PERSPECTIVE published: 16 January 2019 doi: 10.3389/fbioe.2018.00213 Genetic Alterations That Do or Do Not Occur Naturally; Consequences for Genome Edited Organisms in the Context of Regulatory Oversight René Custers 1*, Josep M. Casacuberta 2 , Dennis Eriksson 3 , László Sági 4 and Joachim Schiemann 5 1 VIB, Ghent, Belgium, 2 Centre for Research in Agricultural Genomics (CRAG), Barcelona, Spain, 3 Department of Plant Breeding, Faculty of Landscape Architecture, Horticulture and Crop Production Science, Swedish University of Agricultural Sciences, Alnarp, Sweden, 4 Centre for Agriculture Research, Hungarian Academy of Sciences (MTA), Martonvásár, Hungary, 5 Federal Research Centre for Cultivated Plants, Julius Kühn-Institut, Quedlinburg, Germany The ability to successfully exploit genome edited organisms for the benefit of food security and the environment will essentially be determined by the extent to which these organisms fall under specific regulatory provisions. In many jurisdictions the answer to this Edited by: question is considered to depend on the genetic characteristics of the edited organism, Bruce Budowle, and whether the changes introduced in its genome do (or do not) occur naturally. We University of North Texas Health Science Center, United States provide here a number of key considerations to assist with this evaluation as well as a Reviewed by: guide of concrete examples of genetic alterations with an assessment of their natural Frances Ellen Sharples, occurrence. These examples support the conclusion that for many of the common types National Academy of Sciences, of alterations introduced by means of genome editing, the resulting organisms would United States Gerald Epstein, not be subject to specific biosafety regulatory provisions whenever novelty of the genetic National Defense University, combination is a crucial determinant. United States Keywords: genome editing, regulatory oversight, natural genetic alterations, GMO, classification, future policy *Correspondence: René Custers rene.custers@vib INTRODUCTION Specialty section: The advances presented by genome editing including oligonucleotide-directed mutagenesis (ODM) This article was submitted to Biosafety and Biosecurity, and site-directed nuclease (SDN) technology have been widely recognized as a true revolution a section of the journal in our abilities to alter and improve genomes. One of the fields where these techniques are Frontiers in Bioengineering and predicted to have a significant impact is plant breeding. Humans have selected genotypes more Biotechnology adapted to their needs and have improved agricultural practices ever since the early Neolithic Received: 29 August 2018 period. The use of SDNs, and in particular CRISPR/Cas technology, will allow the introduction Accepted: 21 December 2018 of additional genomic alterations efficiently and with an unprecedented level of precision. This Published: 16 January 2019 technological innovation also presents regulatory challenges and leads to a number of questions. Citation: First, are such genome edited organisms subject to specific regulatory provisions related to Custers R, Casacuberta JM, biosafety? In many jurisdictions around the world, there is still legal uncertainty about this. And Eriksson D, Sági L and Schiemann J second, if the answer to the first question is no, should genome edited organisms nevertheless (2019) Genetic Alterations That Do or be subject to regulatory oversight that is stricter in any aspect than those which apply to Do Not Occur Naturally; conventionally bred organisms? The impact these techniques will have on plant breeding will Consequences for Genome Edited Organisms in the Context of greatly depend on how we answer these questions. To unlock the promise and potential of Regulatory Oversight. genome editing and to take responsibility for its development to benefit society there is an Front. Bioeng. Biotechnol. 6:213. urgent need for a legal clarification based on correct scientific understanding. To support the doi: 10.3389/fbioe.2018.00213 decision-making process we aim to describe what types of genetic alterations do and do not occur Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 1 January 2019 | Volume 6 | Article 213 Custers et al. Regulation of Natural Genome Edits naturally and estimate to what extent various alterations along a of two complete genomes through interspecific crosses, has also range from small to large genetic changes may occur in nature. frequently occurred during plant genome evolution (Wendel, Genome editing also has a lot of potential for the introduction of 2015). epigenetic changes. The scope of this paper, however, is limited to All these fine or crude genetic changes are the raw material alterations in the primary sequence of the genetic material. on which selection operates allowing species adaptation and evolution, both in the wild and under human direction. A look at the mutations that are at the origin of new characters selected GENOMES IN EVOLUTION during crop domestication and breeding shows that they cover a wide range of mutation types and mechanisms (Olsen and Genomes have evolved over millions of years starting from the Wendel, 2013). These include point mutations causing amino first development of living organisms, leading to the evolution of acid changes, premature translation terminations and changes a wide variety of species. Indeed, naturally occurring mutations in transcript splicing or gene regulation. They also include together with natural selection are the key factors driving transposon insertions and large deletions causing changes in evolution. Many organisms have had the capacity to adjust to coding or regulatory sequences in genes involved in plant and specific environmental