GM Crops & Food, 6:223–232, 2015 Ó 2015 Taylor & Francis Group, LLC ISSN: 2164-5698 print / 2164-5701 online DOI: 10.1080/21645698.2015.1134405
Future of breeding by genome editing is in the hands of regulators Huw D Jones#,* Rothamsted Research; Harpenden, UK ABSTRACT. We are witnessing the timely convergence of several technologies that together will have significant impact on research, human health and in animal and plant breeding. The exponential increase in genome and expressed sequence data, the ability to compile, analyze and mine these data via sophisticated bioinformatics procedures on high-powered computers, and developments in various molecular and in-vitro cellular techniques combine to underpin novel developments in research and commercial biotechnology. Arguably the most important of these is genome editing which encompasses a suite of site directed nucleases (SDN) that can be designed to cut, or otherwise modify predetermined DNA sequences in the genome and result in targeted insertions, deletions, or other changes for genetic improvement. It is a powerful and adaptive technology for animal and plant science, with huge relevance for plant and animal breeding. But this promise will be realized only if the regulatory oversight is proportionate to the potential hazards and has broad support from consumers, researchers and commercial interests. Despite significant progress in research and development and one genome edited crop close to commercialization, in most regions of the world it still remains unclear how or whether this fledgling technology will be regulated. The various risk management authorities and biotechnology regulators have a unique opportunity to set up a logical, appropriate and workable regulatory framework for gene editing that, unlike the situation for GMOs, would have broad support from stakeholders.
The term genome editing encompasses a suite of site directed nucleases (SDN) that can be designed to cut, or otherwise modify predetermined DNA sequences in the genome and result in targeted insertions, deletions, or other changes for genetic improvement (Table 1). It is a powerful and adaptive technology for animal and plant science, with huge relevance for plant and animal breeding. Although there are calls from vocal campaign groups for it to be governed as a conventional GMO (e.g. Natural News, 2015; GM Watch, 2014; Kraemer, 2015), in its most basic form, genome editing is equivalent to mutation breeding and logically should
be excluded from GMO regulations. Despite significant progress in research and development and one genome edited crop close to commercialisation, in most regions of the world it still remains unclear how or whether this fledgling technology will be regulated (Jones 2015b, Podevin et al., 2012). The current international governance of biotechnology requires a pre-market risk assessment and approvals process that relies heavily on definitions and concepts enshrined in 2 international instruments; the Cartagena Protocol on Biosafety (Secretariat of the Convention on Biological Diversity (2000)) and the Risk
*Correspondence to: Huw D Jones; Email: [email protected] Received: 08/10/2015; Revised: 12/15/2015; Accepted: 12/15/2015 # Present affiliation: Prof. Translational Genomics for Plant Breeding; Institute of Biological, Environmental & Rural Sciences; Aberystwyth University; Gogerddan; Aberystwyth; Ceredigion, UK Color versions of one or more figures in this article can be found online at www.tandfonline.com/kgmc. 223
224
Jones
TABLE 1. Main genome editing technologies currently used in basic and applied research Category Two-component programmable endonucleases
Gene modulation technique Zinc-finger nuclease (ZFN)
Fok1 nuclease directed to gene target site via a series of protein domains designed to recognize a specific DNA sequence
Transcription activator-like effector nuclease (TALEN) Clustered regularly interspaced short palindromic repeats (CRISPRs)
Non-programmable endonucleases
Meganuclease (MN)
Non-endonuclease dependent homologous recombination
Oligonucleotide-directed mutagenesis (ODM)
Analysis Principles for Foods Derived from Biotechnology (Codex Alimentarius Commission [updated] 2014) supported by numerous outputs from the Organisation for Economic Co-operation and Development (OECD). However, some developing economies are still to formalise any framework for governance of GMOs and of those that have, significant differences already exist in regard to the triggers for GMO regulation and to the data requirements for risk assessment. Worryingly, these inconsistencies are set to widen further as products from emerging biotechnologies such as genome editing reach the market place. The lack of a common trigger for regulating the products of genome editing and the lack of guidance regarding the data package required for risk assessment may result in the same altered traits or identical genetic modifications escaping the GMO regulations in some countries while in others being encompassed by expensive procedures with unpredictable time lines. This lack of consistency risks stifling innovation, exacerbating already difficult international trade issues and more importantly, undermining consumer confidence in both the risk assessment process and the safety of the biotechnology products.
Mechanism
Cas-9 nuclease directed to target site via a short guide RNA. Easy to program for different targets and deactivated or ‘nickase’ variants available to give single-stranded breaks or enhancer/ repressor functions Naturally occurring restriction enzymes which also possess highly specific target sites, but are not programmable to different DNA sequences. Thus they are losing ground to the programmable, 2-component systems above Short, chemically-synthesized DNA/RNA oligonucleotides possessing similarity to the target sequence except for the few bases to be edited that are incorporated into the genome during repair
CURRENT FRAMEWORKS FOR REGULATING GMOS WERE NEVER DESIGNED FOR GENOME EDITING The Cartagena Protocol refers to plants generated via genetic modification as ‘Living Modified Organisms’ and defines then as ‘any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology’. This definition, often supplemented with the concept of ‘recombinant DNA techniques’ is now the foundation of the many national legal frameworks that include the process used to generate the new plant variety as part of the regulatory trigger such as the Argentina, Australia/New Zealand, Brazil, China, EU, Japan, Korea, Russia, Saudi Arabia, Thailand, Taiwan etc. Other countries, notably Canada and to some extent USA, incorporate the concept of a novel trait regardless of process but even these regulations treat crops resulting from recombinant DNA technology as requiring special attention. An emerging challenge for regulators is to accommodate new biotechnologies such as genome editing that do not fall neatly into the definitions of genetic modification laid down in existing
REGULATING GENOME EDITING
legislations. While a few countries have indicated how they intend to treat products of these technologies, many others have not. Uncertainties such as this need urgent clarification, because they have major implications for research, plant breeding and international trade in commodity crops (Jones, 2015b). For the risk assessment of conventional GMOs, there exists a broadly common comparative approach that assumes the non-GM counterpart has a history-of-safe-use and tests the substantial equivalence of the GM crop against its non-GM counterpart. Thus, after assessing the comparative risks of food/feed and environmental safety, if a novel food is found to be substantially equivalent to an existing food with a history-of-safe-use, it can be treated in the same manner and no additional safety concerns would be expected (FAO/WHO, 2000; OECD, 1993). However, even for conventional GMOs there are significant differences in the scope of regulatory oversight between countries. For example, in USA and Canada, GM events stacked by conventional breeding are assumed to be safe if the individual events are already authorised (except where a specific hazard has been identified). E.g. the US Environmental Protection Agency (EPA) requires separate review of the safety of the stack if the parental GMOs carry traits encoding biotoxins because insecticides may result in synergetic or adjuvant effects (EPA, 2001). However, in the EU and other countries including Argentina, Canada, EU, Japan, Korea and the Philippines, each stack, regardless of the traits involved is always considered as a new GMO which needs to be assessed and approved de novo, even though the individual events already have market approval ( EFSA 2011). Other countries take intermediate or case-by-case approaches; e.g., Australia/New Zealand, Brazil, Canada, China (EU FP6 CO-EXTRA Report Summary 2012). These different regulatory requirements reflect as much the policies and politics of the various nations than actual biological risks but result in significant cost implications for breeders, growers and food/feed processors and most importantly, serve to further confuse or undermine the confidence of consumers in the global oversight of biotechnology.
