The slides for the FAO seminar that took place in Budapest, 08.01.2016.

1. Regulatory definition of GMO in relation to
organisms developed through genome editing
Seminar, 8 January 2016
FAO offices Budapest, Hungary,
Piet van der Meer
• Department of Plant Biotechnology and BioInformatics, Faculty of Sciences,
Ghent University, Belgium
• Department of European, Public and International Law, Faculty of Law,
Ghent University, Belgium
• Faculty of Science and Bio-Engineering Sciences,
Free University of Brussels (VUB), Belgium

2. Topics
• Modern Biotechnology and Biosafety – historical highlights
• Biosafety Regulations
• Definitions
• Genome editing

3. Modern Biotechnology – Historical highlights
1850 - 1900: Gregor Mendel and others: the rules of inheritance
1900 - 1920: new techniques, embryo rescue, hybrids,
radiation/chemical induced mutagenesis.
1920 - 1953 Discovery of ‘genes’, their building
blocks, DNA helix.
1950 - 1970: Discovery of restriction enzymes, DNA ligases, etc.
1972: First publications on ‘recombinant DNA’; Jackson, D.; Symons,
R.; Berg, P.
1983: First transgenic plants, University of Gent

4. Conventional breeding and rDNA
Conventional
breeding
x
“elite”
variety
Related
variety
rDNA 
any
gene
source

5. Biosafety – Historical highlights
1974: ‘Berg Letter’ published in Science, anticipated benefits and
potential risks, calling for a voluntary moratorium.
1975: Asilomar Conference: technique not inherently risky, but
potential for novel combinations, safety assessed ‘case by
case’, end of moratorium.
1976: US-NIH Guidelines for rDNA micro-organisms in laboratories
1978: Other countries follow, e.g. UK (1978), Netherlands (1979)
1986: OECD “Blue Book”, rDNA safety recommendations; key
concepts for risk assessment and risk management

6. Biosafety – Historical highlights
1986 - 1990: discussions expand:
• Technical aspects of the discussion broadened to include
– plants and animals
– releases into the environment
– other molecular methods to make novel genetic combinations
• Discussions increasingly international:
– OECD, UN (Agenda 21, CBD), EU, CoE, etc
• Discussions increasingly get a regulatory character

7. Biosafety Regulations
1986: US coordinated framework for regulation of biotechnology
(USDA, EPA, FDA)
1990: EU Directives on Contained Use and Release of GMOs into the
Environment
1996: Canada (CFIA, Health Canada)
2000: Cartagena Protocol on Biosafety - transboundary movement
of LMOs to countries that do not yet have biosafety
frameworks

8. Biosafety Regulation - Cycle
• Developing regulations, e.g.
– Objective, e.g. internal market, safety
– Scope: e.g. contained use, release, placing on the market of
GMOs
– Regulatory mechanism(s): e.g. conditions, authorisations,
notifications
• Implementing, e.g. risk assessment, decision making
• Reviewing, evaluating, and assessing
– Review of legal text: e.g. scope and definition clear vis a vis new
developments such as NBTs?
– Evaluation: is implementation effective and efficient?
– Assessment of impacts on sectors such as R&D and farming

9. Definitions

10. Definitions - History
1986: OECD “Blue Book”, rDNA safety recommendations:
Definition of genetic manipulation and recombinant DNA
varies in detail between countries, but rDNA organisms are
described as constructed by introducing DNA from a “donor”
organism into a “recipient”
E,g: UK: formation of new combinations of heritable material
by the insertion of nucleic acid molecules, produced outside
the cell, via vector system so as to allow their incorporation
into a host organism in which they do not naturally occur but
in which they are capable of continued propagation.

11. Definitions – Historical highlights
Debate as of the mid 80s: which organisms should be subject to prior
risk assessment?
Observations put forward:
• Conventional breeding and rDNA can both result in novel
genetic combinations that can cause adverse effects
• rDNA techniques themselves do not confer risky characteristics
• rDNA can make a broader range of novel combinations
Options discussed:
• Include conventionally produced organisms?
• Focus on ‘risky’ organisms?
• Focus on ‘novel’ organisms ?

