1. New Plant Breeding Techniques: Legal Classification & Regulatory Implications Drew L. Kershen Earl Sneed Centennial Prof. Emeritus Crop Bioengineering Consortium Iowa State University November 5, 2013

2. Site-Directed Nuclease Techniques Meganucleases (MNs) – Restriction enzyme source: many species Zinc-finger Nucleases (ZFNs) – Protein source: many species (including humans) – Synthetic biology – customized ZFNs Transcription Activator-Like Effector Nucleases (TALENs) – Effector source: Xanthomonas genus Clustered regularly interspaced short palindromic repeats (CRISPRs) – Cas9 sources: Streptococcus thermophilus & Staphylococcus aureus Construct components – Promoter (CaMV 35S); Terminator (NOS); nuclear localization signal (SV40 – simian virus 40)

3. United States Law USDA-APHIS Plant Protection Act (2000, as amended), 7 U.S.C. §§ 7701- 7772 Regulated article. Any organism which has been altered or produced through genetic engineering, if the donor organism, recipient organism, or vector or vector agent belongs to any genera or taxa designated in § 340.2 and meets the definition of plant pest, or is an unclassified organism and/or an organism whose classification is unknown, or any product which contains such an organism, or any other organism or product altered or produced through genetic engineering which the Administrator, determines is a plant pest or has reason to believe is a plant pest. Excluded are recipient microorganisms which are not plant pests and which have resulted from the addition of genetic material from a donor organism where the material is well characterized and contains only non-coding regulatory regions. 7 C.F.R. 340.1

4. United States Law USDA-APHIS Plant pest. Any living stage (including active and dormant forms) of insects, mites, nematodes, slugs, snails, protozoa, or other invertebrate animals, bacteria, fungi, other parasitic plants or reproductive parts thereof; viruses; or any organisms similar to or allied with any of the foregoing; or any infectious agents or substances, which can directly or indirectly injure or cause disease or damage in or to any plants or parts thereof, or any processed, manufactured, or other products of plants. 7 C.F.R. 340.1 Organism. Any active, infective, or dormant stage or life form of an entity characterized as living, including vertebrate and invertebrate animals, plants, bacteria, fungi, mycoplasmas, mycoplasma-like organisms, as well as entities such as viroids, viruses, or any entity characterized as living, related to the foregoing. 7 C.F.R. 340.1

5. United States Law USDA-APHIS § 340.2 Groups of organisms which are or contain plant pests and exemptions. (a) Groups of organisms which are or contain plant pests. The organisms that are or contain plant pests are included in the taxa or group of organisms contained in the following list. Within any taxonomic series included on the list, the lowest unit of classification actually listed is the taxon or group which may contain organisms which are regulated. Organisms belonging to all lower taxa contained within the group listed are included as organisms that may be or may contain plant pests, and are regulated if they meet the definition of plant pest in § 340.1 4 4 Any organism belonging to any taxa contained within any listed genera or taxa is only considered to be a plant pest if the organism “can directly or indirectly injure, or cause disease, or damage in any plants or parts thereof, or any processed, manufactured, or other products of plants.” Thus a particular unlisted species within a listed genus would be deemed a plant pest for purposes of § 340.2, if the scientific literature refers to the organism as a cause of direct or indirect injury, disease, or damage to any plants, plant parts or products of plants. (If there is any question concerning the plant pest status of an organism belonging to any listed genera or taxa, the person proposing to introduce the organism in question should consult with APHIS to determine if the organism is subject to regulation.) N OTE : Any genetically engineered organism composed of DNA or RNA sequences, organelles, plasmids, parts, copies, and/or analogs, of or from any of the groups of organisms listed below shall be deemed a regulated article if it also meets the definition of plant pest in § 340.1.

6. United States Law USDA-APHIS § 340.3 Notification for the introduction of certain regulated articles – (b) Regulated articles eligible for introduction under the notification procedure. Regulated articles which meet all of the following six requirements and the performance standards … (6) The plant has not been modified to contain the following genetic material from animal or human pathogens: – (i) Any nucleic acid sequence derived from an animal or human virus, or – (ii) Coding sequences whose products are known or likely causal agents of disease in animals or humans.

7. United States Law USDA-APHIS Regulated Article Letters of Inquiry – http://www.aphis.usda.gov/biotechnology/am_i_reg.shtml http://www.aphis.usda.gov/biotechnology/am_i_reg.shtml – Transformation method – Construct (each element – promoter, gene, terminator and source from which derived) – Recipient Organism – Donor Organism – Trait (phenotype) Nineteen letters of inquiry posted through 13 March 2013 – One letter on ZFN-1; one letter on MN-1 Not regulated articles – Knockout effect – No Letters of Inquiry on TALENs or CRISPRs

8. USDA-APHIS Analysis – The Plant Pest Trigger Transformation method – Agrobacterium tumefaciens (yes); biolistics (no); electroporation (no) TALENs – Binding domain from Xanthomonas – Presumptively a “regulated article” 7 C.F.R. 340.2 CRISPR – Binding domain from Streptococcus thermophilus or pyogenes – not listed in 340.2 Yogurt and cheese – CRISPR – Nuclear Localization signal from Staphylococcus aureus (human pathogen) – not listed in 340.2 but 340.3(b)(6)(ii) – Constructs – several components derived from viruses or bacteria

