NBT(NewBreeding Techniques), or ‘new GMOs,’ it is imperative to strengthen risk analysis. The scientific review published in Environmental Science Europe suggests further exploration of a debate that has been going on under wraps in the Old Continent for at least 4 years.
The debate revolves around whether to progressively liberalize the use of new plant varieties derived from genetic engineering. As is already the case in the U.S., where tens of thousands of them were authorized in just a few months.
Politics is maneuvered by the Big 4, from one side of the Atlantic to the other. But associations representing agroecology and organic production are trying to resist. In the deafening silence of consumer associations(teleguided elsewhere, by the strong powers) and the mainstream media, as is customary. ABC to follow.
Plant breeding and genetic engineering
The concept of plant breeding encompasses a wide range of techniques aimed at obtaining plants with certain genotypic and phenotypic characteristics. Mutagenesis-that is, the mutation of a genome induced by physical (e.g., UV light) or chemical (e.g., ethyl methanesulfonate) agents-dates back to the 1930s.
The discovery of the structure of DNA and RNA and subsequent innovations in genetics and molecular biology enabled the birth of genetic engineering in the 1970s. Genetic engineering techniques involve the introduction of different types of modifications into the genome of an organism in order to obtain specific characteristics. Mediating:
– transgenesis, that is, insertion into the host genome of genetic material from organisms of different species, or
– cisgenesis. The insertion of genes from the same species, knocking-out (removal or inactivation) or silencing and/or overexpression of specific genes.
Figure 1. Comparison of four breeding techniques. Huang S, Weigel D, Beachy RN, Li J. A proposed regulatory framework for genome-edited crops. Nat Genet. 2016;48(2):109-111. doi:10.1038/ng.3484
NBT, New Breeding Techniques
The acronym NBT(New Breeding Techniques) expresses a set of new genetic engineering techniques developed in recent years. They differ from first-generation genomic editing techniques (transgenesis) in that they predominantly use genes that belong to the same target species. Without, however, excluding, in rare cases, the use of genetic material from different species than the one being modified.
NBTs are currently classified into seven categories, further divided into two groups based on whether or not they exploit gene editing techniques. In the second group are agroinfiltration, various epigenetic approaches, site-directed mutagenesis (or oligonucleotide-directed mutagenesis) and RNA interference (RNAi).
Gene editing
Gene editing is a type of genetic engineering that uses nucleases to make targeted changes at precise locations in an organism’s genome. Insertion or removal of specific genes, or other types of modifications. With possible uses in agriculture and floriculture(plant breeding), but also in the medical field. With a view to developing innovative therapies for the treatment of various genetic diseases and cancers.
The main gene editing techniques are ZFN(Zinc Finger) nucleases, TALEN(Transcription Activator-like Effector Nucleases) nucleases and the CRISPR-Cas9 system.
CRISPR-Cas9
The CRISPR-Cas9 system is now the most widely used genomic editing technique because of its relative ease of use and versatility. CRISPR abbreviates the concept of Clustered Regularly Interspaced Short Palindromic Repeats, while Cas9 is the endonuclease used to perform DNA cutting.
This system is derived from a particular defense mechanism discovered in bacteria. Which, although they do not have a true immune system, have developed mechanisms to protect against infection by bacteriophage viruses and plasmids of other bacteria.
The peculiarity of CRISPR-Cas9 is the use of a guide RNA (gRNA) that is synthesized as needed. That is, based on the sequence of the genome you intend to target. This allows cuts to be made, within the genome, with a much higher level of precision than systems that exploit other types of nuclease.
Figure 2. Mechanism of operation of CRISPR-Cas9. Razzaq A, Masood A (2018) CRISPR/Cas9 System: A Breakthrough in Genome Editing. Mol Biol 7: 210. doi: 10.4172/2168-9547.1000210
CRISPR-Cas9 technique and prerogatives
The ‘custom’ synthesized gRNA binds to the Cas9 nuclease and is used as a probe, to identify the exact DNA sequence where to make the cut. In fact, the Cas9 endonuclease binds and runs on the target organism’s DNA until it finds a PAM(Protospacer AdjacentMotif) sequence adjacent to the target sequence. At this point, if the gRNA binds in a complementary manner to the target sequence, the Cas9-gRNA complex undergoes a conformational rearrangement that activates the nuclease domain of the enzyme and allows it to make the cut in the DNA.