225
FITTING GENOME EDITING INTO EXISTING LEGISLATION In the EU, a GMO is defined in Directive 2001/18 as ‘an organism, with the exception of human beings, in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination’. The Directive goes on to exclude organisms obtained through mutagenesis, and cell/protoplast fusion (so long as it does not involve the use of recombinant nucleic acid molecules and the organisms could exchange genetic material through traditional breeding methods). The exclusion of mutation breeding from the GMO regulations is particularly significant when considering genome editing. The use of high-energy radiation or alkylating agents such as ethyl methanesulfonate (EMS) generate multiple genetic legions at random sites in the genome and lead to a range of mutation types that have been used in breeding since the 1940s (Maluszynski, 2001). For instance, the Ruby Red grapefruit variety is part of our daily diets and is just one of the more than 3,000 crop varieties listed in the Joint FAO/IAEA Mutant Variety Database as resulting from mutation breeding. The cellular activities occurring during mutation breeding can be divided into 2 distinct processes; the cleavage of DNA caused by the exogenous mutagen and the repair of that damage by the cell’s own nuclear regeneration machinery. Several factors determine the frequency and type of genetic damage including the species and tissue targeted the specific type and intensity of the mutagen used and the precise stage of the cell-cycle that it is applied. Various mechanisms exist in cells for the restoration of damaged DNA and these are not fully understood. The most cytotoxic form of DNA damage are double-stranded breaks (DSB) which are repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR) depending on whether undamaged homologous DNA is present to act as a repair template. NHEJ serves to ligate together the 2 DNA ends when no template is present but is prone to generating ‘mistakes’ (mutations) at the sites of joining both in the form of single nucleotide polymorphisms or gross
226
Jones
chromosomal rearrangements such as inversions and translocations. An alternative repair mechanism consists of a set of enzyme-controlled pathways that uses a DNA template that flanks the DSB for accurate repair, collectively known as HR. An understanding of the mechanisms of mutation breeding is relevant for 2 reasons. Firstly, as a technique, it is excluded from the scope of GMO regulatory frameworks of many regions of the world including the EU. Secondly, because a SDN can be considered as merely acting as a mutagen, albeit a more benign and highly-targeted one, to break DNA in the same way as high energy radiation or a chemical such as EMS does when used in mutation breeding. Any new plant variety resulting from this simple form of genome editing would contain no recombinant DNA but would possess a mutation undistinguishable from one that could be made through mutation breeding. A significant advantage of doing this with genome editing is that the SDN is highly specific, acting only on the pre-determined target sequence, as opposed to the many thousands of additional mutations that would result from achieving the same goal with mutation breeding. This is particularly evident in hexaploid wheat whose genetic redundancy allows it to carry a large mutation load with little effect. Various wheat populations that were mutated for TILLING experiments possess between 12 and 41 individual mutations per Mb of DNA (Chen et al., 2014) giving an average of 415,000 mutations in each hexaploid wheat plant (Uauy et al., 2009). Even if it was possible, it would take several years of careful crossing and selection to de-convolute these multiple mutations and derive an individual plant that possessed only the 1 or 2 desired alterations. However, by using genome editing, only the desired mutations are generated in the first instance. A recent publication on mildew resistance in wheat nicely illustrates the power of genome editing. MLO is a genetic locus suspected of supressing plant defense against mildew (Jorgensen, 1992). MLO is present in 6 copies in hexaploid wheat, one on each haploid genome, and all 6 must be simultaneously mutated to generate broad-spectrum mildew-resistance.
The probability of achieving this even in a large mutated TILLING population is vanishingly low and it would be laborious to use a backcross program to combine individual null alleles from different plants and remove other deleterious ones. However, Wang et al. (2014) designed TALENs proteins to target a specific site in the MLO sequence and reported the successful knockout of all 6 alleles of MLO in a single plant. This plant was completely resistant to mildew and forms a good candidate for further breeding. If this same goal was attempted by mutation breeding, the plants generated would not be defined a GMO in most countries, despite inevitably possessing many uncharacterised mutations. However, if the mildew-resistant plant made by TALEN-mediated mutations were to be marketed, there is considerable uncertainty whether or not it would be regulated as a GMO (see Fig. 1).
IS GENOME EDITING JUST GENETIC MODIFICATION BY ANOTHER NAME? SDN can be used in 3 different ways and this determines the answer to the question above. In the basic type l mode (SDN-1), a targeted nuclease is used simply as a mutagen to make a directed DSB which the cell’s inherently errorprone DNA repair mechanisms re-join using NHEJ. The exact sequence of final mutations cannot be predicted but several plants can be screened for individuals that possess desired SNPs or INDELs and selfed or backcrossed to remove all transgenes expressing the SND. The most common application of SDN-1 is to switch off a specific gene by screening for mutations resulting in a stop codon or a translational frame-shift. The resulting null segregant contains no recombinant DNA but retains the desired mutation. Although the mutation could be detected by molecular techniques including DNA sequencing, the changes are indistinguishable from the mutations that occur spontaneously or during mutation breeding. Mutation breeding, which is significantly more genetically disruptive, is already excluded from the vast majority of global GMO regulatory
REGULATING GENOME EDITING
oversight. Therefore, it is logical that plants resulting from SDN-1 genome editing should be excluded too. In a refinement to the basic SDN-1 mode, additional DNA in the form of a repair template can be introduced into the cell at the same time as the SDN. This can either be a template designed to introduce a few tens or hundreds of DNA bases which is considered a SDN-2, or a more extreme example where a complete functional transgene is target to a pre-determined safe harbour site in the genome which is designated the ‘SDN-3 mode’. The concept of a safe harbour site for a GMO is useful because it can be chosen to avoid disruption of a native gene and other so-called position effects. Multiple transgenes could then be stacked by re-transformation into the same locus so they segregate together during meiosis and save the significant complexity of breeding with unlinked transgene loci. This concept has been reported in maize using a 2-step process. Firstly, a parent line was generated that possessed a single-copy ‘trait landing pad’ (TLP) site at a convenient location in the maize genome. Then by retransformation, functional intact transgenes were reproducibly added into the TLP at an integration frequency of 5% (Ainley et al., 2013). This demonstrates the feasibility of stacking multiple transgenes into the same safe harbour site avoid unintended positional effects and generate single locus integration for ease of later breeding. SDN-3 applications of genome editing are similar to conventional GM and crop varieties developed using this technology marketed within the EU would clearly fall into the GMO regulations. The European Food Safely Authority (EFSA) has indicated that its current guidelines would be suitable for evaluating products of SDN-3 for risks to food and feed to the environment. They also considered, on a case-by-case basis, lesser amounts of event-specific data may be needed for the risk assessment of plants developed using the SDN3 technique (EFSA 2012). Genome editing in the SDN-2 mode is the most challenging for regulators and ironically will probably be the most useful for plant breeding because it enables editing of existing alleles to redefine their function or new ones to
227
be ‘knocked-in’. The editing could be designed either to copy existing useful alleles already in the gene pool but where introgression into elite commercial germplasm using conventional breeding would be impractical/too costly, or it could be used to create novel traits by completely redesigning gene function by, for instance, altering the substrate specificity of an enzyme. Importantly, both of these can be achieved directly in the elite lines of a breeding scheme saving many generations of backcrossing and avoiding potential problems of linkage drag. Genome editing via homology-dependent template repair of DSB has been achieved in many animal cell systems including human, rat, mouse zebra fish, drosophila C. elegans (reviewed by Gaj et al., 2013; Cox et al., 2015; Sander and Joung, 2014) and in plants (reviewed by Belhaj et al., 2015). There are good examples of genome editing using homology dependent repair templates in plants including the correction and inheritance of a non-functional gus reporter gene in Arabidopsis (Feng et al., 2014). The rice pds gene was successfully edited using a 72bp homologous oligonucleotide combined with a targeted Crispr Cas-9 to insert a 12bp edit in rice protoplasts (Shan et al., 2013). With the rate of research in this area, it is inevitable that agricultural applications of SDN-2 editing will soon be reported. However, there is a genome edited crop variety close to market generated using an alternative method known as oligonucleotide-directed mutagenesis (ODM). ODM does not use the Fok-1 nuclease to cleave DNA but instead uses short, chemically synthesized DNA/RNA oligonucleotides possessing similarity to the target sequence except for the few bases to be edited that are incorporated into the genome during repair. A novel crop Canola variety genome edited by Cibus using their proprietary Genome Repair Oligonucleotide technology to be tolerant to the herbicide Sulfonylurea received regulatory approval from Canadian Food Inspection Agency and Health Canada in March 2014 and is expected to be launched for cultivation in Canada in 2016 (Jones, 2015b). Although there are no commercial crop varieties currently marketed using CRISPR CAS-9, and there remains some way to go before the
228
Jones
intellectual property landscape is fully clarified, this particular technology is proving to be the most adaptable and has the potential to drive rapid advancements in plant breeding (Bortesi 2015, Xiong et al. 2015). In only a few years, CRISPR Cas-9 has been rapidly adopted by the research community as a routine method to knock-in and knock-out DNA sequences in animals and plants (eg Barrangou et al. 2015; Qi et al. 2013). Recent refinements of the basic technology are being reported, including the use of CAS-9 variants that generate singlestranded ‘nicks’ as well as deactivated forms of CAS-9 that modify expression of a targeted native gene by recruiting enhancers or repressors to specific locations within the regulatory regions of genes. Cas-9 nickase can be used to efficiently mutate genes without detectable damage at known off-target sites. This method is applicable for genome editing of any model organism and minimizes confounding problems of off-target mutations (Shen et al., 2014). In a different approach, 2-component transcriptional activator systems have been demonstrated in animal and plant cells consisting of a deactivated Cas-9 fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters (e.g., Cheng et al., 2013; Lowder et al., 2015). This represents a novel way to modulate gene expression by localizing enhancers and repressors to receptive promoter domains while leaving the primary DNA sequence unaltered.