9. USDA-APHIS Analysis – The Classification No Regulated Article Letters of Inquiry – CRISPR appears better than TALENS for regulatory avoidance – CRISPR (Streptococcus t.) compared to CRISPR (Staphylococcus a.) – Need Biolistic transformation & NLS, promoters & terminators from non pathogen sources to avoid the underlying plant from being regulated Null segregants from the construct, not regulated article TALENs – avoidance approaches – Binding domain by synthetic biology (customized chimeric binding domain) – not an organism? – an analog of a listed 340.2 pest? – Null-segregant plants – back crossing with confirmation that no Xanthomonas DNA/RNA Several Letters of Inquiry on null segregant plants – Underlying plant regulated (keep in laboratory) – Offspring plants, not regulated article

10. United States Law Environmental Protection Agency Federal Insecticide Fungicide Rodenticide Act (as amended through 2012), 7 U.S.C. §§ 121-136y PESTICIDE.—The term ‘‘pesticide’’ means (1) any substance or mixture of substances intended for preventing, destroying, repelling, or mitigating any pest, (2) any substance or mixture of substances intended for use as a plant regulator, defoliant, or desiccant, and (3) any nitrogen stabilizer, … 7 U.S.C § 136(u) PLANT REGULATOR.—The term ‘‘plant regulator’’ means any substance or mixture of substances intended, through physiological action, for accelerating or retarding the rate of growth or rate of maturation, or for otherwise altering the behavior of plants or the produce thereof, … 7 U.S.C. § 136(v) Analysis – The intended purpose of the deletion, recombination or insertion, not the SDN method per se – EPA has pondered expanded FIFRA regulation of GE plants, e.g., RNAi and as “new chemicals” using Toxic Substances Control Act (TSCA).

11. United States Law Food & Drug Agency (FDA) Food, Drug & Cosmetic Act, 21 U.S.C. §§ 341-350l-1 (as amended to 2012) – “Voluntary” Consultation – Substantial Equivalence (149 foods to date) Data analysis of the food -- TALENs – Generally Recognized as Safe (GRAS) CRISPR Cas9 and binding domain from a bacterium used to produce yogurts and cheese – assuredly GRAS

12. European Union Law 2001/18/EC – Deliberate Release Article 2 -- Definitions – For the purposes of this Directive: – (1) "organism" means any biological entity capable of replication or of transferring genetic material; – (2) "genetically modified organism (GMO)" means 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; – Within the terms of this definition: – (a) genetic modification occurs at least through the use of the techniques listed in Annex I A, part 1; – (b) the techniques listed in Annex I A, part 2, are not considered to result in genetic modification; Article 3 – Exemptions – This Directive shall not apply to organisms obtained through the techniques of genetic modification listed in Annex 1B.

13. European Union Law 2001/18/EC – Deliberate Release ANNEX I A -- TECHNIQUES REFERRED TO IN ARTICLE 2(2) PART 1: Techniques of genetic modification referred to in Article 2(2)(a) are inter alia: – (1) recombinant nucleic acid techniques involving the formation of new combinations of genetic material by the insertion of nucleic acid molecules produced by whatever means outside an organism, into any virus, bacterial plasmid or other vector system and their incorporation into a host organism in which they do not naturally occur but in which they are capable of continued propagation; – (2) techniques involving the direct introduction into an organism of heritable material prepared outside the organism including micro-injection, macro- injection and micro-encapsulation; – (3) cell fusion (including protoplast fusion) or hybridisation techniques where live cells with new combinations of heritable genetic material are formed through the fusion of two or more cells by means of methods that do not occur naturally. PART 2: Techniques referred to in Article 2(2)(b) which are not considered to result in genetic modification, on condition that they do not involve the use of recombinant nucleic acid molecules or genetically modified organisms made by techniques/methods other than those excluded by Annex I B: – (1) in vitro fertilisation, – (2) natural processes such as: conjugation, transduction, transformation, – (3) polyploidy induction.

14. European Union Law 2001/18/EC – Deliberate Release ANNEX IB--TECHNIQUES REFERRED TO IN ARTICLE 3 – Techniques/methods of genetic modification yielding organisms to be excluded from the Directive, on the condition that they do not involve the use of recombinant nucleic acid molecules or genetically modified organisms other than those produced by one or more of the techniques/methods listed below are: (1) mutagenesis, (2) cell fusion (including protoplast fusion) of plant cells of organisms which can exchange genetic material through traditional breeding methods.