12. Which organisms subject to prior risk assessment?
Considerations:
• Conventional organisms: is there an unmet need to include
them?
• Risky organisms: how to assess which are the risky organisms?
• Novel organisms: How to define “novel”?
– Genotypic and/or phenotypic?
– ‘Does not exist’ or ‘is unlikely to happen’?
• Scope and definitions of existing regulations
>> Different approaches in different systems, most focus on novelty

13. United States Department of Agriculture
Biotechnology Regulatory Services
Animal and Plant Health Inspection Service
 Department of Agriculture (USDA-APHIS-BRS)
• PPA: Protecting against damage from plant pests and noxious
weeds
 Environmental Protection Agency (EPA)
• FIFRA: Regulating the safe use of pesticides
• FFDCA: Setting allowable levels of pesticides in food
• TSCA: Regulating toxic substances
 Food and Drug Administration (FDA)
• FFDCA: Regulating safety of food, drugs, and cosmetics
US Biotechnology Regulation under
the Coordinated Framework

14. Definitions – USA
USDA (1987, based on FPPA and PQA, which were later incorporated
into the Plant Protection Act, which includes also noxious weeds):
Regulatory trigger:
– the use of genetic engineering in combination with
– the use of plant pest as recipient, donor or vector, in the
transformation process.

15. Definitions – USA
FDA: Authority based on Federal Food, Drug and Cosmetic Act
(FFDCA)
Guidance in the Federal Register of 1992
“The 1992 policy applies to foods developed from new plant
varieties, including varieties that are developed using
recombinant deoxyribonucleic acid (rDNA) technology (which is
often referred to as "genetic engineering" or "biotechnology").
This guidance document refers to foods derived from plant
varieties that are developed using rDNA technology as
"bioengineered foods."

16. Definitions – USA
EPA: authority based on the Federal Insecticide, Fungicide and
Rodenticide Act (FIFRA), FFDCA, and Toxic Substances Control Act
(TSCA)
Plant-Incorporated Protectant (PIP, 2001) - a pesticidal substance
that is intended to be produced and used in a living plant, or in the
produce thereof, and the genetic material necessary for the
production of such a pesticidal substance. It also contains any inert
ingredient contained in the plant, or produce thereof."
PIPs developed through conventional breeding will remain exempt
from all FIFRA and FFDCA requirements, with the exception of
adverse effects reporting requirements for manufacturers.

17. Definitions - EU
EU (1990):
GMO means an organism in which the genetic material has been
altered in a way that does not occur naturally;
• genetic modification occurs at least through the use of:
– rDNA techniques;
– direct introduction of heritable material;
– fusion techniques.

18. Definitions – Canada
CFIA (1996, based on the Seeds Act and Feed Act) and
Health Canada (Food and Drug Act)
Plants with novel traits fall under the regulatory trigger of the Seeds
regulations if:
– They have a novel trait that was not present prior to December
1996 in germplasm of the same species in Canada, and
– that plant poses an environmental risk.

19. Definitions – CPB
Cartagena Protocol on Biosafety: (2000)
• "Living modified organism" means any living organism that
possesses a novel combination of genetic material obtained
through the use of modern biotechnology;
• Modern biotechnology" means the application of:
- In vitro nucleic acid techniques, including recombinant
deoxyribonucleic acid (DNA) and direct injection of nucleic acid
into cells or organelles, or
- Fusion of cells beyond the taxonomic family,
that overcome natural physiological reproductive or
recombination barriers and that are not techniques used in
traditional breeding and selection;

20. Definitions - EU
Question whether and to what extent organisms developed through
NBTs such as genome editing fall under the definition of GMO.
Discussion: “Altered in a way…that does not occur naturally…?”
Refers to:
• Process/technique ?
• The product/resulting organisms ?
• Both?
[ See also Callebaut 2015]

21. Definitions - EU
Legal clues: The law itself, explanatory notes, Jurisprudence, doctrine
The final word is with the ECJ.
Interpretation by the ECJ: “the wording, general scheme and spirit”
• Literal interpretation, text of the definition and the annexes
When the wording in itself is not conclusive:
• Systemic interpretation (general scheme), internal and external
consistency (e.g. CPB)
• Dynamic interpretation, e.g. teleological or functional, see ERA,
pushing an interpretation ‘to its limits’
[See also Callebaut 2015]

22. Definitions - EU
Conclusion
For an organisms to fall under the EU definition of GMO, the use of
certain techniques and a certain level of novelty of the resulting
combinations of genetic material are required. E.g. small point
mutations obtained through genome editing would not result in a
GMO.