EMERGING REGULATORY POSITIONS Of the national authorities that include a process-based trigger for biotechnology regulation, only Argentina has so far published detailed guidelines on how they proposed to manage crops made using genome editing. They regard a plant as a GMO when it possesses a new combination of genetic material created by the insertion of one or more genes or DNA sequences that are a part of a genetic construct have been inserted into the plant genome ‘Para que un cambio genetico sea considerado una nueva
combinacion de material genetico, se analizara si se ha producido una insercion en el genoma en forma estable y conjunta de UNO (1) o mas genes o secuencias de ADN que forman parte de una construccion genetica definida’. The means that null segregants possessing genetic changes resulting from NHEJ are not defined as a GMO. Additionally, to remove uncertainty that may stall early R&D in this area, the Argentinian National Advisory Commission on Agricultural Biotechnology (CONABIA) offer to provide a preliminary answer regarding how a new crop variety would be regulated in the design stage of the project (Ministerio de Agricultura, Ganaderia y Pesca, 2015). This is a well-considered and logical approach consistent with the potential hazards and the fact that conventional mutagenesis is excluded from GMO regulations. The USA and Canada, who incorporate the concept of a novel-trait into their regulatory frameworks, have indicated that simple genome editing that resulted in a new crop type containing no recombinant DNA would be regulated using a substantial equivalence approach and not automatically require the level of riskassessment typical of a conventional GMO. For example, the genome edited herbicide tolerant Canola developed by Cibus is the crop closest to commercialization and regulatory approval has been sought in Canada and USA. The Canadian Food Inspection Agency, who are responsible for regulating the environmental release of plant with a novel trait (PNT), scrutinized the Cibus application and determined that ‘this PNT does not present altered environmental risk nor, as a novel feed, does it present livestock feed safety concerns when compared to canola varieties currently grown and permitted to be used as livestock feed in Canada’ (Canadian Food Inspection Agency 2013). The USA has evolved a “Coordinated Framework for Regulation of Biotechnology” by adapting their existing food safety and crop protection laws to also cover biotechnology and incorporate concepts of both process- and trait-based triggers (Jones, 2015a). To date, the US Department of Agriculture have waived the regulations in place for genetically modified organisms for at least 2 products generated
FIGURE 1. Comparison of different breeding methods and whether they are regulated as GMOs in EU. Conventional GMOs (A), mutation breeding (B) and 2 stages of a new variety developed using SDN-1: early R&D phase (Ci) and final marketable variety (Cii)
REGULATING GENOME EDITING 229
230
Jones
using genome editing: a maize line that contains lower levels of the anti-nutritional compound phytate, and the Cibus herbicideresistant canola described above. In Europe, the governance of biotechnology rests at an EU level and the EC has been considering how to regulate new plant breeding techniques including genome editing for over 6 y. They have recently indicated that guidance will be provided during the first quarter of 2016. However, in the absence of any formal guidance from the EC, the competent authorities of several MS (including UK (ACRE), Germany (The Federal Office of Consumer Protection and Food Safety [BVL]), Belgium, Holland and most recently, Sweden have independently stated that they did not consider products of ODM such as the Cibus Canola as a GMO. This triggered protests from NGOs including TestBiotech, Greenpeace and FoE who published an open letter to the EC urging it to ensure (among other things) “that organisms produced by these new techniques will be regulated as genetically modified organisms under existing EU regulations (Directive 2001/18). This means that they will require a full risk assessment before any approval or authorisation is given” (Open letter to the Commission on New Genetic Engineering Methods, 2015). For conventional GMOs the EU has one of the most stringent and costly regulatory frameworks in the world with strict data requirements and high standards of risk assessment. Even so, the final approvals process is slow and unpredictable. The only GM variety currently approved for cultivation in the EU is the insectresistant maize MON810 which was authorized in 1998 under an earlier adoption system. No further approvals have been made despite applications with positive risk assessments from the European Food Safety Authority (EFSA). This is a direct result of the voting patterns of member states (MS) who fail to achieve a qualified majority at the committee stage. We are yet to see the effect of the recently passed national opt-out clause for cultivation but it is likely that it will empower the EC to authorize applications that have been outstanding for many years. There are some MS who consistently vote against adoption of
new GMOs regardless of the positive opinion published by EFSA, e.g., Austria, Denmark, Greece, Lithuania and Luxemburg (Sabalza et al., 2011), and this sends a strong signal that crops made using genome editing techniques would be also considered unacceptable by these MS, perhaps even after a full risk assessment.
IS EXISTING LEGISLATION FIT FOR PURPOSE? Agriculture is facing considerable challenges from many directions. We need safe and nutritious crop types that are predictably-productive under increasingly extreme climatic stresses and pressures from pests and diseases. We must achieve this with sustainable agricultural systems that minimize soil degradation, water use and environmental pollution. Plant breeding has always, and will continue to play a crucial role in meeting these challenges. However, this can only happen with appropriate regulatory oversight that stimulates research and innovation to produce safe and better plant varieties. Plant breeding uses a spectrum of technologies that are themselves evolving at a rapid pace. We need regulatory frameworks that are risk- and evidence-based, proportionate and globally-harmonized. Importantly we need them to be transparent and able to adapt to future changes. For some biotechnology innovations with no major hazards identified, a well-managed post-market system may suffice. Existing legislation that incorporates ambiguous definitions of genetic modification and that excludes from scope, techniques that are more genomically disruptive than techniques it captures, are broadly unfit for future purpose. In the long term, the primary legislation must be redrafted to facilitate a process of hazard identification and risk assessment of the actual crop cultivated and/or the food/feed consumed. The processes by which the new genotype is produced are not irrelevant, but neither should they be a primary trigger for regulation. However, this will take many years of discussion and in the meantime; innovation is already being stifled by uncertainty in some countries while varieties made using new breeding
REGULATING GENOME EDITING
techniques are reaching the marketplace in others. The recent decree by the Argentinian competent authority demonstrates how a process-based legislation can be easily adapted to reasonably accommodate at least the recent developments in genome editing. By mid-2016 we should know whether the EC and perhaps other competent authorities will follow the Argentinian lead or head down a more restrictive path by defining SDN-mediated mutations as GMOs requiring a full risk assessment. Apart from being illogical, the latter course would exacerbate the already complex trade and labeling issues for bulk commodity shipments into EU (Jones, 2015b).
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST Prof. Jones is a scientist using genome editing in functional genomics research. No potential conflicts of interest were disclosed.
