Site-Directed Nucleases and Cisgenesis Maria Fedorova, Ph.D.

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1 Site-Directed Nucleases and Cisgenesis Maria Fedorova, Ph.D. Regulatory Strategy Lead Enabling Technologies DuPont-Pioneer, USA 1

2 New Plant Breeding Techniques 2007 New Techniques Working Group established at EU: In scope: techniques for which it was unclear whether they would result in a GMO What are the risks for human, animals, environment? Is a new risk assessment guidance needed? 2011 ftp://ftp.jrc.es/pub/eurdoc/jrc63971.pdf 2013 EU NTWG Final Report 2

3 List of Techniques (2007) 1. Agro Infiltration 2. Grafting 3. Reverse breeding 4. RNA- dependent DNA methylation (RdDM) 5. Oligonucleotide Directed Mutagenesis (ODM) 6. Synthetic Genomics 7. Cisgenesis (cisgenesis and intragenesis) 8. Zinc Finger Nuclease Technology (ZFN) 3

4 Common Themes Does the technique introduce heritable changes in the genome? Is the rdna transient or present in the final product? Can similar changes be obtained naturally or through conventional breeding? Is the product uniquely identified to be detected? 4

5 Focus of This Presentation 1. Agro Infiltration 2. Grafting 3. Reverse breeding 4. RNA- dependent DNA methylation (RdDM) 5. Oligonucleotide Directed Mutagenesis (ODM) 6. Synthetic Genomics 7. Cisgenesis (cisgenesis and intragenesis) 8. Zinc Finger Nuclease Technology (ZFN) Meganuclease Technology TALEN Technology CRISPR/Cas Technology Site-directed nucleases [SDNs] 5

6 Site-Directed Nucleases 6

7 Site Directed Nucleases (SDNs) Endonucleases directed to specific DNA sequences through a DNA-binding activity Include: ZFNs (Zinc Finger Nucleases) Meganucleases TALEN (Transcription Activator-Like Effector Nucleases) CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats) Protein / Technology Synonyms: Engineered Nucleases Sequence-specific nucleases Targeted genome modification Genome editing technologies 7

8 General Features DNA binding domain and DNA cutting domain SDNs introduces double-strand break in the specific DNA sequence zinc fingers TAL effector array FokI FokI binding domain catalytic domain 8 Illsutration: D. Voytas (2013) Annu Rev Plant Biol 64:

9 d. CRISPR Cas9 grna against the target sequence Illustration: B-R Lee et al.(2013) Mol Cells 35: SDNs are custom designed molecular scissors to introduce break at the DNA sequence of choice SDN technology opens a possibility for various targeted and precise gene manipulations to develop new traits 9

10 SDN Technologies: Mode of Action Nuclease recognizes and binds to the specific DNA sequence Nuclease creates DNA double strand break SDN DNA break is repaired through native cellular mechanisms Type of DNA repair mechanism? Donor DNA for repair? Result is one of the following: - Nucleotides inserted or deleted ( indels ) - Nucleotides substituted - External DNA added

11 SDN classification by end-use SDN1 Targeted random mutations SDN2 Targeted specific mutations SDN3 Targeted transgene insertion 11 Illustration: N. Podevin et al. (2013) Trends in Biotechnology 31(6):

12 Technology Opportunities Targeted mutagenesis of endogenous plant genes (SDN1, SDN2) Knocking down a specific gene to impact associated pathway Precise gene editing to alter gene function > plant phenotype Targeted transgene integration (SDN3) Elimination of unintended effects caused by gene disruption Molecular stacking of several traits into a single breeding locus Advancements in genome sequencing increase precision of SDN tools Off-target effects minimized Predictability of the outcome of the SDN techniques 12

13 Technology Applications SDN1 Maize: Reduced phytate (ZFN), male sterility (Meganuclease) Rice: Improved resistance to blight (TALEN) Rice: chlorophyll content, tiller phenotype (CRISPR) SDN2 Tobacco: ALS mutations for IMI and SU tolerance (ZFN) SDN3 Tobacco, Maize: Targeted transgene integration (ZFN) Cotton: Trait stacking (Meganucleases) 13

14 Considerations for Regulatory Discussions Similarity to mutagenesis SDN-1 SDN-2 SDN-3 yes yes no More precise than mutagenesis: no / less frequent off-target effects Transient presence of Donor DNA and/or SDN transgenes Can be expressed transiently Can be segregated away Delivery via protein or RNA likely possible Introducing a permanent heritable change? Made by the host s own repair mechanism Detection method SDN-1 SDN-2 SDN-3 no no yes 14 14