Double-strand breaks (DSBs) thus introduced into the DNA are readily repaired by the cell itself. Through DNA damage repair mechanisms, which include Non-Homologous End Joining (NHEJ) and homologous recombination (HDR) systems. (1) In this way, the cell uses the DNA provided to it to ‘repair the damage.’ The CRISPR-Cas9 system can then be used to make site-specific sequence alterations, that is, to make deletions or insertions of genetic material in specific stretches of target DNA. Multiple gRNAs can also be used to introduce changes at different sites in the target genome simultaneously or successively.
The changes introduced by this technique are usually stable and therefore heritable, meaning that they are also passed on to the offspring of the target organism. This technique also makes it possible to overcome the natural genome maintenance systems that protect its certain regions from the occurrence of random mutations.
The prerogative of obtaining specific modifications on any part of the genome is impossible to achieve as quickly and effectively through conventional plant breeding techniques. Which instead rely on the induction of random mutations within the genome. Thus, for example, a wheat variety with low gluten content was obtained, resulting in an 85% decrease in immunoreactivity in people with celiac disease.
Old and new GMOs, rules set in European Union
The European Court of Justice (ECJ ) was called upon to decide on the discipline to be applied to NBTs, as noted above, in Case C528/16 brought by Confederation Paysanne and other organizations against the French government.
The Luxembourg judges, for once, disregarded the Advocate General’s conclusions. Affirming – in ruling 25.7.18, in Case C-528/16 – that NBTs must be subject to the discipline already established for ‘conventional’ GMOs. Although formally excluded from the scope of Directive 2001/18/EC on which it is based.
The deliberate release of old and new GMOs into the environment is therefore subject to authorization by the European Commission, in agreement with the representations of the member states. Following appropriate analysis of possible risk to the environment and biodiversity by EFSA.
The use of GMOs and their derivatives in food or feed production is subject to additional authorization. Which may be granted, again at the European level, following an appropriate risk analysis by EFSA. Given the potential impact of consumption of substances derived from or containing GMO and/or its derivatives on human and animal health.
All GMOs released into the environment and on the market in the European Union are in any case subject to precise traceability, food and feed labeling, and monitoring requirements.
NBT, the hypotheses for reforming EU rules.
Break down barriers to the release of NBTs into the environment, liberalize trade in them and any foodstuffs containing them. The Big 4, the global pesticide and seed monopolists, gave the orders to the European Parliament as early as the beginning of the legislature, as it turned out.
European Commissioner for Health and Food Safety, Stella Kyriakides, thus announced the launch of a study on new genomic techniques in spring 2021. With the ill-concealed goal of getting a rationale to the proposed reform of the GMO framework on which, in Brussels, the usual knowns are already working.
In defiance of the ‘transparency’ invoked in the media and social networks, the Commission has long been working-with stakeholders representing only production chains, ça va sans dir-on a document that European citizens and other interested social partners are excluded from knowing. And it is no coincidence that genetic engineering has also been included in the EU Farm to Fork (f2f) strategy, as well.
Precautionary principle and risk analysis
Structural reform of the ecosystem that is foreshadowed in the above technocratic design, however, cannot disregard risk analysis and the precautionary principle on which European policies on the environment, food and feed safety, public health and animal welfare are still based (TFEU, Article 191.1).
The European Network of Scientists for Social and Environmental Responsibility (ENSSER) has repeatedly expressed the need for NBTs to undergo thorough risk assessments. And so do various biologists and molecular geneticists, such as Dr. Michael Antoniou at King’s College London.
Genetic manipulation can in fact cause new combinations of gene functions that are not always as precise as they are presented, nor predictable in subsequent interactions with complex systems. They can produce many unintended effects, not only at off-target sites but also at intended sites of genetic modification.