FUNDING Rothamsted Research receives support from the Biotechnological and Biological Sciences Research Council (BBSRC) of the UK as part of the 20:20 Wheat Ò Programme. Opinions and views expressed in this publication are those of the author and do not necessarily represent those of Rothamsted Research or the European Food Safety Authority. REFERENCES Ainley WM, Sastry-Dent L, Welter ME, Murray MG, Zeitler B, Amora R, Corbin DR, Miles RR, Arnold NL, Strange TL, et al. Trait stacking via targeted genome editing. Plant Biotechnol J 2013; 11:1126–1134; PMID:23953646; http://dx.doi.org/10.1111/pbi.12107 Anonymous. Seeds of change. Nature 2015; 520: 131–132. Barrangou R, Birmingham A, Wiemann S, Beijersbergen RL, Hornung V, Smith Anja VB. Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference Nucleic Acids Res 2015; 43 (7):3407–3419; PMID:25800748; http://dx.doi.org/ 10.1093/nar/gkv226
231
Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V. Editing plant genomes with CRISPR/Cas9. Currnt Opin Biotechnol 2015; 32:76–84; PMID:25437637; http://dx.doi.org/10.1016/j.copbio.2014.11.007 Bortesi L, Fischer R. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 2015; 33:41–52; PMID:25536441; http://dx.doi.org/10.1016/ j.biotechadv.2014.12.006 Canadian Food Inspection Agency. Determination of the safety of Cibus Canada Inc.’s Canola (Brassica napus L.) Event 5715. 2013. Available via http://www.inspection.gc.ca/ plants/plants-with-novel-traits/approved-under-review/deci sion-documents/dd-2013-100/eng/1427383332253/ 1427383674669 Chen L, Hao L, Parry MAJ, Phillips AL, Hu Y-G. Progress in TILLING as a tool for functional genomics and improvement of crops. J Integr Plant Biol 2014; 56:425–443; PMID:24618006; http://dx.doi.org/ 10.1111/jipb.12192 Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan S, Shivalila CS, Dadon DB, Jaenisch, R. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res 2013; 23:1163–1171; PMID: 23979020; http://dx.doi.org/10.1038/cr.2013.122 CO-EXTRA Report Summary. Review on the world wide authorization of gene stacked GMOs in relation to commercial gene stacks and wild gene stacks and the basis for definition of corresponding unique genetic markers. 2012. Deliverable D6.4 EU CORDIS http:// cordis.europa.eu/result/rcn/53012_en.html Codex Alimentarius Commission. PROCEDURAL MANUAL. Joint FAO/WHO Food Standards Programme 22nd edition 2014. FAO/WHO http://www. codexalimentarius.org/ Cox DBT, Platt RJ, Zhang F. Therapeutic genome editing: prospects and challenges. Nat Med 2015; 21:121–131. EFSA, 2011; PMID:25654603; http://dx.doi.org/ 10.1038/nm.3793 EFSA. Guidance on selection of comparators for the risk assessment of genetically modified plants and derived food and feed. EFSA Journal 2011; 9(5):2149 [20 pp.]; Available online: www.efsa.europa.eu/efsajournal EFSA. Scientific opinion addressing the safety assessment of plants developed using Zinc Finger Nuclease 3 and other Site-Directed Nucleases with similar function. EFSA J 2012; 10(10):2943. [31 pp.] . Available online: www.efsa.europa.eu/efsajournal EPA. Environmental Protection Agency, USA. Plantincorporated protectants; final rules and proposed rule. Federal Register 2001; 66(139):37771–37817. http:// www.epa.gov/pesticides/biopesticides/pips/pip_rule. pdf FAO/WHO. Report of a Joint FAO/WHO Expert Consultation on foods derived from biotechnology – safety aspects of genetically modified foods of plant origin.
232
Jones
Geneva, Switzerland: WHO Headquarters; 29 May–2 June 2000. Available from http://www.fao.org/filead min/templates/agns/pdf/topics/ec_june2000_en.pdf Feng Z, Mao Y, Xu N, Zhang B, Wei P, Yang D-L, Wang Z, Zhang Z, Zheng R, Yang L, et al. Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci U S A 2014; 111:4632–4637. Gaj T, Gersbach CA, Barbas CF, III. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trend Biotechnol 2013. 31:397–405; PMID:23664777; http://dx.doi.org/10.1016/j.tibtech.2013.04.004 GM Watch. Genome editing is GM by another name. 2014. http://www.gmwatch.org/news/archive/2014/ 15546-genome-editing-gm-by-another-name Jones HD. Challenging regulations: Managing risks in crop biotechnology. Food Ener Security 2015a; 4:87–91. Jones HD. Regulatory uncertainty over genome editing. Nat Plants 2015b; 1:14011; http://dx.doi.org/10.1038/ nplants.2014.11 Jorgensen JH. Discovery, characterization and exploitation of mlo powdery mildew resistance in barley. Euphytica 1992; 63:141–152; http://dx.doi.org/ 10.1007/BF00023919 Kr€amer, L. (2015) Legal questions concerning new methods for changing the genetic conditions in plants. http://www.testbiotech.org/en/node/1342 Lowder LG, Zhang D, Baltes NJ, Paul JW, Tang X, Zheng X, Voytas DF, Hsieh T-F, Zhang Y, Qi Y. A CRISPR/ Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol 2015; 169 (2):971–985; PMID:26297141; http://dx.doi.org/ 10.1104/pp.15.00636 Maluszynski M. Officially released mutant varieties The FAO/IAEA database. Plant Cell Tissue Organ Cult 2001; 65:175–177; http://dx.doi.org/10.1023/ A:1010652523463 MINISTERIO DE AGRICULTURA, GANADERIA Y PESCA. Resolucion 173/2015 12-may-2015. http://info leg.mecon.gov.ar/infolegInternet/verNorma.do;jses sionid=AC1747549EAABF7CFC3644F02F41B15A? id=246978 Natural News. Biotech companies to use "genome editing" to circumvent GMO regulations. 20150 http:// www.naturalnews.com/048344_GMO_regulations_ge nome_editing_biotech_companies.html OECD. Safety evaluation of foods derived by modern biotechnology; Concepts and principles. Organisation for Economic Co-operation and Development. Paris, France: OECD Publications; 1993. Available from http://www.oecd.org/science/biotrack/41036698.pdf Open letter to the Commission on New Genetic Engineering Methods. 26 January 2015. Available from http://www.test biotech.org/sites/default/files/20150126%20Open%20Let ter%20on%20new%20GM%20technologies.pdf
Podevin N, Devos Y, Davies HV, Nielsen KM. Transgenic or not? No simple answer! New biotechnologybased plant breeding techniques and the regulatory landscape. Embo Rep 2012; 13:1057–1061; PMID: 23154464; http://dx.doi.org/10.1038/embor.2012.168 Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013; 152:1173–1183; PMID: 23452860; http://dx.doi.org/10.1016/j.cell.2013.02.022 Sabalza M, Miralpeix B, Twyman RM, Capell T, Christou P. EU legitimizes GM crop exclusion zones. Nat Biotechnol 2011; 29:315–317; PMID:21478843; http://dx. doi.org/10.1038/nbt.1840 Sander JD, Joung JK. CRIRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 2014. 32:347–355; PMID:24584096; http://dx.doi.org/ 10.1038/nbt.2842 Secretariat of the Convention on Biological Diversity. Cartagena Protocol on Biosafety to the Convention on Biological Diversity: Text and annexes. Montreal, Canada: Secretariat of the Convention on Biological Diversity; 2000. Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu J-L, et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotech 2013; 31:686–688; http://dx.doi.org/ 10.1038/nbt.2650 Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang X, et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Meth 2014; 11(4):399– 402; http://dx.doi.org/10.1038/nmeth.2857 Mutant Varieties database. Joint FAO/IAEA programme nuclear techniques in food and agriculture. International Atomic Energy Agency (IAEA). Vienna International Centre, PO Box 100, A-1400 Vienna, Austria, http://mvd.iaea.org/ Uauy C, Paraiso F, Colasuonno P, Tran RK, Tsai H, Berardi S, Comai L, Dubcovsky J. A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC Plant Biol 2009; 9:115; PMID:19712486; http://dx.doi.org/ 10.1186/1471-2229-9-115 Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qui JL. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology 2014; 32: 947–951; http://dx.doi.org/10.1038/nbt.2969 Whelan AI, Lema MA. Regulatory framework for gene editing and other New Breeding Techniques (NBTs) in Argentina. 2015. GM Crops & Food; http://dx.doi.org/ 10.1080/21645698.2015.1114698 Xiong J-S, Ding J, Li Y. Genome-editing technologies and their potential application in horticultural crop breeding. Horticult Res 2015; 2:15019; PMID: 26504570; http://dx.doi.org/10.1038/hortres.2015.19
Future of breeding by genome editing is in the hands of regulators Huw D Jones#,* Rothamsted Research; Harpenden, UK ABSTRACT. We are witnessing the timely convergence of several technologies that together will have significant impact on research, human health and in animal and plant breeding. The exponential increase in genome and expressed sequence data, the ability to compile, analyze and mine these data via sophisticated bioinformatics procedures on high-powered computers, and developments in various molecular and in-vitro cellular techniques combine to underpin novel developments in research and commercial biotechnology. Arguably the most important of these is genome editing which encompasses a suite of site directed nucleases (SDN) that can be designed to cut, or otherwise modify predetermined DNA sequences in the genome and result in targeted insertions, deletions, or other changes for genetic improvement. It is a powerful and adaptive technology for animal and plant science, with huge relevance for plant and animal breeding. But this promise will be realized only if the regulatory oversight is proportionate to the potential hazards and has broad support from consumers, researchers and commercial interests. Despite significant progress in research and development and one genome edited crop close to commercialization, in most regions of the world it still remains unclear how or whether this fledgling technology will be regulated. The various risk management authorities and biotechnology regulators have a unique opportunity to set up a logical, appropriate and workable regulatory framework for gene editing that, unlike the situation for GMOs, would have broad support from stakeholders.
