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mercredi, 31 août 2016

MOLECULAR SCISSORS

MOLECULAR SCISSORS:

A REVOLUTIONARY TECHNIQUE TO IMPROVE VINE RESISTANCE


Claude Gilois

 

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CRISPR-Cas9 [1], it is under this obscure and slightly barbaric name that this modern technology is known. It first appeared in a publication in the American magazine ‘Science’ in 2012. The concept of CRISPR sequences is not new and originates from the discovery of the ability of bacteria to incorporate a fragment of the DNA virus [2] in its own DNA sequence to defend itself. If the virus with the same DNA sequence is subsequently encountered, the bacteria then produces a protein, Cas9 and a small RNA [3] guides the protein towards the genome of the virus and cuts it into pieces, hence destroying the virus.

 

Since the human race adopted a sedentary life some 10,000 years ago, it has never stopped crossing varieties (animals and plants), to try to produce new ones with better yields, more resistance and improved taste. This process is long as it contains a large proportion of trial and error, as, until recently, man did not understand the genetics behind such crossing. Since 1980, there exist biotechnological techniques to manipulate the genome and to insert or remove elements of it, but these techniques are complicated, expensive and with a relatively low success rate. However, since 2000, a series of new enzymes have appeared which act like scissors to cut, replace, remove or insert a gene at a very precise location. The discovery of a fourth generation of these enzymes called CRISPR-Cas9[4] has constituted a true innovation. The only thing that is required is the RNA guide to identify where the modification has to be made on the genome and it is very easy to produce these RNA guides using robots. When the RNA guide finds its target on the genome, Cas9 cuts the DNA. The genome can then modify as required by removing, inserting or modifying a gene (mutation or correction of a mutation). Cas9 can also be used within the epigenetic’s [5] environment of the gene and, if coupled with the correct enzyme, it can amplify or tone down the activity of the gene. What is revolutionary is that this technique works with all living organisms (plants, mammals, yeasts and amphibians). It only costs about 10 Euros and requires only two weeks preparation. Before the arrival of this technology, it took 18 months to modify a gene of an organism and it could cost between 1,000 and 50,000 Euros and the rate of success was fairly low. With CRISPR-Cas9 it is much easier as no nucleic acid [6] is introduced, only the protein which disappears at a later stage leaving no trace of the modification.

 

IS THERE A DIFFERENCE BETWEEN CRISPR-Cas9 AND GMOs (GENETICALLY MODIFIED ORGANISM)?

 

The European Union has not yet stated its position on this technology and its report is expected sometimes in 2016. Most member states believe that it will classify the new enzymes as GMOs. However, the two technologies differ on a fundamental point. The GMO technology modifies the genome in a very coarse fashion with materials that come mostly from species unrelated to the ones scientists are trying to modify, whereas CRISPR-Cas9 is much softer as it utilises material mostly from the same species even though the technique allows material from different species to be used. To produce its transgenic corn, MON 810, Monsanto used a ‘gene gun’ to introduce the transgene into the genome of the plant. Once the plant had been bombarded, the results were then tested by cultivating it and by observing if the modifications made on the genes had the desired effects. If not, the process of bombarding was repeated until the desired effects were obtained. The CRISPR-Cas9 technology allows modification of the genes identified in a very precise way and its specificity [7] is, today, of 99% and it is in constant progress. With CRISPR-Cas9 the undesired effects [8] are very limited as, previously the DNA was cut twice. It is now only cut once with the new generation of Cas9 and the use of two RNAs to target the correct part of the genome.

If the introduction of GMO technology has largely failed in Europe, it is because the sacrosanct principle of ‘substantial equivalence’ was very difficult to accept conceptually. Under this principle, a genetically modified substance is considered equivalent to the original, and neither external scientific validation nor legislative debate is required. Either the EU classifies the new technology, CRISPR-Cas9 as a GMO and once again it will escape any scientific assessment processes and legislative validation, but its acceptability is likely to cause the same problem that it caused with the MON 810 introduction in Europe as it will be viewed, at best, as another attempt to create another Frankenstein or Frankenfood   or, at worse, to try to patent living organisms, or both.

