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Natural genetic engineering

New plant breeding technologies, and the CRISPR/Cas technique in particular, are making headlines. For the first time in the history of agriculture, these technologies enable the quick and, in particular, precise modification of DNA at a predetermined locus. However, these methods provide authorities with an unexpected headache: are genome-edited plants genetically modified organisms or not?

The CRISPR/Cas system consists of two components: a single guide RNA (sgRNA) with a user-defined 20-nucleotide spacer that is complementary to the genomic target to be modified, and the Cas9 protein, a non-specific CRISPR-associated endonuclease that cuts the two strands of DNA at a specific location in the genome so that DNA fragments can be added or removed. The specificity, i.e. the genomic target of Cas9, can be simply altered by changing the targeting sequence in the sgRNA. The interaction between Cas9 and the genomic DNA is also affected by a short PAM (protospacer adjacent motif) sequence. © KIT

Emmanuelle Charpentier and Jennifer Doudna are considered future Nobel Prize winners as they have shown that Cas9 can be used to make cuts in any DNA sequence whatsoever. In 2012, they published their groundbreaking results on CRISPR/Cas in the renowned journal Science, and it took only four years before the method had become commonplace in countless laboratories around the world. CRISPR/Cas can be used to modify the DNA of humans, animals and crops more easily than ever before – fast, inexpensively and with great accuracy. “The RNA fragment and other components can be purchased for about 20 euros and the process can be carried out in one day. This is a world first. Any laboratory can afford the technology when it is so cheap,” said Holger Puchta, director of the Botanical Institute at the Karlsruhe Institute of Technology (IKT).

CRISPR revolutionises both the field of medicine and the field of agriculture at such a pace that legislation is lagging behind. It is still unclear whether CRISPR-edited crops will be regulated in the EU or not. Are they genetically modified organisms (GMOs) that need to undergo special risk assessments, or are they variants of conventionally bred plants that do not require such testing?

The EU has not yet decided whether genome-edited organisms will be regulated and the decision as to whether these organisms will be considered as GMOs or non-GMOs has been postponed several times. The last decision was scheduled for March 2016 and is still being eagerly awaited. GMO opponents clearly regard the new plant breeding technologies as a form of genetic engineering. If the EU decides to classify gene-edited crops as non-GMO, they fear that genetically modified food that does not need to be labelled as such will be placed on the market. They therefore call for risk assessments of such crops and appropriate labelling so that farmers and consumers can continue to choose between GMO and non-GMO crops/foods.

It is all a question of classification

Many scientists do not regard plants whose genetic traits have been influenced with the new gene- or genome-editing methods as classical GMOs. “The traditional genetic modification of plants can be compared to a heart operation that requires the opening of the entire chest, while genome editing is more like a minimally invasive intervention,” said Detlef Weigel, director of the Max Planck Institute for Developmental Biology in Tübingen. Together with colleagues from China and the USA, Weigel has proposed a regulatory framework for gene-edited crops.

In fact, the old and the new methods for influencing genetic traits differ in essential respects: as far as classical genetic engineering methods are concerned, it is impossible to predict the genomic location where new genes are inserted. The inaccuracy of the method and the insertion of genes from other species are still the two main aspects for the opposition to the introduction of GMOs.

Genome-editing methods are very precise. The CRISPR/Cas9 system which bacteria naturally use to protect themselves against pathogens (viruses for example) is one of the most precise systems of genetic modification that exist. Biologists can use CRISPR/Cas9, which works equally well in plants, animals and humans, to target and modify DNA with outstanding accuracy. In general, all genome-editing methods consist of roughly the same three steps. They all begin with the determination of the locus where a break will then be made in the DNA. The third step involves the repair of the break. Numerous laboratories around the world have shown that the method works in rice, tobacco, tomatoes, corn and wheat. The mutations, i.e. modifications of the letter sequence in the DNA, are inserted stably and passed on to the progeny.