The term genome editing encompasses a suite of site directed nucleases (SDN) that can be designed to cut, or otherwise modify predetermined DNA sequences in the genome and result in targeted insertions, deletions, or other changes for genetic improvement (Table 1). It is a powerful and adaptive technology for animal and plant science, with huge relevance for plant and animal breeding. Although there are calls from vocal campaign groups for it to be governed as a conventional GMO (e.g. Natural News, 2015; GM Watch, 2014; Kraemer, 2015), in its most basic form, genome editing is equivalent to mutation breeding and logically should
be excluded from GMO regulations. Despite significant progress in research and development and one genome edited crop close to commercialisation, in most regions of the world it still remains unclear how or whether this fledgling technology will be regulated (Jones 2015b, Podevin et al., 2012). The current international governance of biotechnology requires a pre-market risk assessment and approvals process that relies heavily on definitions and concepts enshrined in 2 international instruments; the Cartagena Protocol on Biosafety (Secretariat of the Convention on Biological Diversity (2000)) and the Risk
*Correspondence to: Huw D Jones; Email: [email protected] Received: 08/10/2015; Revised: 12/15/2015; Accepted: 12/15/2015 # Present affiliation: Prof. Translational Genomics for Plant Breeding; Institute of Biological, Environmental & Rural Sciences; Aberystwyth University; Gogerddan; Aberystwyth; Ceredigion, UK Color versions of one or more figures in this article can be found online at www.tandfonline.com/kgmc. 223
224
Jones
TABLE 1. Main genome editing technologies currently used in basic and applied research Category Two-component programmable endonucleases
Gene modulation technique Zinc-finger nuclease (ZFN)
Fok1 nuclease directed to gene target site via a series of protein domains designed to recognize a specific DNA sequence
Transcription activator-like effector nuclease (TALEN) Clustered regularly interspaced short palindromic repeats (CRISPRs)
Non-programmable endonucleases
Meganuclease (MN)
Non-endonuclease dependent homologous recombination
Oligonucleotide-directed mutagenesis (ODM)
Analysis Principles for Foods Derived from Biotechnology (Codex Alimentarius Commission [updated] 2014) supported by numerous outputs from the Organisation for Economic Co-operation and Development (OECD). However, some developing economies are still to formalise any framework for governance of GMOs and of those that have, significant differences already exist in regard to the triggers for GMO regulation and to the data requirements for risk assessment. Worryingly, these inconsistencies are set to widen further as products from emerging biotechnologies such as genome editing reach the market place. The lack of a common trigger for regulating the products of genome editing and the lack of guidance regarding the data package required for risk assessment may result in the same altered traits or identical genetic modifications escaping the GMO regulations in some countries while in others being encompassed by expensive procedures with unpredictable time lines. This lack of consistency risks stifling innovation, exacerbating already difficult international trade issues and more importantly, undermining consumer confidence in both the risk assessment process and the safety of the biotechnology products.
Mechanism
Cas-9 nuclease directed to target site via a short guide RNA. Easy to program for different targets and deactivated or ‘nickase’ variants available to give single-stranded breaks or enhancer/ repressor functions Naturally occurring restriction enzymes which also possess highly specific target sites, but are not programmable to different DNA sequences. Thus they are losing ground to the programmable, 2-component systems above Short, chemically-synthesized DNA/RNA oligonucleotides possessing similarity to the target sequence except for the few bases to be edited that are incorporated into the genome during repair
CURRENT FRAMEWORKS FOR REGULATING GMOS WERE NEVER DESIGNED FOR GENOME EDITING The Cartagena Protocol refers to plants generated via genetic modification as ‘Living Modified Organisms’ and defines then as ‘any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology’. This definition, often supplemented with the concept of ‘recombinant DNA techniques’ is now the foundation of the many national legal frameworks that include the process used to generate the new plant variety as part of the regulatory trigger such as the Argentina, Australia/New Zealand, Brazil, China, EU, Japan, Korea, Russia, Saudi Arabia, Thailand, Taiwan etc. Other countries, notably Canada and to some extent USA, incorporate the concept of a novel trait regardless of process but even these regulations treat crops resulting from recombinant DNA technology as requiring special attention. An emerging challenge for regulators is to accommodate new biotechnologies such as genome editing that do not fall neatly into the definitions of genetic modification laid down in existing
REGULATING GENOME EDITING
legislations. While a few countries have indicated how they intend to treat products of these technologies, many others have not. Uncertainties such as this need urgent clarification, because they have major implications for research, plant breeding and international trade in commodity crops (Jones, 2015b). For the risk assessment of conventional GMOs, there exists a broadly common comparative approach that assumes the non-GM counterpart has a history-of-safe-use and tests the substantial equivalence of the GM crop against its non-GM counterpart. Thus, after assessing the comparative risks of food/feed and environmental safety, if a novel food is found to be substantially equivalent to an existing food with a history-of-safe-use, it can be treated in the same manner and no additional safety concerns would be expected (FAO/WHO, 2000; OECD, 1993). However, even for conventional GMOs there are significant differences in the scope of regulatory oversight between countries. For example, in USA and Canada, GM events stacked by conventional breeding are assumed to be safe if the individual events are already authorised (except where a specific hazard has been identified). E.g. the US Environmental Protection Agency (EPA) requires separate review of the safety of the stack if the parental GMOs carry traits encoding biotoxins because insecticides may result in synergetic or adjuvant effects (EPA, 2001). However, in the EU and other countries including Argentina, Canada, EU, Japan, Korea and the Philippines, each stack, regardless of the traits involved is always considered as a new GMO which needs to be assessed and approved de novo, even though the individual events already have market approval ( EFSA 2011). Other countries take intermediate or case-by-case approaches; e.g., Australia/New Zealand, Brazil, Canada, China (EU FP6 CO-EXTRA Report Summary 2012). These different regulatory requirements reflect as much the policies and politics of the various nations than actual biological risks but result in significant cost implications for breeders, growers and food/feed processors and most importantly, serve to further confuse or undermine the confidence of consumers in the global oversight of biotechnology.
