Dr Yves Bertheau and other experts rebut claims that genome-edited products cannot be distinguished from natural products and thus cannot be detected or regulated
GMO proponents lobbying for lax regulation of GM plants and animals produced with "new GM" techniques, including genome editing, argue that living organisms naturally contain many mutations (DNA damage), making them "natural GMOs". They add that it is often impossible to distinguish mutations induced by the new GM techniques from naturally induced mutations and that therefore GMOs produced with these techniques should not be regulated more strictly than conventionally bred varieties. Furthermore, they argue that GMOs produced with these techniques often cannot be distinguished from naturally bred organisms. They conclude that these GMOs cannot be identified or traced – and because traceability is not possible, it is simply not practical to regulate or label them.
However, these claims are challenged in a new methodological paper[1] by Yves Bertheau, director of research at the French National Institute for Agricultural Research (INRA), Versailles, currently at The National Museum of Natural History (MNHN), Paris. The article, which forms part of a scientific Encyclopedia published by Elsevier, is technical, but we have extracted some of the main points and interpret them for the non-specialist reader below.
Dr Bertheau’s article uses the term "New Breeding Techniques" (NBT) to describe the new techniques, but since these are genetic modification techniques and we at GMWatch have always emphasized this fact, we use the term "new GM".
Dr Bertheau’s analysis is backed by the views of other GMO traceability experts, which are summarized at the end of this article.
Incorrect claim no. 1: Many mutations are naturally present in plants, so we shouldn't worry about those caused by new GM techniques.
Truth: Genomes have evolved to be highly stable. Plant and animal cells have various mechanisms to protect their DNA against invasion by introduced DNA and from mutations. For example, cells tend to eliminate mutations during cellular cycles of growth and reproduction.
These protective mechanisms explain the very low rate of mutations transmitted to offspring by even old plants, such as ancient trees.
Cells also have mechanisms in place to repair any damage caused by breaks in the DNA, such as those caused by some genome editing techniques. Some of these repair mechanisms, such as the rapid but error-prone non-homologous end-joining or NHEJ, are exploited in genome editing.
These factors may explain why spontaneous mutations are found at very low rates in both one-celled organisms (prokaryotes) such as bacteria, and complex-celled organisms (eukaryotes) such as animals, plants, algae and fungi.
In light of the very low numbers of spontaneous mutations that are retained, it is wrong to claim that many “natural” mutations persist in plants. Exceptions are in stressful situations such as exposure to radiation, high temperature, drastic drought conditions, tissue culture utilized in genetic engineering processes, and mutagenic chemicals.
Incorrect claim no. 2: Horizontal gene transfer (the acquisition by an organism of genetic information by transfer, for example via a virus, from an organism that is not its parent and is typically a member of a different species) frequently happens in nature and thus we should not worry about this process in genetic engineering.
Truth: “GMO by horizontal gene transfer” is rare and not the norm, especially between different organisms. On the rare occasions when it does occur, its effects are unknown.
Incorrect claim no. 3: Organisms produced with new GM techniques often cannot be identified and/or distinguished from natural organisms. Thus they are not traceable as GMOs and thus it is not possible to subject them to any GM-specific legislation.
Truth: Contrary to this claim, many already-available techniques allow unambiguous detection and identification of a wide range of genetically modified sequences, from the smallest – e.g. a point mutation of a single nucleotide (DNA base unit) – to the largest, e.g. insertion of large genetic sequences. Chromosomal rearrangements and multiple copies of genetic sequences produced by new GM techniques can also be detected and identified. Modifications located in several parts of the genome and epigenome can be detected at the same time (multiplex techniques).
Many of these techniques are already used by breeders and seed companies. They include:
• basic observation of the characteristics of the new GMO (e.g. how a herbicide-tolerant GMO reacts to a herbicide)
• amplification of genetic targets
• whole genome sequencing.
Most use techniques such as PCR (polymerase chain reaction) or NASBA (nucleic acid sequence based amplification).
Methods range from qualitative (identifying if a GM trait is present) to quantitative (measuring how much GM material is present).
These methods are well known to GMO testing laboratories. They could be quickly adopted and validated by the European Union Reference Laboratory for GM Food and Feed (EURL-GMFF), with the support of the European Network of GMO Laboratories (ENGL), and directly applied at low cost by operators at all stages of the supply chain.
To give an idea of costs, sequencing companies recently announced that they will soon be able to provide full genome sequencing at a cost equivalent to current widely used PCR detection tests, i.e. 100 to 250 Euro, depending on the type of test requested.
