounded by a 3 metre-wide wheat pollen barrier and no cereals or grasses will be allowed to grow within 20 metres of the trial.

Could insects be affected by the plants in the trial?

Wheat plants have a range of insect pests in the UK, including thee bird cherry-oat aphid, the grain aphid and the rose grain aphid, as well as the orange wheat blossom midge and the wheat bulb fly. Wheat also interacts with beneficial insects that attack aphid pests, such as the parasitic wasp, Aphidius rhopalosiphi. Interactions with these insects are not expected to be affected in any way by the low asparagine trait carried by the plants.

Will the grains from the GE plants be consumed?

No. Plants and seeds arising from this trial will not enter the food or feed chains.

Is the wheat likely to be more ‘weedy’ than ordinary wheat?

No. Some of the plants do contain a herbicide tolerance gene for now, but phosphinothrycin-based herbicides are not used in the UK anyway and other herbicides could be used to destroy the wheat if necessary. The low asparagine trait is not expected to have any effects on the wheat’s ability to survive in either agricultural or wild settings.

What are the likely effects on biodiversity of this research?

This is a small-scale, highly controlled experiment over the course of five years at the most. We do not anticipate any effects on biodiversity.

How did you make the plants to be used in the trial?

The Cas9 gene has been widely used for gene editing, in plants and other organisms. Colleagues at Rothamsted worked with researchers at the University of Bristol to develop a version that was optimised to work in wheat, and that was the one we used. The Bar gene is also widely used in plant genetic research.  The guide RNA gene had to be made specifically for this project, and was designed to target just the ASN2 gene.  The genes were attached to promoters (effectively gene switches) that determine when and where the gene is active in the plant. These came from rice and maize. The genes were incorporated into circular DNA molecules called plasmids and used to genetically modify wheat variety Cadenza using a technique called particle bombardment. This involves coating microscopic gold particles with DNA and bombarding the particles into wheat embryos. This technique has been used to make genetically modified cereals since the 1980s. Plants were regenerated from the embryos and GM plants were selected using phosphinothrycin. Once the CRISPR machinery was in the plants the nucleotide sequences of the ASN2 genes were analysed to identify edited plants and see what kinds of edits had been induced.

Will your experiment be legal?

Yes. GM/GE experiments are allowed in the UK if strict regulations are followed and this field trial will proceed only if it is authorised by Defra.

Will the public be able to see the trial?

Yes. With Rothamsted’s permission, it will be possible to see the trial.

How big is the trial?

The area of the proposed trial at Rothamsted, including the wheat pollen barrier, will be just under 1500 square metres, or less than one sixth of a hectare or two fifths of an acre.

Where has the funding for the project come from?

The plants were produced by a PhD student within the Biotechnology and Biological Sciences Research Council (BBSRC) South West Biosciences Doctoral Training Partnership.  A two-year project that will include the first year of the field trial was then funded by the BBSRC through its Super Follow-on Fund. We will seek further funding for subsequent years.

Have you received industry funding for this work?

The studentship in which the plants were made was an industrial CASE studentship involving five UK wheat breeders and the Agricultural and Horticultural Development Board, as well as Rothamsted and the University of Bristol. The current project, incorporating the first year of the field trial, is supported by the same group but our work on it is entirely funded by the BBSRC.

Overall, Rothamsted receives about 10% of its funding from industry. Our overarching philosophy is that we need to work with a range of stakeholders, including government policymakers, agrifood businesses and farmers if we are to deliver the knowledge, innovation and new practices that are needed to develop sustainable solutions for the agrifood sector. Working with industry forms a crucial part of this philosophy because only industrial partners have the necessary infrastructure to develop and distribute innovative technologies to those who need it. Nevertheless, we recognise that the UK taxpayer is the main funder of our research, and we have sought the public’s input, as well as that of our stakeholders, in developing guiding principles of how we should work with industry.

Do you or any of the companies involved own patents on the technology?

No. CRISPR patents are held by two consortia, the first comprising the University of California and the University of Vienna, and the second the Broad Institute and Massachusetts Institute of Technology.

Is there a market for this product?

That will depend on how the regulations covering GE crops develop. There is certainly a demand from the food industry for low asparagine grains that will enable them to comply with regulations on the presence of acrylamide in their products while retaining the attributes of baked products that consumers demand.

Are you going to put the knowledge gained from this trial in the public domain?

Yes. The results of the field trial will be published after peer review in appropriate scientific journals and using the open access model, where the papers are free for anyone to download. We will also disseminate data and new knowledge through presentations at scientific conferences. We have extensive contacts with food businesses and will use these contacts, to raise awareness of the project and its outcomes more broadly, as well as taking every opportunity to engage with the more mainstream media.