New genetic engineering techniques such as oligonucleotide-directed mutagenesis may bypass already flimsy GM regulations Dr Eva Sirinathsinghji
New developments in molecular genetics techniques could pose a serious threat to already inadequate regulations on genetically modified organisms (GMOs). One new technique - oligonucleotide-directed mutagenesis (ODM) - is one of many examples. It has been recently trademarked as the Rapid Trait Development System (RTDS) by a small agritech company Cibus LLC that has patented the technology and selling it as a natural and non-transgenic technology.
A partnership between Cibus and BASF has led to the development of sulfonylurea herbicide-tolerant GM canola/rapeseed, which has already been considered not genetically modified by a number of government advisory groups. This would mean bypassing GM labelling in the European Union (EU) as well as the GM bans that are currently in place in many EU member states. UK’s Advisory Committee on Releases to the Environment (ACRE) at Defra (Department for Environment, Food and Rural Affairs) published a report in 2011 stating:
“ACRE considers that herbicide tolerant (HT) oilseed rape plants produced by Cibus LLC have been developed using a form of mutagenesis. It considers that this technique does not involve the use of recombinant nucleic acid molecules. Consequently, the HT oilseed rape plants could be excluded from the GMO Deliberate Release legislation in accordance with Annex 1B of Directive 2001/18/EC” .
The Food Standards Australia New Zealand (FSANZ), as well as equivalent advisory bodies in the US are taking a similar stance , with the US Department of Agriculture (USDA) informing Cibus back in 2004 that crops made using ODM would not warrant review .
Cibus were awarded their first EU patents for the RTDS crop in 2010, which was recently upheld in 2012. They have filed additional patents on RTDS-generated glyphosate-tolerant crops (corn, wheat, rice, barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple, lettuce, peas, lentils, grape, turf grasses and Brassica sp.), and sulfonylurea herbicide-tolerant canola/rapeseed, with the latter currently being targeted at the EU [4-8]. The canola/rapeseed crops developed by Cibus and BASF are a further development of BASF’s Clearfield crops that were generated through conventional and hybrid breeding to be tolerant to BASF’s Cleranda herbicide, which are already grown in the UK. The new RTDS generated crops were aimed for commercialization in 2013, though they do not appear to be on the market just yet.
The RTDS crops are being sold to the public as ‘all-natural’ ‘developed through the process of mutagenesis’, a technique exempt from GM legislation and one that has been used since World War II.
Following the disastrous use of nuclear energy in the atomic bombs, the nuclear industry wanted to put the atomic energy to alternative uses. Classic mutagenesis involves the use of chemicals or ionizing radiation to generate random mutations, a few of which may offer crop advantages depending on where and what type of mutation is generated, though the technique is hardly natural. However, many studies and reports that recommend radiation-induced mutation breeding are sponsored by organizations that promote nuclear energy. There have been an estimated 3 000 varieties of crops generated with mutagenesis techniques, a number dwarfed by the millions generated through conventional breeding techniques. Further, mutagenesis is unpredictable and it is thought that around 70 % of mutations lead to detrimental and not beneficial effects (for a summary of mutagenesis breeding see ). Plants created through mutagenesis are commonly available and not usually restricted by patents. The process is at least free from foreign DNA introduction, though the relative risks of mutagenesis versus genetic engineering techniques is still debatable. Crucially, RTDS technology goes further than mutagenesis with the introduction of synthetic DNA through particle bombardment and furthermore, this process requires the growth of plant cell cultures in vitro. In vitro cell culturing in the lab is well known to lead to random mutations (see below). Particle bombardment is a process of introducing exogenous DNA into cell cultures, using physical force to penetrate the plant cells with the DNA attached to gold particles. This can potentially damage the cell, the exogenous DNA introduced, as well as the target genomic DNA of the plant cell . One study on mammalian cell lines found that the viability of target cells was reduced when high pressures were applied to propel the particles. Damage to the exogenous plasmid DNA was also unavoidable, with the originally circular forms of plasmid DNA breaking into linear forms . Despite these risks Cibus state on their website:
“RTDS™, enables trait development that is quicker to market with less regulatory expense. RTDS™ technology is a non-transgenic approach for providing plant improvements as compared to classical genetic transformation”. Peter Beetham, Cibus’ senior VP of research, describes RTDS as “an all-natural, environmentally safe ‘smart breeding tool’ that helps farmers grow plants with desirable traits to enhance productivity.” .