conditions and the plasticity of genomes inflorescence architecture, seed shattering and dormancy, grain has been one of the factors contributing to the ability to survive size and color, among many other characters that have been changing conditions. Over the years our understanding of the subject to farmer-driven selection (Olsen and Wendel, 2013). The mechanisms underlying this evolution has grown significantly. domestication and genetic improvement of crops to serve human On top of that, modern genome sequencing technology has needs has also necessitated the incorporation of new genes revealed to us what type of alterations have occurred during from other species or even the combination of two complete evolution, domestication and breeding. If we want to determine genomes from different species. In fact, the domestication of what types of combinations of genetic material should be most crops is the result of the combination of many different considered novel and are beyond what can “occur naturally by types of mutagenic events (see for instance Table 1 in Olsen mating and/or natural recombination”—the phrase used in the and Wendel, 2013). As an example, bread wheat domestication EU GMO definition—it is important to have a closer look at these required many independent mutations, including those at the alterations. two genes controlling seed shattering in the wild emmer wheat Genetic information has to be faithfully transmitted during (Avni et al., 2017), different introgressions from wild related each cell division to allow the correct development and species, a whole genome duplication event in emmer wheat, and functioning of each organism. It also needs to be faithfully an interspecific cross between the domesticated emmer wheat transmitted to the offspring in order to maintain species and the wild goatgrass (Aegilops tauschii) (Gornicki and Faris, boundaries and biodiversity. However, some level of change 2014). In general, crop domestication has required a significant needs to be generated to endow genetic information with number of mutations and genome rearrangements accumulated the plasticity required for organisms and species adaptation in genomes, and this has also been true during the whole history to a changing environment. For this reason, most organisms of crop breeding. have evolved mechanisms to ensure a high but imperfect Whereas, all these individual mutations happened in nature fidelity in DNA replication, causing spontaneous mutations at spontaneously, they were artificially selected and combined by a low rate, and equally efficient and imperfect mechanisms humans, who ultimately have made possible the large phenotypic for repairing the DNA when damaged by endogenous or diversity of today’s crops and the radical differences crops present environmental mutagenic agents such as UV or radiation when compared to wild plants. One may even argue that the (Kunkel and Erie, 2015). This leads to a certain amount of natural specific combinations of enhanced traits in all our crops are mutations continuously being introduced into the genomes of something that never would have occurred nor maintained all organisms. In the annual model plant Arabidopsis thaliana, without human intervention. with a genome size that is about 24 times smaller than the Plant breeding has been widely influenced by scientific human genome, this mutation rate has been estimated to be progress during modern history, which has allowed expanding 7 × 10−9 base substitutions per site per generation (Ossowski the range of techniques incorporated and boosted its et al., 2010), which is approximately one substitution per sophistication. One of the key factors during this process genome per generation. In addition to these continuously arising was the increase in genetic variation available for breeding which mutations, genomes are equipped with repetitive and mobile was achieved both by expanding the gene pool that could be used genetic elements that promote additional mutations and genome for breeding and by enlarging the variability within the species rearrangements, which are much more discontinuous during by mutagenesis. The expansion of the gene pool was obtained by evolution (Lisch, 2013). Finally, genomes are not completely forcing crosses with increasingly distant species with the help, isolated within the species boundaries and different species among others, of in vitro culture techniques allowing the rescue can exchange genetic information in nature through horizontal of the offspring of otherwise sterile crosses. Embryo rescue plays transfer, as it has been shown for rice to millet (Diao et al., an important role in plant breeding programmes and is expected 2006) or for the Poa to Festuca grass genera (Vallenback et al., to retain or even broaden its significance, since plants obtained 2008), or even between kingdoms such as from Agrobacterium by embryo rescue do not have to be considered as genetically to sweet potato (Kyndt et al., 2015). Moreover, the combination modified (Winkelmann et al., 2010). Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 2 January 2019 | Volume 6 | Article 213 Custers et al. Regulation of Natural Genome Edits The increase in variability within species through mutagenesis conventional breeding approaches would not be considered to has been particularly successful and during the past 70 years result in the formation of a specifically regulated organism. more than 3,200 new crop varieties have been developed through The first two examples in Table 1 are about point mutations mutation programs (cf. IAEA/FAO mutant variety database, of which we state that the likelihood of occurrence is very high. https://mvd.iaea.org/), predominantly using γ-ray irradiation, This can be easily substantiated by performing sequence analyses but also other physical or chemical mutagens (Jankowicz-Cieslak to estimate the occurrence of single nucleotide polymorphisms and Till, 2015). (SNPs) in genes in different crops. In most genes one will find It is clear that during evolution, domestication and plant dozens if not hundreds of SNPs. The more varieties of a crop are breeding a wide variety of genetic alterations have occurred subject to the analysis, the more SNPs are generally found. In a and are still being introduced and further exploited. But not separate appendix to this paper we provide such an analysis for every type of alteration does or is likely to occur naturally. the acetolactate synthase (ALS) gene in Arabidopsis, wheat and Alterations that cannot occur naturally are considered novel. It rice. This can be seen as an illustrative example for the occurrence is for instance highly unlikely that organisms that are unrelated of SNPs in any gene in a crop. Similarly, one can perform at any higher taxonomic level exchange large amounts of genetic sequence analyses for determining the natural occurrence of the material, although the examples given above show that horizontal other types of genetic alterations that we describe in Table 1, as gene transfer (HGT) is known to occur also among sexually well. incompatible eukaryotic/plant species. THE NOVELTY CRITERION IN GMO EMERGING CONCLUSIONS AND REGULATORY REGIMES PRINCIPLES From what we have described above it is apparent that quite a The Cartagena Protocol on Biosafety to the Convention lot of alterations are already occurring naturally. If we then go on Biological Diversity serves as a framework promoting over the more concrete examples of genetic alterations (Table 1), international harmonization in the legislation of GMOs in many occasions we get a rather clear picture. When such (Cartagena Protocol, 2000). This Protocol does not use the alterations are deliberately introduced by means of techniques term GMO but defines a “living modified organism” (LMO) that do not occur naturally, and the regulatory framework uses as “any living organism that possesses a novel combination of novelty of the genetic combination as a criterion, then the genetic material developed through modern biotechnology.” In resulting organisms either would clearly classify as a specifically the Cartagena Protocol, the mere use of a technique of modern regulated organism or would clearly not classify as such. This is biotechnology is not enough to trigger regulatory oversight. for instance the case for single point mutations or for deletions of The resulting organism additionally needs to possess a novel any size, which in that context would not be considered to result combination of genetic material. The combination of genetic in the formation of a specifically regulated organism, or for the material needs to be beyond what can occur naturally by mating introduction of a transgene, which does result in the formation of and/or natural recombination. We deliberately use these latter such an organism. The deliberate and simultaneous introduction words because also the EU GMO definition uses a similar of a very large amount of specific point mutations would under phrasing. But what does “beyond what can occur naturally by such legal approach also be considered to result in the formation mating and/or natural recombination” actually mean? As a next of a specifically regulated organism, even though each individual step we will therefore go over a list of concrete examples of mutation can occur naturally. genetic alterations in different species and determine, based on But there are also areas that are less clear. Exactly how many our current understanding of biology, whether these alterations point mutations can be simultaneously introduced before the do occur naturally. organism would become a specifically regulated organism? And how many sequential base pairs of exogenous or recombinant ALTERATIONS BEYOND WHAT CAN DNA, stemming from a non-crossable source, can be introduced OCCUR NATURALLY before the organism becomes a specifically regulated organism? For sure, the more point mutations and the longer the stretch In Table 1 we provide 15 concrete examples of genetic alterations. of exogenous DNA, the less likely it is that this would occur We do not specify by what means these alterations have been spontaneously in nature. Drawing a line will inevitably be introduced, but in line with the Cartagena Protocol definitions arbitrary, however for regulatory purposes this will be necessary. they would only result in the formation of a specifically regulated Concerning the number of point mutations one could organism, if—besides having resulted in the formation of a perhaps debate why this is important to discuss in detail. genetic combination that does not occur naturally by mating Yet, it might still be relevant to determine how many point and/or natural recombination—they also have been achieved by mutations are allowed to happen because it will largely determine a method that does not occur naturally. We propose that the whether the resulting organism would have to be classified as wording “does not occur” should be interpreted such that the a specifically regulated organism or not. Are 10 simultaneous alterations are extremely unlikely to occur. Those that are more point mutations acceptable? For sure the introduction of 40 likely to occur in nature or as a result of human intervention via SNPs is acceptable if they are introduced by means of an Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 3 January 2019 | Volume 6 | Article 213 TABLE 1 | Genetic alterations and the proposed classification of organisms in the context of GMO regulatory oversight. Organism Description of the Does it occur in nature? Likeliness of occurrence Alteration Motivation alteration beyond what Custers et al. does occur naturally by mating and/or natural recombination* 1 Any plant Single point mutation YES Very likely NO The mutation rate has been estimated in different plants to be on average 1 mutation per genome per generation, meaning that every single plant will contain, at least, one new mutation. See also the Appendix in supplementary material. 