The objectives of the new genome-editing methods do not differ from those of traditional genetic engineering methods or traditional breeding – they all have the objective of adapting crops to the needs of people, making them more productive, more resistant to pathogens and less sensitive to drought. In view of the changing climate and the ever-growing population, such crops can make enormous contributions to increased global food production. But why is it so difficult to define the legal status of the new methods?

Repair decides about extent of modification

The crucial aspect is the way a break in the DNA is mended by the cells’ repair machinery. Biologists distinguish between three types of repairs. Type I leads to a point mutations where one nucleotide, i.e. one letter of the DNA sequence, is replaced by a different one. Type II repairs involve the modification of a rather small number of letters, and type III involves the insertion of a larger piece of foreign DNA at the site where the cut has been made.

Type III DNA repairs clearly fall under the Genetic Engineering Act as such an organism would not develop naturally. There is still disagreement in the classification of type I and type II breaks whose repair only involves the exchange of one or a few DNA constituents. “Type I and type II repairs do not lead to GMOs because these genetic modifications are point mutations that can also arise naturally as a result of crossing and/or natural recombination,” wrote the German Federal Office of Consumer Protection and Food Safety in a statement in November 2015.

Arabidopsis seeds: seeds of a wild-type plant (left) and seeds of a plant in which the transparent testa4 gene was switched off by inserting a mutation using the CRISPR/Cas sytem. Seeds carrying the mutation are unable to form the pigment that is typical for wild-type seeds. © KIT

In nature, mutations, i.e. minor genetic DNA modifications, occur all the time, they are in fact the driver of evolution. Even conventional breeding methods lead to changes in the DNA. Mutation breeding is one such method and involves exposing seeds to chemicals or radioactive radiation. “This treatment generates countless mutations, but they are not directed, i.e. nobody knows where they occur. Most of these mutations are deleterious. Plants developed via mutagenic processes are regarded as natural and can be placed on the market without undergoing safety testing,” said Dr. Puchta. He asked: “Why should plants whose genome is minimally modified at a predetermined locus have a less favourable legal status than mutation breeding crops? Why should genetically edited crops be regulated, but not those produced by mutation breeding?”

The reason for this is down to government regulations for genetically modified foods. EU rules consider the genetic engineering process used to make food, rather than the final product (as for example in the USA), which means that all GM foods are regulated because they are produced with processes that differ from those used for making conventional foods. Therefore, according to current EU regulations, genetic engineering is when heritable material that is prepared outside the organism is introduced into a cell – this is the finding of two legal opinions that were commissioned by NGOs such as Greenpeace. This definition would then automatically make all plants and animals that are modified using CRISPR/Cas or other genome editing methods GM crops/animals.

Proof is not possible

However, this interpretation has a flaw: “Genome-edited plants cannot be differentiated from their natural counterparts,” said Puchta, explaining that this is not due to the lack of methods that would be able to detect such mutations, but simply due to the fact that there is no difference to be detected. “How are we going to control the use of a technology if we are unable to prove that it has actually been used?” asked Puchta.

Canada or the US, for example, apply the same regulations to GM and conventional foods because the final products are considered to be similar despite the different processes that were used to make them. At the end of April 2016, the regulatory authorities in the USA decided not to regulate a corn variety genetically modified with the gene-editing tool CRISPR/Cas9. The decision means that when the corn is placed on the American market in about 5 years’ time, there is also a good chance that it will end up on European plates without the possibility to prove that it was modified using CRISPR/Cas.

Weigel and his colleagues have come up with a number of recommendations as guiding principles when considering the regulation of genome-edited crops; one of these recommendations is to analyse and document DNA sequence changes at the target sites, another one is the recommendation to demonstrate the absence of foreign sequences. Genome-edited crops would then have to be considered non-GMOs and given the same status as crops produced with traditional breeding methods. It remains to be seen what final decision the EU will make.

References

Sanwen Huang, Detlef Weigel, Roger N Beachy & Jiayang Li; A proposed regulatory framework for genome-edited crops. Nature Genetics. DOI 10.1038/ng.3484

Bortesi, L. and Fischer, R. (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol. Adv. 33, 41–52.

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