 

If the EU classifies the new technology as chemical substances, they will fall under the ‘REACh’[9] program of the EU, which is attempting to control the diffusion of all chemical substances more tightly within the Union. The term substantial equivalent is far more suited to CRISP-CAS9 than to GMOs especially when the genetic modifications are made with material of the same organism and it mimics what is really happening in nature, as modification of living organisms are very often made by mutation. In this case, attempting to patent such modification appears difficult. However, if a gene that codes for a particular enzyme is modified to produce something other than it was intended for in the first place, then patenting appears to be justified. However, most improvements on the genome will be carried out with genes of the same species. Furthermore, CRISPR-Cas9 is the fruit of fundamental research and not from applied science as was MON 810. This should reduce the attempt from biotechnological businesses to try to patent the living.

 

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WHAT ARE THE FIELDS OF APPLICATION OF THIS TECHNOLOGY

 

They are vast and the most important will be in the medical field if the specificity of the technology can reach 100% (scientists are confident on this point) in particular to correct the genome of incurable diseases that have occurred over time usually by a single mutation in the human genome [10].  However, this topic is beyond the scope of this paper. In agriculture, a 99% chance of success is more than enough and the technology is already being used. The 1% failure can be easily identified and eliminated in agriculture. The French INRA [11] has been conducting research over the last 35 years on a wild variety of Vitis vinifera, called Mucadine, which is resistant to odium and mildew. After 20 years of experimentation and hybridisation, the resistant gene has been successfully transferred into a grape variety. With CRISP-Cas9 technology it can be expected that a giant leap forward will take place in producing disease-free plants and that the resistance will not limit itself to mildew, odium or botrytis and it will encompass viral diseases such as leaf roll virus (so pre-eminent in South Africa), Pierce disease (in California) and the diseases of the trunk (Black Dead Arm, Esca disease and Eutypiosis). It is anticipated that the grafting of vines on American rootstocks to produce phylloxera resistance to the aphid could even be eliminated with this technology. Hybridisation and promising evanescent techniques such as ‘Genomic Selection by Makers Assisted’ [12], which I described in this blog a few months ago could become rapidly obsolete. This technology should allow a marked reduction of chemical input in vineyards and in organic farming to reduce or eliminate the treatment with copper sulphate, as it is known to have an effect on amphibians. Furthermore, this technique is also likely to become an important weapon in the context of climatic changes as varieties more resistant to heat and drought can be developed.

 

But, a word of caution is necessary in order not to repeat the mistakes made during the introduction of the GMOs in Europe as another powerful civil reaction could develop, as was the case a few years ago.

 

[1] ‘Clustered Regularly Interspaced Short Palindromic Repeats’

[2] Often referred as phage.

[3] Its primary function is to convert the information stored in DNA into proteins.

[4] It is estimated that 3000 papers will be published on this subject in 2016.

[5] Epigenetic: the different ways that a gene can manifest itself without the sequence of the DNA being changed. A gene can have a different ‘strength’ of expression from one plant or from one individual to another.

[6] DNA, RNA or large bio-molecules.

[7] 99% chance that the cut will be made in the correct position

[8]  They are also called ‘off target ‘or unintentional effects

[9] Control system of the European Union for chemical substances which covers fields such as genetic toxicity, toxicity by repeated administration, toxicity on the foetus, toxicity on reproduction, carcinogenic effects, long- term aquatic toxicity, biodegradation and bioaccumulation.

 

[10] Diseases such Sickle Cell anaemia and Thalassemia, Haemophilia, Cystic Fibrosis and Down’s Syndrome could be eliminated. The full list can be consulted on https://en.wikipedia.org/wiki/List_of_genetic_disorders

[11] French ‘Institut National pour la Recherche Agronomique’

[12] Marker assisted selection or marker aided selection (MAS) is an indirect selection process where a trait of interest is selected based on a marker (morphological, biochemical, DNA/RNA variation) linked to a trait of interest (e.g. productivity, disease resistance, abiotic stress tolerance and quality), rather than on the trait itself, This process is used in plant and animal breading. Source Wikipedia.

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