15. The Cartagena Protocol Super-imposed on European law Binding in 167 countries Regulates “Modern biotechnology” – “In vitro nucleic acid techniques, including recombinant deoxyribonucleic acid (DNA) and direct injection of nucleic acid into cells or organelles” What makes the CP so nefarious is that nothing says the nucleic acid must remain in the cell – Null segregants are still covered

16. 2001/18/EC Analysis No mechanism in the EU directly comparable to USDA- APHIS Regulated Article Letters of Inquiry – Working groups (advisory) – Discussion of ZFNs, but not TALENs or CRISPR Annex IA Part One – “inter alia” – TALENs and CRISPR probably covered New introduced genetic material Technique used for insertion of genetic material prepared outside the organism Null segregant plants – very unclear under EU law – Covered by Cartagena Protocol if will be released to the environment Like mutagenesis? Especially SDN-1 techniques – Precautionary Principle Whereas Clause (8)

17. European Union Law Reg. (EC) No. 1829/2003 (22 Sept 2003) Whereas Clause 16) -- This Regulation should cover food and feed produced ‘from’ a GMO but not food and feed ‘with’ a GMO. The determining criterion is whether or not material derived from the genetically modified source material is present in the food or in the feed. Processing aids which are only used during the food or feed production process are not covered by the definition of food or feed and, therefore, are not included in the scope of this Regulation. Article 2: Definitions -- 10. ‘produced from GMOs’ means derived, in whole or in part, from GMOs, but not containing or consisting of GMOs;

18. Reg. (EC) No. 1829/2003 Analysis EFSA opinion of October 2012 – p. 2 “With respect to the genes introduced, the SDN-3 technique does not differ from transgenesis or from the other genetic modification techniques currently used with respect to any hazards associated with introduced genes. The SDN-3 technique can be used to introduce transgenes, intragenes, or cisgenes.” Null segregant plants – produced “with” (not covered) or “from” (covered by regulations)? – Wine (GM yeast) is “with”; Wine (GM grapes) is “from”; Wine from GM rootstalk but non-GM scion is unclear and undecided – My opinion on null segregant plants: likely produced “from” GMO plants (covered) Reg. (EC) No. 1830/2003 – labeling and traceability – Applies once 1829/2003 determines something to be a GMO food or feed

19. Exceptions Vitamins extracted from GM soy are covered Vitamins extracted from GM microorganisms are exempt The point is that Europeans do make exceptions when convenient for them

20. Legal Issues arising from differences between EU & US laws/regulations Regulatory costs to get multiple approvals Asynchronous approval Legal Liability for impact on international trade with Europe – Voluntary Contracts – Coexistence World Trade Organization Treaties – Sanitary and Phytosanitary Treaty (SPS) – Technical Barriers to Trade Treaty (TBT)

21. Policy Issue New Plant Breeding Techniques NAS (1987): “Several conclusions can be drawn from this review of the relationship between traditional genetic manipulation techniques and the R-DNA techniques developed during the last 15 years, and of the experience gained from the application of each: – There is no evidence that unique hazards exist either in the use of R-DNA techniques or in the movement of genes between unrelated organisms. – The risks associated with the introduction of R-DNA engineered organisms are the same in kind as those associated with the introduction of unmodified organisms and organisms modified by other methods. – Assessment of the risks of introducing R-DNA engineered organisms into the environment should be based on the nature of the organism and the environment into which it is introduced, not on the method by which it was produced.”

22. Policy Issue New Plant Breeding Techniques ACRE (2013): Executive Summary – “Our understanding of genomes does not support a process-based approach to regulation. The continuing adoption of this approach has let to, and will increasingly lead to, problems. This includes problems of consistency, i.e. regulating organisms produced by some techniques and not others irrespective or their capacity to cause environmental harm.” – “Our conclusion, that the EU’s regulatory approach is not fit for purpose for organisms generated by new technologies, also applies to transgenic organisms produced by ‘traditional’ GM technology. … the potential for inconsistency is inherent because they may be phenotypically identical to organisms that are not regulated.”

23. References Lusser, M. & Davies, H.V., (2013) Comparative regulatory approaches for groups of new plant breeding techniques, New Biotechnol. (in press) Minikel, E. (2013) TALENs and ZFNs, www.cureffi.org www.cureffi.org Pennisi, E. (2013) The CRISPR Craze, Science 341:833-836 Podevin, N. et. al, (2013) Site-directed nucleases: a paradigm shift in predictable, knowledge-based plant breeding, Trends in Biotechnology, 31:375- 383.

24. References European Commission, Directorate-General Environment, Working Group on the Establishment of a List of Techniques Falling under the Scope of Directive 2001/18/EC (unpublished, available as a leaked document, August 2013) Lusser, M. et al., (2011) New Plant Breeding Techniques: State-of-the-art and prospects for commercial development (JRC Scientific and Technical Reports). EFSA Panel on GMOs: Scientific opinion addressing the safety assessment of plants developed using ZFNs-3 and other SDN with similar functions, EFSA Journal 2012, 10(10:2943 (31 pp.)

25. References National Academy of Science (USA), Introduction of Recombinant DNA- Engineered Organisms into the Environment: Key Issues (1987) Advisory Committee on Releases to the Environment (ACRE), Report 2: Why a modern understanding of genomes demonstrates the need for a new regulatory system for GMOs. (Sept. 2013)

26. Wayne Parrott, Ph.D., Professor, Plant Breeding and Genomics, Crop & Soil Sciences, University of Georgia is coauthor of this PowerPoint. Thank you. dkershen@ou.edu