NBT (New breeding techniques), new GMOs in disguise. The propaganda of 'safe' biotechnology masks the grave uncertainties widely demonstrated in the scientific literature.
Genetic engineering - anything but similar to natural evolution - therefore deserves further study. It is enough to understand the functioning of DNA and cells to understand the risks also linked to NBTs.
New GMOs, the birth of new techniques editing
In 2012, two researchers (Emmanuelle Charpentier and Jennifer A. Doudna, awarded the 2020 Nobel Prize in Chemistry for their discovery) found that the system called CRISPR-Cas9, with which bacteria defend themselves from invasions by their viruses by cutting their DNA into pieces, could constitute a universal system of editing (modification) of DNA.
These techniques, this gene (o genome) editing and often abbreviated to NBT, New Breeding Techniques, they use a system consisting of two main components: (A) a guide RNA (gRNA) derived from the bacterial sequence called CRISPR, which directs (B) a protein called Cas9 towards a specific DNA sequence. Here, Cas9 cuts both strands of DNA.
These double breaks produced in DNA by Cas9 are then repaired, with variable precision, by the mechanisms inherent in the cell subjected toediting.
An easy and cheap way
One of the advantages recognized techniques of editing it is the fact that modification can occur within species. Genetic elements of foreign origin, i.e. coming from a different species, are not inserted (at least in theory) into the DNA of the organism to be modified. For this reason theediting it is referred to as a cisgenesis and not a transgenesis technique.
Before 2012Other molecular tools (eg the Zinc Finger [ZFN] and TALEN nucleases) were used to modify the base sequences in DNA, which involved lengthy and complicated procedures. Thanks to simplicity, speed and low costs, the CRISPR-Cas9 system has become a tool of editing within the reach of almost all laboratories and for this reason it had a very rapid spread all over the world, with a dizzying increase in experiments of editing on plants and animals.
The (misplaced) certainties of SIGA
The SIGA, Italian Society of Agricultural Genetics, is one of the main proponents of a biotechnological approach to solving problems in agriculture. And it 'blindly' supports the NBTs, demanding that their products be removed from the regulation of GMOs and placed on the market without any particular pre- and post-market control.
In the manifesto 'Genes first - Let's free the future of agriculture'(SIGA, 2017), we read:
'Precisely because only one character changes, perhaps the one that can make the plant resistant to a parasite or climate change, or more nutritious, genome editing can help us to preserve a typical variety exactly as it is and how we like it today.'.
The legend of punctual precision
'Genetic modifications obtained with genome editing are absolutely punctual, and unlike all the techniques used in the last century - traditional and GMO - they are not accompanied by any other change in the genome of the plant, therefore by any other type of effect', continues the SIGA.
'The improvement [genetic] it becomes in fact of absolute precision, because it manages to change only the stretch of DNA to be improved and no other (therefore better than any traditional technique such as crossing and mutagenesis) and without introducing foreign DNA (as in GMOs or in hybridizations between different species). The absence of other changes in the rest of the genome is the best guarantee of the absence of undesirable effects, but also of the protection of the typicality of the product '.
'In practice, the CRISPR-cas9 system is an extremely precise molecular “scalpel”… with genome editing only the desired mutation is produced, without obtaining many others, unwanted and randomly distributed throughout the genome.'
Nbt with the use of vectors
Blind trust of SIGA, among others, requires an in-depth study on the alleged accuracy of these new techniques. Let's start by investigating the technique used in an absolutely predominant way until not so long ago, that is theediting vector mediated.
In this technique, in order to enter the cells and act, the two components of the CRISPR-Cas9 system must be 'mounted' on vectors. Typically these are circular DNA molecules derived from bacteria (called plasmids) or engineered viruses.
Unexpected effects
Numerous scientific studies have highlighted unwanted effects of this technology, which can be divided into:
- Effects "off-target", that is, off target, as they occur in points of the genome other than the one to be modified, often even distant. The problem with these unwanted mutations is that they can potentially lead to the production of toxins, anti-nutrients and allergens - compounds that are harmful to health and the environment.
- Effects "on target" (on the target) unwanted, because the technique sometimes produces unexpected effects even at the level of the target sequence on which one intervenes with theediting.
- Creation of GMOs in the classical sense, i.e. of transgenic organisms, following the incorporation, in the DNA of the edited organism, of the entire vector (or its fragments) that transported the components of theediting inside the cells.
As such carriers they are of bacterial or viral origin, their integration into the DNA of the edited organism makes it a GMO according to the standard definition.
Let's see some examples of these three different cases.
Effects off-target, a problem with no solution
Many scientific works highlight the occurrence of mutations off-target in edited DNA, most often in animal cell experiments (Skryabin et al ,. 2020), but also in plants (Zhang et al., 2018; Hahn and Nekrasov, 2019).
It is of a very common problem, which can be mitigated but cannot be eliminated. It often goes unnoticed, also due to the limitations of standard identification methods. Yet genetic mutations should be identified, studied and evaluated, considering the potential consequences on the environment and on people.
The German study
A study of the German Federal Institute of Biosafety of Plant Biotechnology (Modrzejewski, Hartung et al., 2019) feeds fears. The researchers probed the majors database Scientific online to locate all articles onediting of plants published between January 1996 and May 2018.
Of the 1.032 studies conducted with CRISPR-Cas (77,7% of editing studies), only 22% contain an in-depth analysis of possible off-target, unwanted mutations in the edited DNA. It is obvious to conclude that, if you do not look for them, then you will certainly find no trace of these mutations.
