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Agriculture Division of DowDuPont™
OECD Conference
Application of genome editing in Crop Plants: 2
Next-generation waxy co...
Agriculture Division of DowDuPont™ OECD Conference
BROAD R&D INVESTIGATIONS
NEAR-TERM PRODUCTS TO MARKET
WAXY CORN HYBRIDS...
Agriculture Division of DowDuPont™ OECD Conference
3
Listening to a Full Range of Stakeholders
• Recognize that all new te...
Agriculture Division of DowDuPont™ OECD Conference
Waxy corn characteristics
A long history of safe use as a specialty cro...
Agriculture Division of DowDuPont™ OECD Conference 5
Waxy Maize Cultivation and Processing: A “closed loop”
• Cultivated i...
Agriculture Division of DowDuPont™ OECD Conference
Waxy corn alleles
Numerous insertions and deletions have been described...
Agriculture Division of DowDuPont™ OECD Conference
Conventional wx1 mutation in current Pioneer products
30 bp deletion @ ...
Agriculture Division of DowDuPont™ OECD Conference
Conventional Waxy products vs.
Minimum of 8 cycles to produce commercia...
Agriculture Division of DowDuPont™ OECD Conference
CRISPR-waxy allele
Drop-out deletion (SDN1) of the entire Waxy coding s...
Agriculture Division of DowDuPont™ OECD Conference 10
Process to Generate CRISPR-Cas Waxy Maize
Robust
Design
Elite
Transf...
Agriculture Division of DowDuPont™ OECD Conference
CRISPR-Cas & Genomic Design Principles
from Haplotype Blocks to Allele ...
Agriculture Division of DowDuPont™ OECD Conference
CRISPR-waxy allele
Drop-out deletion of the entire Waxy coding sequence...
Agriculture Division of DowDuPont™ OECD Conference
2016
A breakthrough in cereal transformation methods
Agriculture Division of DowDuPont™ OECD Conference
No exogenous DNA in the final waxy maize line(s)
Targeted transformatio...
Agriculture Division of DowDuPont™ OECD Conference
0
20
40
60
1 2 3 4 5 6 7 8 9 10 11 12
4-kb
Wx1 deletion
Targeted transf...
Agriculture Division of DowDuPont™ OECD Conference
Precise confirmation of CRISPR changes
Example set of 7 repair site var...
Agriculture Division of DowDuPont™ OECD Conference
Precise confirmation of CRISPR changes
Deletion repair site variants fo...
Agriculture Division of DowDuPont™ OECD Conference 18Confidential Business Information
CRISPR-Cas Waxy Maize: Absence of P...
Agriculture Division of DowDuPont™ OECD Conference
Precise confirmation: NO extraneous DNA
Agriculture Division of DowDuPont™ OECD Conference
Phenotypic characterization
CRISPR-waxy endosperm is just like starch f...
Agriculture Division of DowDuPont™ OECD Conference
Phenotypic characterization
Starch from CRISPR-waxy is indistinguishabl...
Agriculture Division of DowDuPont™ OECD Conference
Agronomic characterization
CRISPR-waxy hybrids out-yield their conventi...
Agriculture Division of DowDuPont™ OECD Conference
Summary
Gene editing directly in elite maize produces improved products...
Agriculture Division of DowDuPont™ OECD Conference
Next Generation Waxy Corn via CRISPR-Cas
Merci beaucoups, OECD
Morrie B...
Agriculture Division of DowDuPont™ OECD Conference
 Genome editing generates products similarly possible through spontane...
Agriculture Division of DowDuPont™ OECD Conference
Genome Editing and Possible Unintended Changes Considerations
 How dif...