Risk analysis on NBT, scientific review
The scientific review published in Environmental Science Europe by Andreas Bauer-Panskus et al. (2020) points out that the protocols adopted by EFSA for risk analysis related to NBT need to be revised. As inadequate to adequately consider all the consequences that may result from the introduction and persistence of these organisms in the environment. (2)
Researchers at the Institute for Independent Impact Assessment of Biotechnology in Munich therefore insist on the need to consider the next-generation effects that may arise from the transmission of new genetic material, including through interaction with environmental conditions.
‘Consequently, the biological characteristics of the original events cannot be considered sufficient to draw conclusions about the dangers that may emerge in subsequent generations. Potential hazards identified by the European Food Safety Authorization (EFSA) include aggravation of pest problems, displacement and even extinction of native plant species.
However, there are reasons for concern that might escape environmental risk assessment (ERA). Because EFSA takes into account only the characteristics of the original events, leaving aside unintended or unanticipated next-generation effects that emerge from spontaneous propagation and gene flow.’
Risk analysis and the precautionary principle
Scientific review of available publications and analysis of risk assessments performed lead researchers to conclude that risk assessment of spontaneous persistence and propagation in the environment of NBTs is subject to significant spatiotemporal complexity that causes many uncertainties. To address this problem, ‘cut-off criteria’ should therefore be established in risk assessment that consider the practical limitations of current knowledge.
The proposed exclusion criteria should be applied at a further and specific stage of risk assessment, namely ‘spatio-temporal controllability’. Making use of well-defined biological features to delineate a boundary between known (and/or predictable) and unknown (and/or unpredictable) effects. This additional stage of risk assessment, according to the authors, will promote the robustness of the process and can substantially benefit the overall reliability and completeness of risk assessment. As well as on the decision-making process regarding deliberate releases of NBTs into the environment.
After all, the European regulation of GMOs is based on theprecautionary principle (Precautionary Principle). (3) This principle is characterized in the prevention of risks in the face of scientific uncertainty, with the goal of avoiding harm before a danger arises.
Environmental risks not to be underestimated
The Munich researchers focus in particular on the reproductive success(fitness) of plants obtained by gene editing, which in some cases is better than that of corresponding natural varieties. An appreciable achievement-where intentional, from the perspective of commercial development-which at the same time deserves attention with regard to ecosystem balances.
The increase in fitness may in fact exceed intentions and depend on the interaction of the newly introduced genetic material with the rest of the genome or on a combination of the interaction of the new genetic material and the environmental conditions in which the plant is growing.
Genetic material introduced by gene editing can also be passed on to wild plants belonging to the same species, with several possible consequences for hybrids. Such as increased fitness or other effects on the genome that could manifest in changes in plant physiology and metabolism. So finally on biodiversity, whose promised protection cannot be separated from these assessments.
Dario Dongo, Riccardo Clerici, Silvia Comunian
Notes
(1) The NHEJ system is prone error and often causes base listings or cancellations (indels) that alter the starting nucleotide sequence, and thus if the double helix break occurred within the coding sequence of a gene or the regulatory sequence of a gene this repair system can alter the functionality of that specific gene. The homologous repair system usually intervenes when the NHEJ fails to repair the double-strand break and takes advantage of the homologous regions of DNA to repair the damage. This system can also be exploited to introduce nucleotide substitutions or to introduce insertions of specific sequences or to replace specific sequences within the target DNA simply by providing the cell with exogenous DNA with regions homologous to those adjacent to the cut
(2) Bauer-Panskus, A., Miyazaki, J., Kawall, K. et al. Risk assessment of genetically engineered plants that can persist and propagate in the environment. Environ Sci Eur 32, 32 (2020). doi:10.1186/s12302-020-00301-0
(3) Dir. 2001/18/EC, Article 1. TFEU (Treaty on the Functioning of the European Union), Article 191.2
(4) Garnett K, Parsons DJ (2016). Multi-case review of the application of the precautionary principle in European Union law and case law: application of the precautionary principle. Risk Anal 37:502-516. https://doi.org/10.1111/risa.12633
Dario Dongo, lawyer and journalist, PhD in international food law, founder of WIISE (FARE - GIFT - Food Times) and Égalité.
High school diploma, studies Biotechnology at the University of Milan Bicocca
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