Effects on target unwanted
The experiments di editing with CRISPR-Cas9 on human and animal cells they also produced unexpected genetic mutations with obscure consequences at the target site.
'Genomic damage caused by CRISPR-Cas9 editing observed in mitotic cells [the most common type of cell division, in which two daughter cells genetically identical to the parent cell and to each other derive from a single cell] it can have pathogenic consequences', conclude the authors of the study published in Nature Biotechnology (Kosicki et al., 2018).
In another study published Nature Methods (Smits et al., 2019), the researchers tested in human cells whether the target gene ofediting with CRISPR-Cas9 it is really knocked out and ceases to produce its usual protein. The result is that in about one third of the target sites the protein remains at normal levels, while sometimes the synthesis of abnormal proteins emerges, the short- and long-term effects of which remain unknown.
The results of the two studies cited are also relevant for plants. Especially since 87% of the studies of editing of plants has as its purpose the knock out of a particular gene. But the verification of the possible residual functionality of the protein has NEVER been done.
Unintended insertions of foreign DNA
An exemplary case of these 'collateral damage' ofediting is that of calves born without horns, after their progenitor was edited with TALEN in 2013. A result touted too hastily as a living demonstration of the prodigies ofediting.
In 2019, the FDA (Food and Drug Administration) discovered (Norris et al., 2020) in the DNA of these calves the presence of the DNA of the entire vector (a bacterial plasmid) used for theediting native to. Since the plasmid is made up of DNA of various bacterial species, including genes for resistance to antibiotics (of which it would be good to limit the possible spread), these calves clearly fall within the classic definition of GMOs. The FDA therefore decided in February 2020 that edited animals, and their products, must be subjected to in-depth pre-market analysis and subject to the same regulation as new drugs.
The case of rice
In an article published by Nature (Banakar et al., 2019), the results of an experiment are reported editing of a rice gene, applying the CRISPR-Cas9 system with three different methods. All three methods involve the construction of numerous, complicated vectors, starting from bacterial plasmids also containing bacterial genes for resistance to antibiotics or herbicides.
The authors found unexpected insertions of bacterial vector DNA and chromosomal DNA from fragmented and rearranged rice at the Cas9 target sites. A sensational event, but as a rule not sought or communicated. "The specialized literature often does not report the presence of these unwanted inserts, or does not provide detailed data”, Warn the authors of the study.
NBT DNA-free, that is, without the use of vectors
Aware of the fact that NBTs involve a high risk of integration of the vector into the DNA of the edited organism (with a consequent high risk of falling under the European regulations on GMOs), many researchers working on plants are increasingly turning to a method of editing genetic said DNA-free, as it does not involve the use of vectors.
The components of the CRISPR-Cas9 system are synthesized and pre-assembled in vitro and are then conveyed ready-made, in the form of ribonucleoproteins (RNP), into plant cell protoplasts, by means of nanoparticles or by infusion in a polyethylene glycol solution ( PEG).
Different technique, same risks
In an extensive review, which aims to examine methods for risk assessment in the light of new techniques editing (Agapito-Tenfen et al., 2018), the authors find that the non-use of vectors, while eliminating the problem of integrating foreign DNA, does not eliminate the problem of the formation of unwanted mutations. on- And off-target. Therefore not even this method allows to exclude the unexpected production of toxins, anti-nutrients and allergens, which pose a risk to the environment and human or animal health.
In an experiment conducted on human pluripotent stem cells (Ihry et al., 2018), applying the vector-free method, the researchers found that the Cas9-induced double-stranded breaks were toxic, and resulted in the death of most of the cells.
Such an answer has been related to P53, a protein controlled by a tumor suppressor gene and called "the guardian of the genome", as it protects DNA from mutations. Although it was obtained on human cells, this result is also very significant for other species, including plants.
Conclusions
From this brief review of the scientific data available today regarding the accuracy and potential risks associated with new DNA modification technologies (NBT), some conclusions are clear:
- there is still a substantial ignorance of the mechanisms of action of the components of the CRISPR-Cas9 system, in particular of the cutting action of the Cas9 protein. Therefore also the so-called methods DNA-free they do not protect against the possibility of DNA rearrangements and mutations with unpredictable effects;
- there is insufficient capacity to control unwanted effects;
- there are still many limitations in the methods for identifying the unwanted effects generated byediting;
- there is the possibility that unexpected and unknown proteins are formed, with potential toxic or allergenic effects.
The precautionary principle reaffirmed by the European Court
The technique it is still in an experimental phase and it is more than justified, from a scientific point of view, to adhere to the precautionary principle with regard to its possible practical or even commercial applications. It is necessary that the products of these techniques are subjected to careful investigations and specific evaluations before, during and after the genetic modification.
Thus after all, the European Court of Justice decided in 2018, which ruled that even the bodies obtained with the new techniques of editing genomic should be considered genetically modified (GMO). As such, the new organisms are regulated by the European directive 2001/18 / EC which establishes to authorize a GMO only after a thorough assessment of the risks for the environment and human health, and makes their traceability, labeling and tracking.
This article is an extract from the document "Building the future: Taking care of agricultural and natural biodiversity", An integral part of the project"Biodiversity and peasant seeds”, Presented to the Emilia-Romagna Region by the Food Sovereignty Network and by CRESER (Regional Coordination for Solidarity Economy in Emilia-Romagna).
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