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Next-generation waxy corn – a flagship case of SDN-1/NHEJ genome editing via CRISPR/Cas9

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Next-generation waxy corn – a flagship case of SDN-1/NHEJ genome editing via CRISPR/Cas9

  1. 1. Agriculture Division of DowDuPont™ OECD Conference Application of genome editing in Crop Plants: 2 Next-generation waxy corn – a flagship case of SDN-1/NHEJ genome editing via CRISPR/Cas9 Dr. Robert Meeley – Senior Research Scientist Thursday, 28 June 2018 1
  2. 2. Agriculture Division of DowDuPont™ OECD Conference BROAD R&D INVESTIGATIONS NEAR-TERM PRODUCTS TO MARKET WAXY CORN HYBRIDS • Foundational for future product development • First commercial agricultural product • To market by end of decade Leadership in CRISPR Technology for Agricultural Applications Product concepts & benefits described here will not be offered for sale or distribution until completion of field testing and applicable regulatory reviews. DISEASE RESISTANCE YIELD & YIELD STABILITY DROUGHT TOLERANCE OUTPUT TRAITS MATURITY CORN • • • • SOY • • • CANOLA • • • RICE • • • • WHEAT • • SUNFLOWER • • 2
  3. 3. Agriculture Division of DowDuPont™ OECD Conference 3 Listening to a Full Range of Stakeholders • Recognize that all new technologies require a “social license” • Better engagement with the customer • Applying insights to our own plans as well as sharing with others in the industry • On-going dialogue…
  4. 4. Agriculture Division of DowDuPont™ OECD Conference Waxy corn characteristics A long history of safe use as a specialty crop  Known since 1908  Commercially cultivated since 1940’s  Sold by Pioneer since 1980’s
  5. 5. Agriculture Division of DowDuPont™ OECD Conference 5 Waxy Maize Cultivation and Processing: A “closed loop” • Cultivated in the US since the 1940’s; tapioca substitute • Recessive trait – grown in an identity-preserved system • Typically produced by growers under contracts • processed into starch by wet-milling industry • Small market ~0.5% of US corn acres annually • Very limited exports for feed in the livestock, dairy, and poultry industries; Japan, Mexico
  6. 6. Agriculture Division of DowDuPont™ OECD Conference Waxy corn alleles Numerous insertions and deletions have been described  Classical locus for maize geneticists  Extensive literature & sourcing of wx1 mutations  Mutations include insertions, deletions, translocations From: Wessler and Varagona (1985) PNAS 82: 4178
  7. 7. Agriculture Division of DowDuPont™ OECD Conference Conventional wx1 mutation in current Pioneer products 30 bp deletion @ Exon/Intron boundary producing a frameshift / null allele
  8. 8. Agriculture Division of DowDuPont™ OECD Conference Conventional Waxy products vs. Minimum of 8 cycles to produce commercial hybrid seed lots • unintended content from Donor A, even with selection CRISPR Innovation Fewer cycles / Greater precision Marker Assisted Selection
  9. 9. Agriculture Division of DowDuPont™ OECD Conference CRISPR-waxy allele Drop-out deletion (SDN1) of the entire Waxy coding sequence 2 guide RNAs were designed to introduce 5’ and 3’ DNA breaks
  10. 10. Agriculture Division of DowDuPont™ OECD Conference 10 Process to Generate CRISPR-Cas Waxy Maize Robust Design Elite Transformation Precise Confirmation Agronomic Testing  Confirm intended CRISPR change  Confirm NO integration of extraneous DNA
  11. 11. Agriculture Division of DowDuPont™ OECD Conference CRISPR-Cas & Genomic Design Principles from Haplotype Blocks to Allele Models  Leveraging >90 years of Pedigree history  Depth of Next Generation Sequencing  Reference Gene Models
  12. 12. Agriculture Division of DowDuPont™ OECD Conference CRISPR-waxy allele Drop-out deletion of the entire Waxy coding sequence Waxy guideRNAs are 100% conserved across >85% of North American elite germplasm
  13. 13. Agriculture Division of DowDuPont™ OECD Conference 2016 A breakthrough in cereal transformation methods
  14. 14. Agriculture Division of DowDuPont™ OECD Conference No exogenous DNA in the final waxy maize line(s) Targeted transformation in elite maize • 12 inbreds were co-bombarded with the same six plasmids “Developmental Gene Package” to facilitate plant regeneration Selectable marker
