NBT, the dark side of new editing techniques. Scientific Review

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NBT(new breeding techniques), new GMOs in disguise. The propaganda of ‘safe’ biotechnology masks the serious uncertainties amply demonstrated in the scientific literature.

Therefore, genetic engineering-anything but similar to natural evolution-deserves further study. One only needs to understand how DNA and cells work to understand the risks associated with NBTs as well.

New GMOs, the emergence of new editing techniques

In 2012, two researchers (Emmanuelle Charpentier and Jennifer A. Doudna, recipients of the 2020 Nobel Prize in Chemistry for their discovery) found that the system called CRISPR-Cas9, by which bacteria defend themselves against invasions by their viruses by cutting their DNA into pieces, could constitute a universal editing (modification) of DNA.

These techniques, called gene (or genome) editing and often abbreviated to NBT, New Breeding Techniques, make use of 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 toward a specific DNA sequence. Here it junta, Cas9 cuts both strands of DNA.

These double breaks produced in the DNA by Cas9 are then repaired, with variable precision, by the mechanisms inherent in the cell undergoingediting.

An easy and inexpensive way

One of the recognized advantages of editing techniques is the fact that editing can take place within species. No genetic elements of foreign origin, that is, from a different species, are inserted (at least in theory) into the DNA of the organism to be modified. For this reason,editing is referred to as a cisgenesis technique and not transgenesis.

Prior to 2012, other molecular tools (e.g., Zinc Finger nuclease [ZFN] and TALEN) were used to modify base sequences in DNAs, which involved time-consuming and complicated procedures. Because of its simplicity, speed, and low cost, the CRISPR-Cas9 system has become an editing tool within the reach of almost any laboratory, and because of this, it has had a very rapid spread around the world, with a dramatic increase in editing experiments on plants and animals.

SIGA’s (misplaced) certainties.

SIGA, the Italian Society of Agricultural Genetics, is among the leading proponents of a biotechnological approach to solving problems in agriculture. And it ‘blindly’ supports NBTs, demanding that their products be removed from GMO regulation and placed on the market without any special pre- and post-market controls.

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 pest or climate change, or more nutritious, genome editing can help us preserve a typical variety exactly as it is and how we like it today.’

The legend of pinpoint accuracy

Genetic modifications achieved by genome editing are ‘absolutely spot on, and unlike all the techniques used in the last century-traditional and GMO-are not accompanied by any other changes in the plant’s genome, thus no other kind of effect,’ the SIGA continues.

‘The improvement [genetico] indeed becomes of absolute precision, because it succeeds in changing only the stretch of DNA to be improved and no other (thus better than any traditional technique such as crossing and mutagenesis) and without introducing foreign DNA (as in GMOs or interspecies hybridization). 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 product’s typicality’.

‘In practice, the CRISPR-cas9 system is an extremely precise molecular “scalpel” … with genome editing, only the desired mutation is produced, without also obtaining many other, unwanted, randomly distributed mutations throughout the entire genome.’

Nbt using carriers

SIGA’s blind trust, among others, calls for further investigation into the alleged accuracy of these new techniques. Let us begin by investigating the absolutely predominant technique used until not so long ago, namely vector-mediatedediting.

In this technique, in order to enter cells and act, the two components of the CRISPR-Cas9 system must be ‘mounted’ on vectors. These are usually circular DNA molecules derived from bacteria (called plasmids) or engineered viruses.

Unexpected effects

Numerous studies have shown unintended effects of this technology, which can be distinguished into:

Off-target” effects, i.e., off-target, in that they occur at locations in the genome other than the one intended to be modified, often even distant. The problem with these unintended mutations is that they can potentially lead to the production of toxins, anti-nutrients, and allergens-that is, compounds that are harmful to health and the environment.

– Unintended on-target ” effects (on the target), because the technique sometimes produces unintended effects even at the level of the target sequence beingedited.

Creation of GMOs in the classical sense, i.e., transgenic organisms, following the incorporation, into the DNA of the edited organism, of the entire vector (or fragments thereof) that carried theediting components into the cells.

Since such vectors 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 look at some examples of these three different cases.

Off-target effects, a problem without a solution

Many scientific papers highlight the occurrence of off-target mutations in edited DNAs, most often in animal cell experiments (Skryabin et al,. 2020), but also in plants (Zhang et al., 2018; Hahn and Nekrasov, 2019).

This is a very common problem that can be mitigated but not eliminated. It often goes unnoticed, partly because of the limitations of standard detection methods. Yet, genetic mutations should be detected, studied and evaluated, given the potential consequences on the environment and people.

The German study

A study by the German Federal Institute of Plant Biosafety Biotechnology (Modrzejewski, Hartung et al., 2019) fuels fears. Researchers surveyed major online scientific databases to locate all articles on plantediting published between January 1996 and May 2018.