Oligonucleotide-directed mutagenesis (ODM) is a method of gene targeting where a specific DNA sequence in the gene is modified. This is distinct from conventional transgenesis where a foreign gene is randomly integrated into the target genome. Gene targeting has been performed for many years with various methods that aim for homologous recombination, the recombination between similar DNA sequences. Gene targeting via homologous recombination has been responsible for the generation of thousands of genetically modified knock-out and knock-in mice, where deletions or additions of DNA sequences have been performed on the mouse genome to create heritable mouse lines primarily used in research for studying the function of genes or the pathological mechanisms underlying human disease mutations. It has also been used to generate in vitro cell lines as well as other genetically modified lab animals. However, despite the workable frequency of targeted events in mammalian systems, the frequency of non-targeted random integration events elsewhere in the genome have been shown to occur at 1 000-fold the frequency of targeted events . This disparity can be reduced by the introduction of positive and negative selection markers, though this does not eliminate random integration events. Random versus targeted integration can be analysed to a limited degree through techniques such as Southern blotting where radioactive probes bind to the integrated DNA constructs, which can then be visualized when the genomic DNA (previously cut with enzymes that cut the DNA to give a predicted digest pattern if the targeting worked correctly) is resolved on an agarose gel. However, in the case of rearrangements of the DNA construct, the probes may no longer be able to recognize it and therefore would not pick up such rearrangement events. Only complete genomic sequencing would be able to determine genomic and construct DNA integrity, something rarely done in practice. As such, introduction of exogenous DNA constructs can cause chromosomal rearrangements as well as duplications, tandem repeat integrations and rearrangements of the DNA constructs.
Homologous recombination has lower targeting frequency in plants than mammals such as mice, which has led to the development of techniques either to improve the recombination frequency by for example using site-specific nucleases to nick DNA in the target region, or through other mechanisms of gene repair.
ODM is one new technique believed to involve other endogenous gene repair pathways. It uses small chimeric DNA/RNA oligonucleotide sequences containing roughly 25 base pairs of homologous sequence to that of the target, apart the intended mutation that may be a single base change. The homologous region is encompassed by RNA molecules that are thought to stabilise the molecule in the cells as well as increase the frequency of recombination. It was first performed in mammalian cells [15, 16] and since then, several studies have been published showing modifications of plant crops including tobacco, rice, maize and wheat [17-21].
Critical in assessing the safety of this new technology is the mechanism underlying ODM; but that has remained unknown despite the glossy summaries provided by those promoting the technique. So much so that even the senior vice-president of Cibus Peter Beetham has described the mechanism as “elusive” [see 20]. It has been suggested that the process relies on the endogenous DNA repair machinery that removes single base mismatches in DNA or damaged bases. Mismatch repair is ordinarily used by the cell following mistakes in DNA replication or recombination as well as in DNA damage. It relies on enzymes that recognise the mismatch by comparing the strand to a template strand’s homologous region, during which, specific structural conformations appear, the DNA strands forming a specialised structure of homologous pairing i.e., D-loop; after which the DNA mismatched sequence is cleaved out, the correct bases synthesised and the DNA re-ligated back together. This is a highly complex process essential to the integrity of the DNA and the cell. However, this remains a speculation, and others have suggested that homologous recombination, transcription as well as DNA replication processes are involved .
There appears to be no real effort devoted to understanding ODM before pushing the products onto the market, which is crucial, not only in determining whether or not these crops should be defined as GMOs; but also in anticipating and identifying non-target effects (see below) that may make the product unsafe.
Even if homologous recombination between the oligonucleotide and the target sequence does take place, there is in fact an introduction of foreign DNA material from the oligonucleotide, thus rendering the crop a GM crop as defined by the EU directive (2001/18/EC Annex IA) which states that: “techniques involving the direct introduction into an organism of heritable material prepared outside the organism...”.
And even if there is no introduction of foreign DNA, EU GM legislation defines GMOs not only by the end product itself, but the process whereby the crops are generated. So, specific techniques are included as GM such as the use of DNA plasmid vectors, while mutagenesis defined as excluding the use of recombinant DNA, is exempt (Directive 90/219/EEC Annex II Part A). However, not defined are a number of new techniques including ODM. Though it may/may not include the introduction of foreign DNA, it does involve the use of recombinant molecules which therefore makes it GM and distinct from mutagenesis.
Cibus aims to use these techniques to bypass legislation, going as far as ghost authoring a commentary published in a peer-reviewed journal stating their case against GM legislation in Europe . Despite only researchers of European public institutions appearing as authors, the paper is in fact advertised on Cibus’s website as being written by the company .
Off-target effects of the technique should not be ruled out. In generating crops with ODM techniques, plant cells to be modified are cultured in vitro, a process that systematically induces DNA abnormalities (somaclonal variations). Further, ODM itself has been shown to cause non-target modifications, as published by Cibus, including the introduction of a wrong base into the genome, and as published by Pioneer Hi-Bred International: 6 of 40 clones generated had alternative mutations to the single-base mutation they were attempting to generate [17, 25]. They concluded that the target sequence or the structural formation of the RNA/DNA oligonucleotide with the target sequence may activate “error-prone” mismatch repair, and that the phenomenon could be used to “to assay the poorly characterized mismatch repair pathways in plants”.