  15. 15. Agriculture Division of DowDuPont™ OECD Conference 0 20 40 60 1 2 3 4 5 6 7 8 9 10 11 12 4-kb Wx1 deletion Targeted transformation in elite maize Morphogenic regulators enable elite inbred transformation Regeneration frequency (plants per embryo, %) Inbreds Frequency Example: Inbred 1  500 embryos  65 T0 plants (13%)  7 T0 wx1 (1.4%) Wx1 whole gene deletion achieved in one dozen elite inbreds
  16. 16. Agriculture Division of DowDuPont™ OECD Conference Precise confirmation of CRISPR changes Example set of 7 repair site variants in Inbred 1
  17. 17. Agriculture Division of DowDuPont™ OECD Conference Precise confirmation of CRISPR changes Deletion repair site variants for 9 inbreds advanced for hybrid agronomic testing
  18. 18. Agriculture Division of DowDuPont™ OECD Conference 18Confidential Business Information CRISPR-Cas Waxy Maize: Absence of Plasmid DNA - Overview Southern-by-Sequencing (SbS) • Uses sequence capture technology + deep sequencing • Capture Probes cover entire sequences of all plasmids used • Analysis checks for novel junctions between plasmid + plant genome • Extremely sensitive limit of detection • Individual plants are analyzed; only clean (“SBS-Green”) variants advanced
  19. 19. Agriculture Division of DowDuPont™ OECD Conference Precise confirmation: NO extraneous DNA
  20. 20. Agriculture Division of DowDuPont™ OECD Conference Phenotypic characterization CRISPR-waxy endosperm is just like starch from conventional waxy corn Wx1/Wx1/Wx1 wx1/wx1/wx1 Representative CRISPR-wx kernels on a diffusion light box T0 pollen iodine stain Dark blue/black: Wx1/Wx1/Wx1 = Wild Type Amber/reddish: wx1/wx1/wx1 = waxy
  21. 21. Agriculture Division of DowDuPont™ OECD Conference Phenotypic characterization Starch from CRISPR-waxy is indistinguishable from conventional waxy TI, Trait Introgression = conventional waxy Inbred 1
  22. 22. Agriculture Division of DowDuPont™ OECD Conference Agronomic characterization CRISPR-waxy hybrids out-yield their conventional hybrid checks
  23. 23. Agriculture Division of DowDuPont™ OECD Conference Summary Gene editing directly in elite maize produces improved products faster and with no linkage drag compared to conventional production Year: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1st commercial sales in year 4 1st commercial sales in year 9 EDITING BREEDING TRANSGENICS 1st commercial sales in year 13-20Dr. Maria Fedorova Regulatory Product Strategy Lead Corteva
  24. 24. Agriculture Division of DowDuPont™ OECD Conference Next Generation Waxy Corn via CRISPR-Cas Merci beaucoups, OECD Morrie BryantSutirtha Basu Scott Betts Brian Dahlke Jeffry Farrell Masha Fedorova Mark Gadlage Renée LafitteHuirong Gao
  25. 25. Agriculture Division of DowDuPont™ OECD Conference  Genome editing generates products similarly possible through spontaneous or induced mutagenesis deployed in traditional breeding  No foreign DNA is present in the gene edited end-product  Corteva Agriscience confirms that CRISPR-Cas Waxy Maize is produced without the creation of a novel combination of genetic material  Potential for unintended changes in the gene-edited varieties needs to be viewed in the context of conventional breeding  Every new seed product developed through conventional methods is expected to have hundreds to thousands of novel uncharacterized mutations  History of safe use of plant varieties developed through conventional breeding demonstrates that mutations do not inherently represent a safety risk  Gene editing can replicate the outcomes of conventional breeding but in a targeted and precise manner 25 CRISPR as Breeding Innovation: Considerations
  26. 26. Agriculture Division of DowDuPont™ OECD Conference Genome Editing and Possible Unintended Changes Considerations  How different from processes occurring in nature or conventional breeding practices?  What is a likelihood of any given mutation creating a biosafety risk? • Gene editing can replicate variations occurring in nature • Changes Happen! Genomic diversity is exceedingly common • Rate of spontaneous mutations in plants • Untargeted classical mutagenesis with a history of safe use • Traditional breeding causes genetic changes • Advantages of gene editing being a targeted mutagenic technique 26

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