225
FITTING GENOME EDITING INTO EXISTING LEGISLATION In the EU, a GMO is defined in Directive 2001/18 as ‘an organism, with the exception of human beings, in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination’. The Directive goes on to exclude organisms obtained through mutagenesis, and cell/protoplast fusion (so long as it does not involve the use of recombinant nucleic acid molecules and the organisms could exchange genetic material through traditional breeding methods). The exclusion of mutation breeding from the GMO regulations is particularly significant when considering genome editing. The use of high-energy radiation or alkylating agents such as ethyl methanesulfonate (EMS) generate multiple genetic legions at random sites in the genome and lead to a range of mutation types that have been used in breeding since the 1940s (Maluszynski, 2001). For instance, the Ruby Red grapefruit variety is part of our daily diets and is just one of the more than 3,000 crop varieties listed in the Joint FAO/IAEA Mutant Variety Database as resulting from mutation breeding. The cellular activities occurring during mutation breeding can be divided into 2 distinct processes; the cleavage of DNA caused by the exogenous mutagen and the repair of that damage by the cell’s own nuclear regeneration machinery. Several factors determine the frequency and type of genetic damage including the species and tissue targeted the specific type and intensity of the mutagen used and the precise stage of the cell-cycle that it is applied. Various mechanisms exist in cells for the restoration of damaged DNA and these are not fully understood. The most cytotoxic form of DNA damage are double-stranded breaks (DSB) which are repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR) depending on whether undamaged homologous DNA is present to act as a repair template. NHEJ serves to ligate together the 2 DNA ends when no template is present but is prone to generating ‘mistakes’ (mutations) at the sites of joining both in the form of single nucleotide polymorphisms or gross
226
Jones
chromosomal rearrangements such as inversions and translocations. An alternative repair mechanism consists of a set of enzyme-controlled pathways that uses a DNA template that flanks the DSB for accurate repair, collectively known as HR. An understanding of the mechanisms of mutation breeding is relevant for 2 reasons. Firstly, as a technique, it is excluded from the scope of GMO regulatory frameworks of many regions of the world including the EU. Secondly, because a SDN can be considered as merely acting as a mutagen, albeit a more benign and highly-targeted one, to break DNA in the same way as high energy radiation or a chemical such as EMS does when used in mutation breeding. Any new plant variety resulting from this simple form of genome editing would contain no recombinant DNA but would possess a mutation undistinguishable from one that could be made through mutation breeding. A significant advantage of doing this with genome editing is that the SDN is highly specific, acting only on the pre-determined target sequence, as opposed to the many thousands of additional mutations that would result from achieving the same goal with mutation breeding. This is particularly evident in hexaploid wheat whose genetic redundancy allows it to carry a large mutation load with little effect. Various wheat populations that were mutated for TILLING experiments possess between 12 and 41 individual mutations per Mb of DNA (Chen et al., 2014) giving an average of 415,000 mutations in each hexaploid wheat plant (Uauy et al., 2009). Even if it was possible, it would take several years of careful crossing and selection to de-convolute these multiple mutations and derive an individual plant that possessed only the 1 or 2 desired alterations. However, by using genome editing, only the desired mutations are generated in the first instance. A recent publication on mildew resistance in wheat nicely illustrates the power of genome editing. MLO is a genetic locus suspected of supressing plant defense against mildew (Jorgensen, 1992). MLO is present in 6 copies in hexaploid wheat, one on each haploid genome, and all 6 must be simultaneously mutated to generate broad-spectrum mildew-resistance.
The probability of achieving this even in a large mutated TILLING population is vanishingly low and it would be laborious to use a backcross program to combine individual null alleles from different plants and remove other deleterious ones. However, Wang et al. (2014) designed TALENs proteins to target a specific site in the MLO sequence and reported the successful knockout of all 6 alleles of MLO in a single plant. This plant was completely resistant to mildew and forms a good candidate for further breeding. If this same goal was attempted by mutation breeding, the plants generated would not be defined a GMO in most countries, despite inevitably possessing many uncharacterised mutations. However, if the mildew-resistant plant made by TALEN-mediated mutations were to be marketed, there is considerable uncertainty whether or not it would be regulated as a GMO (see Fig. 1).
IS GENOME EDITING JUST GENETIC MODIFICATION BY ANOTHER NAME? SDN can be used in 3 different ways and this determines the answer to the question above. In the basic type l mode (SDN-1), a targeted nuclease is used simply as a mutagen to make a directed DSB which the cell’s inherently errorprone DNA repair mechanisms re-join using NHEJ. The exact sequence of final mutations cannot be predicted but several plants can be screened for individuals that possess desired SNPs or INDELs and selfed or backcrossed to remove all transgenes expressing the SND. The most common application of SDN-1 is to switch off a specific gene by screening for mutations resulting in a stop codon or a translational frame-shift. The resulting null segregant contains no recombinant DNA but retains the desired mutation. Although the mutation could be detected by molecular techniques including DNA sequencing, the changes are indistinguishable from the mutations that occur spontaneously or during mutation breeding. Mutation breeding, which is significantly more genetically disruptive, is already excluded from the vast majority of global GMO regulatory
REGULATING GENOME EDITING
oversight. Therefore, it is logical that plants resulting from SDN-1 genome editing should be excluded too. In a refinement to the basic SDN-1 mode, additional DNA in the form of a repair template can be introduced into the cell at the same time as the SDN. This can either be a template designed to introduce a few tens or hundreds of DNA bases which is considered a SDN-2, or a more extreme example where a complete functional transgene is target to a pre-determined safe harbour site in the genome which is designated the ‘SDN-3 mode’. The concept of a safe harbour site for a GMO is useful because it can be chosen to avoid disruption of a native gene and other so-called position effects. Multiple transgenes could then be stacked by re-transformation into the same locus so they segregate together during meiosis and save the significant complexity of breeding with unlinked transgene loci. This concept has been reported in maize using a 2-step process. Firstly, a parent line was generated that possessed a single-copy ‘trait landing pad’ (TLP) site at a convenient location in the maize genome. Then by retransformation, functional intact transgenes were reproducibly added into the TLP at an integration frequency of 5% (Ainley et al., 2013). This demonstrates the feasibility of stacking multiple transgenes into the same safe harbour site avoid unintended positional effects and generate single locus integration for ease of later breeding. SDN-3 applications of genome editing are similar to conventional GM and crop varieties developed using this technology marketed within the EU would clearly fall into the GMO regulations. The European Food Safely Authority (EFSA) has indicated that its current guidelines would be suitable for evaluating products of SDN-3 for risks to food and feed to the environment. They also considered, on a case-by-case basis, lesser amounts of event-specific data may be needed for the risk assessment of plants developed using the SDN3 technique (EFSA 2012). Genome editing in the SDN-2 mode is the most challenging for regulators and ironically will probably be the most useful for plant breeding because it enables editing of existing alleles to redefine their function or new ones to
227
be ‘knocked-in’. The editing could be designed either to copy existing useful alleles already in the gene pool but where introgression into elite commercial germplasm using conventional breeding would be impractical/too costly, or it could be used to create novel traits by completely redesigning gene function by, for instance, altering the substrate specificity of an enzyme. Importantly, both of these can be achieved directly in the elite lines of a breeding scheme saving many generations of backcrossing and avoiding potential problems of linkage drag. Genome editing via homology-dependent template repair of DSB has been achieved in many animal cell systems including human, rat, mouse zebra fish, drosophila C. elegans (reviewed by Gaj et al., 2013; Cox et al., 2015; Sander and Joung, 2014) and in plants (reviewed by Belhaj et al., 2015). There are good examples of genome editing using homology dependent repair templates in plants including the correction and inheritance of a non-functional gus reporter gene in Arabidopsis (Feng et al., 2014). The rice pds gene was successfully edited using a 72bp homologous oligonucleotide combined with a targeted Crispr Cas-9 to insert a 12bp edit in rice protoplasts (Shan et al., 2013). With the rate of research in this area, it is inevitable that agricultural applications of SDN-2 editing will soon be reported. However, there is a genome edited crop variety close to market generated using an alternative method known as oligonucleotide-directed mutagenesis (ODM). ODM does not use the Fok-1 nuclease to cleave DNA but instead uses short, chemically synthesized DNA/RNA oligonucleotides possessing similarity to the target sequence except for the few bases to be edited that are incorporated into the genome during repair. A novel crop Canola variety genome edited by Cibus using their proprietary Genome Repair Oligonucleotide technology to be tolerant to the herbicide Sulfonylurea received regulatory approval from Canadian Food Inspection Agency and Health Canada in March 2014 and is expected to be launched for cultivation in Canada in 2016 (Jones, 2015b). Although there are no commercial crop varieties currently marketed using CRISPR CAS-9, and there remains some way to go before the
228
Jones
intellectual property landscape is fully clarified, this particular technology is proving to be the most adaptable and has the potential to drive rapid advancements in plant breeding (Bortesi 2015, Xiong et al. 2015). In only a few years, CRISPR Cas-9 has been rapidly adopted by the research community as a routine method to knock-in and knock-out DNA sequences in animals and plants (eg Barrangou et al. 2015; Qi et al. 2013). Recent refinements of the basic technology are being reported, including the use of CAS-9 variants that generate singlestranded ‘nicks’ as well as deactivated forms of CAS-9 that modify expression of a targeted native gene by recruiting enhancers or repressors to specific locations within the regulatory regions of genes. Cas-9 nickase can be used to efficiently mutate genes without detectable damage at known off-target sites. This method is applicable for genome editing of any model organism and minimizes confounding problems of off-target mutations (Shen et al., 2014). In a different approach, 2-component transcriptional activator systems have been demonstrated in animal and plant cells consisting of a deactivated Cas-9 fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters (e.g., Cheng et al., 2013; Lowder et al., 2015). This represents a novel way to modulate gene expression by localizing enhancers and repressors to receptive promoter domains while leaving the primary DNA sequence unaltered.