No publication has looked at genome wide stability or off-target integration or modification, making it impossible to predict the wider effects that this technology may have on the entire genome, transcriptome, proteome or metabolome of the target crops. Procedures to characterize the genome, transcriptome, proteome and metabolome are now becoming routine, and should be included in any characterization of organisms resulting from a new proprietary technique such as ODM. Far too little research has been done thus far to assess all the genetic possibilities of introducing these chimeric nucleotides into genomic DNA. If, as is the case with classic gene targeting by homologous recombination techniques, that multiple non-specific events can occur, it would be outrageous to let ODM techniques slip through the regulatory framework and force us to unwittingly eat foods that are modified to a completely unknown degree.
This technique is being sold as a novel, all natural, non-transgenic technology that will answer critics of GM. However, all that has been done is applying a newer technology to generate the same old herbicide tolerant crops that already exist, either through the process of traditional breeding techniques, as is the case with sulfonylurea-tolerant crops, or through classic GM technologies, as is the case with glyphosate-tolerant crops. It is already widely acknowledged that such technologies lead to increased herbicide use and therefore increased environmental and human exposure to these herbicides (see  Study Confirms GM Crops Lead to Increased Pesticide Use, SiS 56 and  Ban GMOs Now - Special I-SIS Report). Further, these technologies have limited value in terms of crop protection and yields due to the widespread evolution of herbicide resistant weeds. There is over 23 species of glyphosate-resistant weeds and 132 species resistant to acetolactate synthase (ALS) inhibitors, the class of herbicide to which sulfonylurea herbicides belong. With such mutations occurring naturally in weeds as well as crops there seems little point in using this technology aside from making a GM company a lot of money through the application of patents and the sale of more toxic herbicides.
Article first published 02/09/13
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Madeleine Love Comment left 2nd September 2013 15:03:50
Oligonucleotide-directed mutagenesis was used to alter the DNA of the GOX protein for Monsanto's Roundup Ready Canola (GT73), reported in 1994. The crop had two genes - one being the CP4 EPSPS, and the other post-mutagenesis GOX variant 247. The GOX enzyme could degrade glyphosate - they employed mutagenesis to make it more effective. They'd wanted to change one nucleotide but ended up with five differences. The nuclear DNA of the plant wasn't exposed at all though.
Eva Comment left 3rd September 2013 10:10:59
Thanks for an interesting point, Madeleine. Indeed, the use of mutagenesis as a technique is not that new, and was used to modify the glyphosate tolerant gene (EPSPS) to identify the active sites within the gene. The difference is that the new crops are being made through mutagenesis of their endogenous genes directly. With the GT73 Canola, the EPSPS glyphosate-tolerance gene was modified in the bacterial vector, prior to it being inserted as a transgene into the crop. What I think is most important here, is that the new modifications are avoiding the use of transgenes altogether and thus may be able to avoid regulatory restrictions that are placed on GM crops.
Eva Comment left 3rd September 2013 10:10:17
Hi John, the RNA/DNA chemeric molecule is chemically synthesised in the lab and can therefore be the length they desire and does not derive from another organism such as a virus. Indeed, this is foreign DNA that is being introduced into the organism, but unlike traditional GM crops, there is no vector system such as the SV40 retroviruses, with instead the DNA/RNA being added directly to the plant cells. Those arguing in favour of this technique use the lack of a vector system as a means of excluding it as a GM technique, despite the crop being modified unnaturally, with an undefined mechanism and unknown side-effects. It is still unknown how the introduction of these chimeric molecules works, or whether it has effects on the rest of the genome and as such, should not go through without thorough long-term tests on how the crops are being effected and what this might mean for our health and the environment - something that definitely would not happen if they are excluded from GM regulation.
John Fryer Comment left 2nd September 2013 22:10:45
I have to say I no nothing about what is called ODM but if it truly is just irradiating seeds and observing the effects then that is fine. But they talk of extremely short sequences of DNA wrapped up in RNA. Isnt this GMO? GMO is broken bits of DNA and then using the retrovirus SV40 and SV40 is either linked to cancers, linked to AIDS and / or linked to contaminated vaccines that affected millions of people decades ago. What are the RNA they use and how do they snip the DNA into such short lengths?
Rory Short Comment left 4th September 2013 17:05:16
Humans are naturally curious consequently we have developed the ability to meddle with genes and that is to be expected. Individual humans also naturally fall at particular points on the scale of human greed from totally altruistic to totally self-serving. The combination of the fruits of curiosity with self-serving greed is toxic as it is inevitably hugely socially destructive. Our problem is that as societies we have created institutions, like patents for example, which promote this toxic combination rather than counter it.