Legal and technical frameworks are all in place to ensure that the test material is provided and analytical traceability of new GM products is quickly implemented, as requested by consumers.
Incorrect claim no. 4: Targeted mutations due to new GM techniques are "natural" and therefore indistinguishable from those spontaneously arising. Thus the identification of the products of these techniques would be impossible and traceability questionable.
Truth: This is more of a political assertion than a scientific truth. Claiming that a mutation is "natural" is similar to asserting that current climate change is natural and not due to man-made changes.
The probability of mutations occurring and being maintained in the absence of environmental selection pressures is so low that human intervention is necessary to accelerate genetic diversity – as is recognized by all plant breeders. The sheer increase in the frequency of mutations through the application of new GM techniques is part of the artificial character of these techniques and therefore must be considered in any discussion of how "natural" the mutations are.
In addition, mutations cannot be looked at in isolation from their wider genetic context. As is usual in logic, we must consider all available contextual information, such as
• intended and unintended modifications to genomes and epigenomes – the latter meaning the level of gene regulation via chemical modification of DNA, or the proteins (histones) with which the DNA is associated
• RNA modifications.
The frequencies and types of these modifications must be taken into account.
By analogy, when it comes to identifying humans, biometric systems use various factors, such as fingerprints, iris recognition, and facial recognition. Thus several factors converge toward the identification of an individual. In the case of GMOs, detection and identification techniques rely on unambiguous signatures and/or convergent clusters of evidence. Such a multi-targeted approach is already used by risk assessors in various fields under the name of “weight of evidence”. In the field of GMO detection, this approach is known as the “matrix approach”. A similar approach is used by breeders in marker assisted selection, a breeding technique that relies upon identifying genes of interest or other related sequences and selecting progeny in which those traits are expressed.
The data gathered in the matrix approach for the identification of unapproved and/or unknown GMOs, whether developed using old or new GM techniques, could be collected by the enforcement agencies and private laboratories, which could act as the technical and scientific “police”. The data could then be easily and accurately evaluated using statistical and other analytical tools, and/or fed into computer-based decision support and artificial intelligence systems in order to reach a conclusion as to whether the product is a GMO.
Identifying “scars”
New GM techniques involve the use of the same techniques as are employed to produce transgenic GMOs, for example:
• the preparation of plant protoplasts (cells that have had their walls removed) or tissues
• tissue culture
• the vectorization of molecules so that they reach the target cell nucleus
• selection of the genetically transformed (engineered) cells followed by the elimination of the selection factors
• plant regeneration for non-recalcitrant species (meaning the few plant species that are able to respond to tissue culture manipulations by growing into new plantlets). The “recalcitrance” (non-responsiveness to such manipulations) of some plant species explains why new GMOs are still restricted to the old GM crop species range and why the majority of “orphan” crops cannot be modified.
These are all stressful techniques for the plant and leave detectable scars in the genomes and epigenomes, in the form of mutations and epimutations. Epimutations are abnormal changes in the chemical modification of DNA or the proteins with which it is associated, or RNAs leading to alterations in the overall profile of gene expression.
In animals, equivalent scars are caused by stresses such as in vitro fertilization and can result in abnormalities in the offspring.
In plants, the remaining scars (collectively called somaclonal variation – the variation seen in plants that have been produced by plant tissue culture), have long been valued by seed producers at the stage of varietal selection as they can act as markers for genes of interest. However, once this stage is over and the breeders want to propagate the selected line, they try to reduce the number and effects of these scars.
This is attempted by backcrossing. GM crop varieties are backcrossed with elite non-GM varieties in an attempt to "clean up" the unintended changes and mutations. But in spite of this, numerous changes intentionally or unintentionally induced by new GM techniques can be transmitted to offspring and appear in the final marketed seed variety.
Indeed, the number of backcrosses performed with elite varieties for marketed products is generally below the six that are theoretically necessary to obtain the desired minimum 95% level of genomic “purification”.
Identifying new GM signatures
New GM techniques not only induce scars such as chromosomal rearrangements and certain patterns of indels (insertion or deletion of bases, or basic units of DNA in the genome of an organism), but also provide specific signatures for the new GM technique used.
For instance, in the case of the CRISPR system, the sequence of the RNA that guides the CRISPR site-directed nuclease complex to the site within the host genome to be modified (the guide RNA) possesses a specific invariant sequence component, which is technically known as the PAM – the protospacer adjacent motif. “Invariant” means that guide RNAs of a given CRISPR system designed to target different sites in the genome will possess the same PAM as part of their sequence.