Of the 1,032 studies conducted with CRISPR-Cas (77.7% of all editing studies), only 22% contained in-depth analysis of possible off-target, unintended mutations in the edited DNA. It is obvious to conclude that, if you do not look for them, certainly these mutations then cannot be found.

Unintended on-target effects

Editing experiments with CRISPR-Cas9 on human and animal cells have also produced unexpected genetic mutations at the target site with obscure consequences.

‘Genomic damage caused by editing with CRISPR-Cas9 observed in cells in mitosis’ [the most common type of cell division, in which two daughter cells genetically identical to the mother cell and to each other arise from a single cell] can have pathogenic consequences, conclude the authors of the study published in Nature Biotechnology (Kosicki et al., 2018).

In another study published in Nature Methods (Smits et al., 2019), researchers tested on human cells whether the gene targeted forediting with CRISPR-Cas9 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 abnormal protein synthesis emerges, the short- and long-term effects of which remain unknown.

The results of the two studies mentioned above are also relevant to plants. Especially since 87% of plant editing studies aim to knock out a particular gene. But verification of any residual functionality of the protein has NEVER been done.

Unintended insertions of foreign DNA

An exemplary case of this ‘collateral damage’ ofediting is that of calves born without horns, after one of their progenitors was edited with TALEN in 2013. An achievement touted too hastily as the living demonstration of the wonders ofediting.

In 2019, theFood and Drug Administration (FDA) discovered (Norris et al., 2020) in the DNA of these calves the presence of the whole vector DNA (a bacterial plasmid) used for the originalediting. Since the plasmid is composed of DNA from various bacterial species, including also genes for resistance to antibiotics (the possible spread of which it would be good to limit), these calves clearly fall under the classic definition of GMO. The FDA therefore decided in February 2020 that edited animals, and their products, must undergo extensive premarket testing and be subject to the same regulation as new drugs.

The case of rice

In an article published in Nature (Banakar et al., 2019), we report the results of an experiment to edit a rice gene by applying the CRISPR-Cas9 system with three different methods. All three methods involve the construction of numerous, complicated vectors from bacterial plasmids that also contain bacterial antibiotic or herbicide resistance genes.

The authors found unexpected insertions of bacterial vector DNA and fragmented and rearranged rice chromosomal DNA in the target sites of Cas9. A sensational event, but as a rule not researched or reported. “Specialized literature often does not report the presence of these unintended inserts, or does not provide detailed data“, warn the study authors.

NBT DNA-free, i.e., without the use of vectors

Aware of the fact that NBTs carry a high risk of vector integration into the DNA of the edited organism (resulting in a high risk of falling under European GMO regulations), 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 carriers.

The components of the CRISPR-Cas9 system are synthesized and pre-assembled in vitro and are then delivered ready-made, in the form of ribonucleoproteins (RNPs), inside plant cell protoplasts by means of nanoparticles or by infusion into a polyethylene glycol (PEG) solution.

Different technique, same risks

In an extensive review, which aims to examine methods for risk assessment in light of the new techniques of editing (Agapito-Tenfen et al., 2018), the authors find that while not using vectors eliminates the problem of foreign DNA integration, it does not eliminate the problem of unintended mutation formation on– e off-target. Thus, even this method cannot rule out 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 vectorless method, the researchers found that Cas9-induced double-strand breaks had proven to be toxic, and had caused most of the cells to die.

This response has been related to P53, a protein controlled by an oncosuppressor gene and called “the guardian of the genome” because it protects DNA from mutations. Although this result was obtained on human cells, it 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 related to new DNA editing technologies (NBTs), some conclusions become apparent:

– there is still substantial ignorance of the mechanisms of action of the components of the CRISPR-Cas9 system, particularly the cutting action of the Cas9 protein. Therefore, even so-called DNA-free methods do not repair from the possibility of DNA rearrangements and mutations with unpredictable effects;

– there is insufficient ability to control unintended effects;

– there are still many limitations in the methods for detecting unintended effects generated byediting;

– there is the possibility of the formation of unanticipated and unknown proteins with potential toxic or allergenic effects.

The precautionary principle reaffirmed by the European Court.

The technique is still at an experimental stage, 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 undergo careful investigation and specific evaluation before, during and after genetic modification.

So after all, the European Court of Justice ruled in 2018 that organisms obtained through new genomic editing techniques should also be considered genetically modified (GMOs). As such, the new organisms are regulated by European Directive 2001/18/EC, which stipulates that a GMO should be authorized only after a thorough assessment of the risks to the environment and human health, and makes traceability, labeling and monitoring mandatory.


This article
is an excerpt from the document “Building the Future: Caring for Agricultural and Natural Biodiversity,” part of the project “Biodiversity and Peasant Seeds,” submitted to the Emilia-Romagna Region by the Food Sovereignty Network and CRESER (Regional Coordination for the Solidarity Economy in Emilia-Romagna).