15 Cisgenesis and Intragenesis 15

16 Cisgenesis Cisgenesis is a genetic modification of a recipient plant with a natural gene from a crossable - sexually compatible organism. The gene includes its introns and is flanked by its native promoter and terminator in the normal sense orientation. Any suitable technique used for production of transgenic organisms may be used. Genes must be isolated, cloned, or synthesized and transferred back into a recipient where stably integrated and expressed. <EU NBT WG report> Involves gene cloning and genetic transformation All genetic elements from the same gene of the same species Sense orientation of the coding sequence Native introns included? Agrobacterium LB and RB allowed Cisgenesis with T-DNA borders Selectable markers to be segregated out 16

17 Intragenesis Intragenesis is a genetic modification of a recipient organism that involves the insertion of a reorganized, full or partial coding region of a gene frequently combined with a promoter and/or terminator from another gene of the same species or a crossable species. These may be arranged in a sense or antisense orientation compared to their orientation in the donor organism. <EU NBT WG report> Involves gene cloning and genetic transformation Agrobacterium LB and RB allowed Cisgenesis with T-DNA borders Selectable markers to be segregated out All genetic elements from the same gene species Sense or antisense orientation, full or partial coding region 17

18 Illustration: I.B. Holme et a. (2013) Plant Biotechnology J 11:

19 Technology Opportunities Trait improvement for: Difficult to breed, complex genetics crops Crops with long breeding cycle Crops with limited natural allelic variation Avoidance of linkage drag Fine-tuning trait gene expression: Enhancing expression [cisgenesis and intragenesis] Lowering expression [intragenesis] 19

20 Technology Applications Disease resistance Scab resistant apple Late blight resistant potato Fungal resistant grapevine Quality traits High amylopectin potato Reduced lignin alfalfa Durum wheat with improved baking quality Agronomic Traits, Phenotype Drought tolerant perennial ryegrass Poplar with different growth types 20

21 Considerations for Regulatory Discussions Similarity to conventional breeding Cisgenesis yields plants that can be also obtained by breeding or via normal reproduction; Intragenic plants cannot be obtained by traditional breeding Similarity to products of natural variation, genome plasticity High degree of natural plasticity and variability between genomes; Naturally occurring mechanisms for gene duplication, shuffling, and translocation can generate cisgenics and even intragenic plants Non-novelty Transferred genes have a history of safe use Detection method Possible, with prior knowledge from the producer 21

22 Why SDN, cisgenesis? Innovative, rapidly developing modern plant breeding technologies to produce plants with new, valuable traits in a precise, fast, efficient manner Products of many technology applications share commonalities with products of conventional breeding, natural variation Science based regulatory approach: plants with no safety concerns beyond the baseline of conventional breeding products should not be regulated 22

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24 Appendix 24

25 Technology Applications References - SDNs SDN1 Maize: Reduced phytate (ZFN) Shukla et al (2009) Nature 459 (7245): Djukanovic et al (2013) Plant J: Rice: Improved resistance to blight (TALEN) Li et al (2012) Nature Biotechnology 30 (5): Rice: chlorophyll content, tiller phenotype (CRISPR) Miao et al (2013) Cell Research 23: SDN2 Tobacco: ALS mutations for IMI and SU tolerance (ZFN) Townsend et al (2009) Nature 459 (7245): SDN3 Tobacco, Maize: Targeted transgene integration (ZFN) Ainley et al (2013) Plant Biotechnology J. online 19AUG2013 DOI: /pbi Cai et al (2009) Plant Mol Biol 69: Cotton: Trait stacking (Meganucleases) D Halluin et al (2013) Plant Biotechnology J. 11 (8):

26 Technology Applications References cis/intra-genesis Disease resistance Scab resistant apple Vanblaere et al (2011) J. Biotechnol. 154: Late blight resistant potato Havekort et al (2009) Potato Res. 52: Fungal resistant grapevine Dhekney et al (2011) In Vitro Cell Dev Biol. Plant 47: Quality traits High amylopectin potato de Vetten et al (2003) Nature Biotechnology 21: Reduced lignin alfalfa - Weeks et al (2008) Transgenic Res. 17: Durum wheat with improved baking quality Gadaleta et al (2008) J. Cereal. Sci. 48: Agronomic Traits, Phenotype Drought tolerant perennial ryegrass - Bajaj et al (2008) 6 th Canadian plant genomics workshop, Abstract p.62 Poplar with different growth types Han et al (2011) Plant Biotechnology J. 9: