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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
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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 • •
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3. Agriculture Division of DowDuPont™ OECD Conference
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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. 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

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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. 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. 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. 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. 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

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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. 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. 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. Agriculture Division of DowDuPont™ OECD Conference
2016
A breakthrough in cereal transformation methods

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. 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. Agriculture Division of DowDuPont™ OECD Conference
Precise confirmation of CRISPR changes
Example set of 7 repair site variants in Inbred 1

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. 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

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Precise confirmation: NO extraneous DNA

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. 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. Agriculture Division of DowDuPont™ OECD Conference
Agronomic characterization
CRISPR-waxy hybrids out-yield their conventional hybrid checks

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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. 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. 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
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CRISPR as Breeding Innovation: Considerations

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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
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