2 Any plant 2,000 point mutations Perhaps only in a nuclear Extremely unlikely YES This is the example of classical mutagenesis using a fall-out radioactive source to induce random mutations. The organisms resulting from this intervention are exempted from the scope of the EU GMO directive on the condition that no recombinant nucleic acid molecules or GMOs have been used. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 3 Any plant Short deletions (a few bp) YES Very likely NO Such one base pair deletions occur on a regular basis. Mostly after the occurrence of a double-strand break. 4 Any plant Larger deletions (a few kbp) YES It does happen NO Larger deletions also do occur, but with a lower frequency. In viruses such deletions occur rather frequently during processes of attenuation. 4 5 Any plant Short insertions (a few bp) YES Very likely NO Small insertions also occur on a regular basis. Similar as with small deletions such insertions may occur, for example after a double-strand break. 6 Maize 50 kb exogenous DNA YES Very unlikely YES Such size of insertions do occur in very rare occasions, insertion but the larger the insertion the more unlikely they become. 7 Any plant 2 kb exogenous DNA YES Very unlikely YES Even though there are rare events of horizontal gene insertion from a non-related transfer, it is very unlikely that such an event occurs. This source (a transgene) is also what the legislation truly intended to have covered by the GMO legislation. 8 Any plant A cisgene flanked by the NO Extremely unlikely YES? There are no known examples of such types of T-DNA left and right borders alterations occurring in nature. Where T-DNA has been introduced spontaneously, it was with sequences stemming from A.tumefaciens in between. 9 Any plant An allele swap YES It does happen NO Allele swaps do occur, especially as a result of recombination during meiosis. 10 Any plant Cisgene addition YES It does happen NO/YES The difference with an allele swap is that the cisgene may be present anywhere in the genome, not necessarily on its ’natural’ location. It is known however that in plants genes on a regular basis get duplicated, resulting in an extra copy of the gene being inserted at another location in the genome, away from its original ‘natural’ location. (Continued) Regulation of Natural Genome Edits January 2019 | Volume 6 | Article 213 TABLE 1 | Continued Organism Description of the Does it occur in nature? Likeliness of occurrence Alteration Motivation alteration beyond what Custers et al. does occur naturally by mating and/or natural recombination* 11 Any plant Intragene addition ? Extremely unlikely YES The rearrangement of regulatory sequences does occur in nature and is actually important in evolutionary terms. But it is extremely unlikely to occur within a few generations in the context of plant breeding. 12 Plants that cannot T-DNA insertion NO Extremely unlikely YES When a plant cannot naturally be infected by A. naturally be tumefaciens, then the insertion of T-DNA cannot occur. infected by A. tumefaciens 13 Plants that can be T-DNA insertion YES Unlikely, but it has NO A. tumefaciens can naturally infect a wide variety of naturally infected happened plants, but this usually is a local infection resulting in the Frontiers in Bioengineering and Biotechnology | www.frontiersin.org by A. tumefaciens formation of crown galls, without the T-DNA being such as Nicotiana inserted into the reproductive DNA. However, this has spp., sweet potato happened during evolution of for example sweet potato. and Linaria spp 14 Any plant Genome duplication YES It does happen NO Genome duplications do spontaneously happen in nature but have even more been exploited by man to 5 strengthen certain characteristics of crops. 15 Any plant Transposon insertion YES It does happen NO Transposons mobile genetic elements that account for an important fraction of all plant genomes. Its movement generates mutations that are at the origin of many traits selected by humans to domesticate wild plants of breed new varieties. *As proposed in the article organisms should only be considered as GMO subject to specific regulatory oversight when they fulfill two criteria: (1) the alteration must have been achieved by means of a technical intervention that does not occur naturally, and (2) the genetic combination formed must be beyond what does occur naturally by mating and/or natural recombination. Regulation of Natural Genome Edits January 2019 | Volume 6 | Article 213 Custers et al. Regulation of Natural Genome Edits allele swap, where one existing allele is replaced by another proposal put forward by the Netherlands (Eriksson et al., 2018). allele originating from the organisms’ gene pool. Another This would allow including genome editing techniques in the list question is whether the introduction of different mutations in of techniques exempted from the regulation. Another option that consecutive rounds of intervention at a certain point would has also been proposed would be to revert the trend of the last 15 trigger the legislation. In other words: would the simultaneous years limiting the latitude of scientist and risk assessors applying introduction of 200 point mutations be considered to lead to the case-by-case approach to the risk analysis of GMOs