23. Definitions - EU
Views of others and other views:
• European Commission:
– formulating views by taking into account reports of a WGNBT
(2012), EFSA, JRC and Legal Services
– Confirmed in reply to MEPs, both process and product relevant
• D and SE authorities have formally concluded that certain
organisms developed through ODM and CRISPR are not GMOs
• D, UK, IRL and have confirmed in a letter to the EC that the EU
GMO definition relies on both the technique and the resulting
organism.
• Some NGOs adhere to a process based interpretation arguing
that “these technologies are considered likely to bring negative
impacts on human and/or environmental health”.

24. Definitions - EU
EU (2001):
GMO means an organism in which the genetic material has been
altered in a way that does not occur naturally;
• genetic modification occurs at least through the use of:
– rDNA 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;
– fusion 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.

25. What does genome editing do?
René Custers

26. ‘New’ modification technologies
1. Oligo-directed mutagenesis
2. ZFN and other SDNs
3. Cisgenesis/intragenesis
4. RdDM
5. Grafting
6. Reverse breeding
7. Agro-infiltration
Genome editing

27. A
T
G C
T
G
A T
G C
A
T
G G A C T A
A T
C
mutagenic oligonucleotide
Chemically differing from DNA
plant protoplast
Oligo-directed mutagenesis - ODM
Adapted from Keygene

28. Oligonucleotide lifetime in the cell
After mutagenesis
the oligo is degraded
and disappears from the cell
Source: Keygene

29. From single protoplast to plant
Source: Keygene

30. ODM compared to classical mutagenesis
Natural random
mutation
Induced random
mutagenesis
Directed
mutagenesis
UV light,
chemicals
Chemical treatment
or radiation
Oligo
treatment
1100 5,000
4000 BC 20th century 21st century
Source:Keygene

31. Conclusions ODM
• Only in crops with established plant regeneration from protoplasts
• Current efficiency ≈ 2%
• Mutagenic oligonucleotide disappears within 48 hours
• Extremely specific
• Single point mutation introduced in a precise and directed manner,
compared to dozens to thousands of random mutations in natural and
classical mutagenesis

32. Site-Directed Nuclease technology (SDN)
For instance:
• Zink Finger Nucleases (ZFN)
• TAL Effector Nuclease (TALEn)
• CRISPR/Cas9
• (Meganuclease)

33. Zink Finger Nucleases

35. CRISPR/Cas9

36. A Double-strand break

37. Double-strand break repair
INDELs
Homologous
gene with edit
Full (trans)gene
addition

38. Double-strand break repair
INDELs
‘SDN-1’ ‘SDN-2’ ‘SDN-3’

39. SDN-1 generated INDELs
Generally between -20 to +20 bp

40. Off-target effects using CRISPR/Cas9
Craddick et al, Nucleic Acids Research, 2013
Off-target effects:
• May introduce one or a few additional INDELs
• Locus dependent and guide RNA sequence specific
• Smart guide RNA design can limit off-target effects

41. Cas9 nickase to enhance specificity
Ran et al, Cell, 2013
Can result in larger deletions (up to ≈50 bp)
Or multiple small deletions in one locus

42. SDN-2

43. SDN-2 not 100% efficient
Competition

44. SDN-2 generated edits
Any type of edit you want (already existing in nature or not):
• Point mutation ACGTCCGGCATTC -> ACGTCCTGCATTC
• Frameshift mutation ACGTCCGGCATTC -> ACGTCCTGGCATTC
• Stop codon insertion ACGTCCGGCATTC -> ACGTCCTAAGGCATTC
• Small deletion ACGTCCGGCATTC -> ACGT---GCATTC
• Small insertion ACGTCCGGCATTC -> ACGTCCTATCGGCATTC
• Etc, etc..

45. Reversal back to wild type using SDN-2
Donor DNA: wild-type gene
Cas9 DS break
mutation

46. SDN-2 can also achieve allele replacement
Donor DNA containing
replacement allele

47. SDN-3: insertion of full genes
Double-strand break
Full gene added, either:
• Transgene
• Intragene
• Cisgene

48. To conclude: Genetic alterations achieved by
genome editing (ODM and SDN-1 and -2)
1. Generation of already existing combinations of genetic material
o Allele replacement
o Introduction of already existing mutations
o Reversion of existing mutations back to wild-type
2. Introduction of small alterations
o Point mutations
o Frameshift mutations
o Introduction of stop codons
o Small deletions (1 to ≈50 bp)
o Larger deletions (>100 bp) (if two Cas9s are used to generate two DS breaks)
o Multiple small deletions in one and the same locus (nickase technology)
o Small insertions (up to 20 bp)
o Small INDELS in different loci (resulting from off-target effects)