EMERGING REGULATORY POSITIONS Of the national authorities that include a process-based trigger for biotechnology regulation, only Argentina has so far published detailed guidelines on how they proposed to manage crops made using genome editing. They regard a plant as a GMO when it possesses a new combination of genetic material created by the insertion of one or more genes or DNA sequences that are a part of a genetic construct have been inserted into the plant genome ‘Para que un cambio genetico sea considerado una nueva
combinacion de material genetico, se analizara si se ha producido una insercion en el genoma en forma estable y conjunta de UNO (1) o mas genes o secuencias de ADN que forman parte de una construccion genetica definida’. The means that null segregants possessing genetic changes resulting from NHEJ are not defined as a GMO. Additionally, to remove uncertainty that may stall early R&D in this area, the Argentinian National Advisory Commission on Agricultural Biotechnology (CONABIA) offer to provide a preliminary answer regarding how a new crop variety would be regulated in the design stage of the project (Ministerio de Agricultura, Ganaderia y Pesca, 2015). This is a well-considered and logical approach consistent with the potential hazards and the fact that conventional mutagenesis is excluded from GMO regulations. The USA and Canada, who incorporate the concept of a novel-trait into their regulatory frameworks, have indicated that simple genome editing that resulted in a new crop type containing no recombinant DNA would be regulated using a substantial equivalence approach and not automatically require the level of riskassessment typical of a conventional GMO. For example, the genome edited herbicide tolerant Canola developed by Cibus is the crop closest to commercialization and regulatory approval has been sought in Canada and USA. The Canadian Food Inspection Agency, who are responsible for regulating the environmental release of plant with a novel trait (PNT), scrutinized the Cibus application and determined that ‘this PNT does not present altered environmental risk nor, as a novel feed, does it present livestock feed safety concerns when compared to canola varieties currently grown and permitted to be used as livestock feed in Canada’ (Canadian Food Inspection Agency 2013). The USA has evolved a “Coordinated Framework for Regulation of Biotechnology” by adapting their existing food safety and crop protection laws to also cover biotechnology and incorporate concepts of both process- and trait-based triggers (Jones, 2015a). To date, the US Department of Agriculture have waived the regulations in place for genetically modified organisms for at least 2 products generated
FIGURE 1. Comparison of different breeding methods and whether they are regulated as GMOs in EU. Conventional GMOs (A), mutation breeding (B) and 2 stages of a new variety developed using SDN-1: early R&D phase (Ci) and final marketable variety (Cii)
REGULATING GENOME EDITING 229
230
Jones
using genome editing: a maize line that contains lower levels of the anti-nutritional compound phytate, and the Cibus herbicideresistant canola described above. In Europe, the governance of biotechnology rests at an EU level and the EC has been considering how to regulate new plant breeding techniques including genome editing for over 6 y. They have recently indicated that guidance will be provided during the first quarter of 2016. However, in the absence of any formal guidance from the EC, the competent authorities of several MS (including UK (ACRE), Germany (The Federal Office of Consumer Protection and Food Safety [BVL]), Belgium, Holland and most recently, Sweden have independently stated that they did not consider products of ODM such as the Cibus Canola as a GMO. This triggered protests from NGOs including TestBiotech, Greenpeace and FoE who published an open letter to the EC urging it to ensure (among other things) “that organisms produced by these new techniques will be regulated as genetically modified organisms under existing EU regulations (Directive 2001/18). This means that they will require a full risk assessment before any approval or authorisation is given” (Open letter to the Commission on New Genetic Engineering Methods, 2015). For conventional GMOs the EU has one of the most stringent and costly regulatory frameworks in the world with strict data requirements and high standards of risk assessment. Even so, the final approvals process is slow and unpredictable. The only GM variety currently approved for cultivation in the EU is the insectresistant maize MON810 which was authorized in 1998 under an earlier adoption system. No further approvals have been made despite applications with positive risk assessments from the European Food Safety Authority (EFSA). This is a direct result of the voting patterns of member states (MS) who fail to achieve a qualified majority at the committee stage. We are yet to see the effect of the recently passed national opt-out clause for cultivation but it is likely that it will empower the EC to authorize applications that have been outstanding for many years. There are some MS who consistently vote against adoption of
new GMOs regardless of the positive opinion published by EFSA, e.g., Austria, Denmark, Greece, Lithuania and Luxemburg (Sabalza et al., 2011), and this sends a strong signal that crops made using genome editing techniques would be also considered unacceptable by these MS, perhaps even after a full risk assessment.
IS EXISTING LEGISLATION FIT FOR PURPOSE? Agriculture is facing considerable challenges from many directions. We need safe and nutritious crop types that are predictably-productive under increasingly extreme climatic stresses and pressures from pests and diseases. We must achieve this with sustainable agricultural systems that minimize soil degradation, water use and environmental pollution. Plant breeding has always, and will continue to play a crucial role in meeting these challenges. However, this can only happen with appropriate regulatory oversight that stimulates research and innovation to produce safe and better plant varieties. Plant breeding uses a spectrum of technologies that are themselves evolving at a rapid pace. We need regulatory frameworks that are risk- and evidence-based, proportionate and globally-harmonized. Importantly we need them to be transparent and able to adapt to future changes. For some biotechnology innovations with no major hazards identified, a well-managed post-market system may suffice. Existing legislation that incorporates ambiguous definitions of genetic modification and that excludes from scope, techniques that are more genomically disruptive than techniques it captures, are broadly unfit for future purpose. In the long term, the primary legislation must be redrafted to facilitate a process of hazard identification and risk assessment of the actual crop cultivated and/or the food/feed consumed. The processes by which the new genotype is produced are not irrelevant, but neither should they be a primary trigger for regulation. However, this will take many years of discussion and in the meantime; innovation is already being stifled by uncertainty in some countries while varieties made using new breeding
REGULATING GENOME EDITING
techniques are reaching the marketplace in others. The recent decree by the Argentinian competent authority demonstrates how a process-based legislation can be easily adapted to reasonably accommodate at least the recent developments in genome editing. By mid-2016 we should know whether the EC and perhaps other competent authorities will follow the Argentinian lead or head down a more restrictive path by defining SDN-mediated mutations as GMOs requiring a full risk assessment. Apart from being illogical, the latter course would exacerbate the already complex trade and labeling issues for bulk commodity shipments into EU (Jones, 2015b).
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST Prof. Jones is a scientist using genome editing in functional genomics research. No potential conflicts of interest were disclosed.