The guide RNA, including the PAM, constitutes a recognition sequence that is unique to the given CRISPR system being used. A detection test can be applied that looks for the recognition sequence of a given CRISPR complex at the site of alteration of the genome. If the recognition sequence is found, it identifies the mutation, at on-target or off-target sites, as arising from the editing tool. Single or multiple CRISPR-induced mutations with different PAM sequences can be identified in this way.
The genomic signatures of other gene-editing tools can be similarly identified. In the case of the ZFN and TALEN gene-editing tools, these work as a separate pair of protein complexes. Each of the ZFN or TALEN pair is able to recognize and bind to a specific DNA base unit sequence within the DNA of the organism being targeted. This means that each of the ZFN or TALEN pairs possesses its own unique recognition sequence. Once both components of the ZFN or TALEN pair bind to their respective recognition sequence sites, which are usually spaced close by, they will create a double-strand break of the DNA helix. Looking for one or both of the ZFN or TALEN pair recognition sequences around an on-target or off-target mutation site will uniquely identify the mutation as induced by these editing tools and not by some random natural mutational event.
From the point of view of the consumer and regulator, all these scars and “signatures” allow the identification of products and techniques to their origin. There are also likely to be used by the developer companies to protect their patents.
In vegetatively propagated plants (e.g. potatoes or fruit trees) or those resulting from micropropagation (the propagation of plants by growing plantlets in tissue culture and then planting them out), none of the mutations and changes introduced by the genetic engineering processes used to produce the plant are removed by backcrossing.
There is no organism for which stresses, especially those produced through in vitro processes, do not induce heritable scars that can be identified and traced. The data collected can be analyzed and interpreted, for example, using computer-based aids and routine laboratory detection practices. This applies equally to new GMOs.
For the above reasons, it is not valid to argue that new GMOs, and/or their offspring, will not be identifiable as GMOs.
Traceability and labelling of new GMOs is technically possible
Dr Bertheau’s article concludes that the proof of concept of the ability to identify the particular new GM technique that gave rise to any given product should be readily available as soon as the European Commission decides to provide the means – just as it did in the late 1990s with research programmes on GMOs produced by the old transgenesis methods.
And just as with transgenic GMOs thirty years ago, the analytical traceability and labelling of new GM products is technically possible. Whether or not the means are put in place is thus a matter of political will and will be influenced by the balance of power between stakeholders.
Commission’s science service JRC confirms detection possible
Dr Bertheau’s conclusions are supported by a report by the EU Commission’s science service, the Joint Research Centre (JRC).[2] The JRC report was in turn developed from a 2011 ENGL working group report on the detection of unknown GMOs. The working group co-chair was Dr Bertheau.[3]
The JRC looked at GMOs as a whole, without differentiating the techniques used to generate them. It concluded that detection of any given GMO is feasible provided that enough information on the GMO is provided by the developer company.
Thus since 2011, the European enforcement laboratories have had available the tools and concepts to identify and detect all kind of GMOs, whatever the methods used to produce them.
However, the JRC warned that it has always been difficult to detect some unauthorized GMOs [UGM], for which the identifying data has not been notified to authorities: “The less [that] is known about a UGM (e.g. not officially registered in order to obtain an authorization for food/feed use), the more difficult it will be to assess its safety, the higher the perceived risk, and the more difficult (unlikely) it will be to detect it by the currently applied standard analytical methods.”
This view is in line with the advice that GMWatch has received from scientists. This says that provided the GMO developer company makes available sufficient identification information on its GMO, via patents and the EU’s GMO registration and authorization process, detection of the GMO is both possible and straightforward. Dr John Fagan, chief scientist at the Health Research Institute, an Iowa, USA-based organization that provides testing services, pioneered the development of DNA tests for GM foods from their introduction in the 1990s. He commented on the potential for testing for new GMOs:
"Descriptions can be formulated that make it sound like the new GMOs are special, different, and difficult or impossible to test for. In practice they are not more difficult to test for than the old fashioned GMOs. Enough information will be available in the patents and applications for approvals so that with just a little bit of molecular biological detective work, tests can be designed. And because these are commercial products, as soon as they enter the market, it will be possible, in one way or another, to obtain a sample that can be used to verify the tests designed. This is exactly how labs have been developing GMO tests since 1996.
"Whether you call it genetic engineering or gene editing, the fact remains that these methods change the DNA sequence. The changed sequences are discoverable. And with that, a test can be developed."