as laid the formation of a novel combination of genetic material and down in Directives 90/220/EEC and 2001/18/EC (Casacuberta therefore become a specifically regulated organism, whereas and Puigdomènech, 2018). Whatever the path followed, the the accumulated introduction of 200 single point mutations principles developed in this article should help to adjust the would not? These intricate but realistic questions need pragmatic legal framework to each use of genome editing techniques and answers. perform a more proportionate risk assessment of genome edited organisms. RECOMMENDATIONS It is important to note that being classified or not as an LMO under the Cartagena Protocol, a GMO under EU legislation, a The scheme and the concrete examples that we have presented regulated article under the USDA-APHIS plant pest legislative will help to clarify the regulatory status of genome edited framework, is not per se a safety related issue. However, it is true organisms within different regulatory frameworks. Several that especially the EU GMO regulatory framework is foremost competent authorities in the world have recently clarified the applied to organisms that contain novel genetic combinations scope of their legislation. USDA-APHIS has stated that organisms beyond what does occur naturally by mating and/or natural with single point mutations, deletions of any size, and in which recombination, and subject these to pre-market risk assessment genes from compatible species have been introduced are not a under the pretext that these organisms may carry with them regulated article under its biotechnology regulations. In their a risk due to the genetic novelty per se. Mutations, whether view such organisms do not require specific regulatory scrutiny naturally occurring or man-made, such as limited nucleotide because they could otherwise have been developed through changes could also result in phenotypic changes presenting traditional breeding techniques. Conventionally bred varieties a hazard and the legislator has not seen a need to place are considered by the US regulators to present a risk level one them under additional scrutiny. This is because the regulator should not be forced to go below. In Argentina and Brazil has considered the products of traditional breeding techniques regulatory procedures have been introduced through which including mutagenesis to present a risk level that is acceptable. the regulatory bodies can determine on a case-by-case basis Breeders are not required by law to perform a pre-market whether something would be regulated as a GMO (Whelan risk assessment and get a market authorization for new varieties and Lema, 2015). They use the novelty criterion from the based on the use of conventional breeding techniques including Cartagena Protocol LMO definition as their guiding principle. conventional mutagenesis. But their products nevertheless need The outcomes of these procedures so far show that also they do to be safe for human consumption and the environment. If they not regard organisms with point mutations or (small) deletions, are not, other food and environmental legislation such as the or any other that could have occurred through conventional US legislation on food safety, the EU general food law and the breeding or by natural, spontaneous mutations, as GMOs that EU environmental liability legislation enters into force to correct require specific scrutiny. them and, if necessary, hold the developer accountable (De Jong In the EU, the recent ruling of the Court of Justice of et al., 2018). the European Union considers genome edited organisms as GMOs that do not fall under the existing exemption for AUTHOR CONTRIBUTIONS organisms resulting from conventional mutagenesis (CJEU).1 The motivation of the ruling leaves very little room for a more All authors have jointly contributed to the manuscript, with product-oriented interpretation of the phrase “has been altered particular contribution of JC to the text on genomes in evolution in a way that does not occur naturally by mating and or natural and LS to the appendices. recombination” in the EU GMO definition. The Court has not used the novelty criterion, even though it could have. This implies FUNDING that organisms that have edits that can or do occur naturally, will have to follow the same regulatory procedures as GMOs, This work was supported by the Swedish Foundation for Strategic including a detailed analysis of possible risks. This does not Environmental Research (Mistra) through the Mistra Biotech comply with the principles set out in the Cartagena Protocol research program. on Biosafety and would be disproportionate and scientifically unsound. In the last few months, different proposals to solve this situation have been proposed. First, the existing EU GMO SUPPLEMENTARY MATERIAL legal framework could be modified following for instance the The Supplementary Material for this article can be found 1 http://curia.europa.eu/juris/document/document.jsf?text=&docid=207002& online at: https://www.frontiersin.org/articles/10.3389/fbioe. pageIndex=0&doclang=EN&mode=req&dir=&occ=first&part=1&cid=6947981 2018.00213/full#supplementary-material Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 6 January 2019 | Volume 6 | Article 213 Custers et al. Regulation of Natural Genome Edits REFERENCES Lisch, D. (2013). How important are transposons for plant evolution? Nat. Rev. Genet. 14, 49–61. doi: 10.1038/nrg3374 Avni, R., Nave, M., Barad, O., Baruch, K., Twardziok, S. O., Gundlach, H., Olsen, K. M., and Wendel, J. F. (2013). A bountiful harvest: genomic insights et al. (2017). Wild emmer genome architecture and diversity elucidate wheat into crop domestication phenotypes. Annu. Rev. 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