FUNDING Rothamsted Research receives support from the Biotechnological and Biological Sciences Research Council (BBSRC) of the UK as part of the 20:20 Wheat Ò Programme. Opinions and views expressed in this publication are those of the author and do not necessarily represent those of Rothamsted Research or the European Food Safety Authority. REFERENCES Ainley WM, Sastry-Dent L, Welter ME, Murray MG, Zeitler B, Amora R, Corbin DR, Miles RR, Arnold NL, Strange TL, et al. Trait stacking via targeted genome editing. Plant Biotechnol J 2013; 11:1126–1134; PMID:23953646; http://dx.doi.org/10.1111/pbi.12107 Anonymous. Seeds of change. Nature 2015; 520: 131–132. Barrangou R, Birmingham A, Wiemann S, Beijersbergen RL, Hornung V, Smith Anja VB. Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference Nucleic Acids Res 2015; 43 (7):3407–3419; PMID:25800748; http://dx.doi.org/ 10.1093/nar/gkv226
231
Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V. Editing plant genomes with CRISPR/Cas9. Currnt Opin Biotechnol 2015; 32:76–84; PMID:25437637; http://dx.doi.org/10.1016/j.copbio.2014.11.007 Bortesi L, Fischer R. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 2015; 33:41–52; PMID:25536441; http://dx.doi.org/10.1016/ j.biotechadv.2014.12.006 Canadian Food Inspection Agency. Determination of the safety of Cibus Canada Inc.’s Canola (Brassica napus L.) Event 5715. 2013. Available via http://www.inspection.gc.ca/ plants/plants-with-novel-traits/approved-under-review/deci sion-documents/dd-2013-100/eng/1427383332253/ 1427383674669 Chen L, Hao L, Parry MAJ, Phillips AL, Hu Y-G. Progress in TILLING as a tool for functional genomics and improvement of crops. J Integr Plant Biol 2014; 56:425–443; PMID:24618006; http://dx.doi.org/ 10.1111/jipb.12192 Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan S, Shivalila CS, Dadon DB, Jaenisch, R. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res 2013; 23:1163–1171; PMID: 23979020; http://dx.doi.org/10.1038/cr.2013.122 CO-EXTRA Report Summary. Review on the world wide authorization of gene stacked GMOs in relation to commercial gene stacks and wild gene stacks and the basis for definition of corresponding unique genetic markers. 2012. Deliverable D6.4 EU CORDIS http:// cordis.europa.eu/result/rcn/53012_en.html Codex Alimentarius Commission. PROCEDURAL MANUAL. Joint FAO/WHO Food Standards Programme 22nd edition 2014. FAO/WHO http://www. codexalimentarius.org/ Cox DBT, Platt RJ, Zhang F. Therapeutic genome editing: prospects and challenges. Nat Med 2015; 21:121–131. EFSA, 2011; PMID:25654603; http://dx.doi.org/ 10.1038/nm.3793 EFSA. Guidance on selection of comparators for the risk assessment of genetically modified plants and derived food and feed. EFSA Journal 2011; 9(5):2149 [20 pp.]; Available online: www.efsa.europa.eu/efsajournal EFSA. Scientific opinion addressing the safety assessment of plants developed using Zinc Finger Nuclease 3 and other Site-Directed Nucleases with similar function. EFSA J 2012; 10(10):2943. [31 pp.] . Available online: www.efsa.europa.eu/efsajournal EPA. Environmental Protection Agency, USA. Plantincorporated protectants; final rules and proposed rule. Federal Register 2001; 66(139):37771–37817. http:// www.epa.gov/pesticides/biopesticides/pips/pip_rule. pdf FAO/WHO. Report of a Joint FAO/WHO Expert Consultation on foods derived from biotechnology – safety aspects of genetically modified foods of plant origin.
232
Jones
Geneva, Switzerland: WHO Headquarters; 29 May–2 June 2000. Available from http://www.fao.org/filead min/templates/agns/pdf/topics/ec_june2000_en.pdf Feng Z, Mao Y, Xu N, Zhang B, Wei P, Yang D-L, Wang Z, Zhang Z, Zheng R, Yang L, et al. Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci U S A 2014; 111:4632–4637. Gaj T, Gersbach CA, Barbas CF, III. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trend Biotechnol 2013. 31:397–405; PMID:23664777; http://dx.doi.org/10.1016/j.tibtech.2013.04.004 GM Watch. Genome editing is GM by another name. 2014. http://www.gmwatch.org/news/archive/2014/ 15546-genome-editing-gm-by-another-name Jones HD. Challenging regulations: Managing risks in crop biotechnology. Food Ener Security 2015a; 4:87–91. Jones HD. Regulatory uncertainty over genome editing. Nat Plants 2015b; 1:14011; http://dx.doi.org/10.1038/ nplants.2014.11 Jorgensen JH. Discovery, characterization and exploitation of mlo powdery mildew resistance in barley. Euphytica 1992; 63:141–152; http://dx.doi.org/ 10.1007/BF00023919 Kr€amer, L. (2015) Legal questions concerning new methods for changing the genetic conditions in plants. http://www.testbiotech.org/en/node/1342 Lowder LG, Zhang D, Baltes NJ, Paul JW, Tang X, Zheng X, Voytas DF, Hsieh T-F, Zhang Y, Qi Y. A CRISPR/ Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol 2015; 169 (2):971–985; PMID:26297141; http://dx.doi.org/ 10.1104/pp.15.00636 Maluszynski M. Officially released mutant varieties The FAO/IAEA database. Plant Cell Tissue Organ Cult 2001; 65:175–177; http://dx.doi.org/10.1023/ A:1010652523463 MINISTERIO DE AGRICULTURA, GANADERIA Y PESCA. Resolucion 173/2015 12-may-2015. http://info leg.mecon.gov.ar/infolegInternet/verNorma.do;jses sionid=AC1747549EAABF7CFC3644F02F41B15A? id=246978 Natural News. Biotech companies to use "genome editing" to circumvent GMO regulations. 20150 http:// www.naturalnews.com/048344_GMO_regulations_ge nome_editing_biotech_companies.html OECD. Safety evaluation of foods derived by modern biotechnology; Concepts and principles. Organisation for Economic Co-operation and Development. Paris, France: OECD Publications; 1993. Available from http://www.oecd.org/science/biotrack/41036698.pdf Open letter to the Commission on New Genetic Engineering Methods. 26 January 2015. Available from http://www.test biotech.org/sites/default/files/20150126%20Open%20Let ter%20on%20new%20GM%20technologies.pdf
Podevin N, Devos Y, Davies HV, Nielsen KM. Transgenic or not? No simple answer! New biotechnologybased plant breeding techniques and the regulatory landscape. Embo Rep 2012; 13:1057–1061; PMID: 23154464; http://dx.doi.org/10.1038/embor.2012.168 Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013; 152:1173–1183; PMID: 23452860; http://dx.doi.org/10.1016/j.cell.2013.02.022 Sabalza M, Miralpeix B, Twyman RM, Capell T, Christou P. EU legitimizes GM crop exclusion zones. Nat Biotechnol 2011; 29:315–317; PMID:21478843; http://dx. doi.org/10.1038/nbt.1840 Sander JD, Joung JK. CRIRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 2014. 32:347–355; PMID:24584096; http://dx.doi.org/ 10.1038/nbt.2842 Secretariat of the Convention on Biological Diversity. Cartagena Protocol on Biosafety to the Convention on Biological Diversity: Text and annexes. Montreal, Canada: Secretariat of the Convention on Biological Diversity; 2000. Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu J-L, et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotech 2013; 31:686–688; http://dx.doi.org/ 10.1038/nbt.2650 Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang X, et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Meth 2014; 11(4):399– 402; http://dx.doi.org/10.1038/nmeth.2857 Mutant Varieties database. Joint FAO/IAEA programme nuclear techniques in food and agriculture. International Atomic Energy Agency (IAEA). Vienna International Centre, PO Box 100, A-1400 Vienna, Austria, http://mvd.iaea.org/ Uauy C, Paraiso F, Colasuonno P, Tran RK, Tsai H, Berardi S, Comai L, Dubcovsky J. A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC Plant Biol 2009; 9:115; PMID:19712486; http://dx.doi.org/ 10.1186/1471-2229-9-115 Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qui JL. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology 2014; 32: 947–951; http://dx.doi.org/10.1038/nbt.2969 Whelan AI, Lema MA. Regulatory framework for gene editing and other New Breeding Techniques (NBTs) in Argentina. 2015. GM Crops & Food; http://dx.doi.org/ 10.1080/21645698.2015.1114698 Xiong J-S, Ding J, Li Y. Genome-editing technologies and their potential application in horticultural crop breeding. Horticult Res 2015; 2:15019; PMID: 26504570; http://dx.doi.org/10.1038/hortres.2015.19