JRC/industry “ghost note” confirms new GMOs can be detected
Following the July 2018 ruling by the European Court of Justice that the products of certain new GM techniques are GMOs and must be risk assessed and labelled as such, the discussion about detectability was enlivened by the appearance on the Internet of an “explanatory note” titled, “Challenges for the detection of genetically modified food or feed originating from genome editing”.[3] The “note” was co-authored by members of the JRC and Wim Broothaerts, a former JRC member who is employed by the GMO developer company Dupont Pioneer.
The note subsequently vanished from the Internet, but not before the French NGO Inf’OGM captured and posted it on its website.
The authors of what Inf’OGM termed the “ghost note” claimed, “Many of the mutations induced by new mutagenesis techniques cannot be unequivocally distinguished from natural mutations [or] from those introduced by conventional mutagenesis techniques” used in conventional breeding programmes.
However, as Inf’OGM pointed out, the authors only considered one of many existing detection methods. They also chose to focus only on the intended mutation, ignoring the other mutations, including the off-targets, which appear from the application of the new GM techniques, including “mutagenesis” techniques (as clarified by Dr Bertheau’s analysis).
Moreover, the authors of the note immediately went on to contradict themselves, admitting that detection and traceability are indeed possible: “Products of genome editing could only be detected and identified [...] when prior knowledge on the altered genome sequence, a validated detection method… and certified reference materials are available”.
Such information is precisely that required by the existing European legislation before authorizing a GMO to enter the market. As Inf’OGM noted, “The detection of unknown GMOs suddenly appears possible for the authors even if they underline that ‘emerging sequencing-based analysis for the detection of unknown [GMOs] would require significantly more time and resources [which would] affect the timely clearance of goods entering the EU market]’.”
Converging analyses
The motivation of the authors of the “ghost note” is brought into question by this admission. It appears to be in line with the economic interests of the industry rather than the biosafety interests of just about everyone else.
However, when this bias is accounted for, it is clear that the authors of the note agree with the official JRC report and Dr Bertheau’s analysis that the products of new GM techniques can be identified, provided the developer company makes available the necessary data and reference material (that is, the original gene-edited plant material generated by the developer).
These converging analyses are reassuring for members of the public who want to know how their food was produced. Now it is the duty of the EU institutions to task the GMO companies with providing data and reference material on their GMOs and the EU’s GMO Laboratories (ENGL national laboratories supporting the EURL-GMFF Reference Laboratory) with validating the required tests.
References
1. Bertheau, Yves. (2019). New Breeding Techniques: Detection and identification of the techniques and derived products. In: Melton L et al (eds.) (2019). Encyclopedia of Food Chemistry. Reference Module in Food Science. Elsevier. 320-336. 10.1016/B978-0-08-100596-5.21834-9. https://www.sciencedirect.com/science/article/pii/B9780081005965218349?via%3Dihub
2. JRC (2017). Detection, interpretation and reporting on the presence of authorised and unauthorised genetically modified materials. JRC Technical Report: European Network of GMO Laboratories Working Group Report. https://www.gmwatch.org/en/67-uncategorised/18694-jrc-technical-report-european-network-of-gmo-laboratories-working-group-report-2
3. JRC (2012). Overview on the detection, interpretation and reporting on the presence of unauthorised genetically modified materials. http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm.
4. JRC (2018). Explanatory note: Challenges for the detection of genetically modified food or feed originating from genome editing. https://www.infogm.org/IMG/pdf/comeur_note-detection-nveaux-ogm_nov2018.pdf
Glossary
Genes: Regions of DNA sequences that either code for proteins through intermediary RNA or RNA molecules that directly participate in cellular processes, especially in the regulation of gene functions that affects the expression of traits.
Epigenome: A collection of chemical compounds that regulate gene function and expression. They include:
(i) chemicals that modify the base units of DNA (ii) proteins (histones) with which DNA is associated
(iii) small RNAs: small non-coding RNA molecules, including messenger RNA (mRNA) and microRNA (miRNA). mRNA conveys genetic information from the DNA to protein-making structures. miRNA regulates gene expression.
Dr Yves Bertheau is an expert in GMO traceability. A list of his relevant publications is here. He coordinated Co-Extra, the largest EU-funded project on GMO and non-GM) supply chain coexistence and traceability. The results were collated in the report, “Genetically modified and non-genetically modified food supply chains: Co-existence and traceability”.
See also our GMWatch article: Old lobby, new language: The row over whether genome-editing techniques should be regulated as genetic modification has given rise to a new terminology of "SDNs". We explain what it means and why we need to know.