New CRISPR-Cas Gene Editing: Technology Opportunities and … · 2018. 1. 19. · Maize Lethal...
Transcript of New CRISPR-Cas Gene Editing: Technology Opportunities and … · 2018. 1. 19. · Maize Lethal...
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CRISPR-Cas Gene Editing:
Technology Opportunities and Regulatory Policies to Enable Innovation
Dr. Kevin Diehl, Mr. Kulani Machaba
Oligonucleotide-Directed Mutagenesis (ODM) • Zinc Finger Nucleases
• Meganucleases
• TALENs
• CRISPR-CasSite-directed Nucleases (SDN)
Cisgenesis and Intragenesis
Reverse breeding
RNA-dependent DNA methylation (RdDM)
Virus-Induced Transient Gene Expression or Gene Silencing
Accelerated Plant Breeding
Employing a GM Inducer Plant
Agro-infiltration
Grafting onto a GM Rootstock
https://croplife.org/wp-content/uploads/2015/03/Technical-Summary-of-NBTs_final.pdf
GeneEditing
Platforms
Terminology:
New Breeding Techniques (NBT) or
Precision Breeding Innovations (PBI)
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2007 – EU New Techniques Working Group
In scope: techniques for which it was unclear whether they fall in the scope of GMO regulation
What are the risks for human, animals, and environment?
Is a new risk assessment guidance needed?
Diverse Techniques. What’s in Common?
➢ Intermediate product contains (may contain) recombinant DNA but it is absent in the end-product
➢ End-product is similar to an outcome from mutagenesis
RdDMReverse Breeding
SDN1 and SDN2
SDN1 and SDN2 ODM
Accelerated Breeding
ExamplesHistory
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Building on Legacy of Crop Improvement Innovation
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5
Plant Breeding Develops Product Diversity
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CRISPR-Cas Scientific Publications
0
500
1000
1500
2000
2500
2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 YTD*
*As of Sep 2017
CRISPR-Cas are custom-designed molecular scissors
to introduce a break at the target DNA sequence
Cas9 enzyme binds to the targeted
DNA sequence and makes the
DNA double strand break
DNA break is repaired through
endogenous cellular mechanism
Guide RNA is designed to bring Cas9
enzyme to the targeted DNA sequenceCas9 nuclease
(DNA cutting enzyme)
Guide RNA homologous
to the target DNA
sequence
CRISPR-Cas
applications7
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Use of CRISPR-Cas for Gene Editing
The plant does not have desired output.The plant does have desired output.
DELETE
SEARCH / REPLACE
EDITThe plant has tolerance to drought.
The plant has high tolerance to drought.
The plant is susceptible to disease.The plant is resistant to disease.
Gene editing utilizes genetic material from the target plant/crop -no DNA from a different organism in the final plant
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• No unintended plasmid DNA in the final product
• Change similarly possible through conventional breeding
• Targeted mutagenesis
Process to Generate Gene Edited Plants
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Waxy Corn Is High Amylopectin Starch Corn
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Starch
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Current Pioneer Waxy Corn Product
• Commercial wx1 allele originates from >100 year old conventional corn variety
• Specialty corn, produced under contracts with wetmilliers
• Challenges
• Time lag to introgress wx1 allele into elite inbreds
• Some yield penalty, likely due to linkage drag
Solution:
Disrupt Wx1 gene directly in elite lines using CRISPR-Cas Gene-DELETE technology
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Gene Off>97%
amylopectinWAXY MAIZE
waxy
maize30 bp deletion
Current product
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• Targeted mutation of the Wx1 gene, as in the current product
• Wx1 mutation is directly introduced into elite corn inbreds using CRISPR-CasGene-DELETE technology
• Expected intended phenotype, no unintentionally integrated plasmid DNA, and undergoes several years of field assessments according to common breeding practices
Next Generation Waxy Corn
CRISPR-Cas Accelerates Breeding of Traits
Conventional Breeding by Backcrossing
Stress sensitiveGood agronomics
Stress tolerantPoor agronomics
Stress tolerance geneGood agronomics
5…5…
INBRED A
INBRED B100% A
100% A
Requires many generations
25%
7…
12%
88%
7
%93%
3
%97%
CRISPR-Cas Enabled Breeding
Move STRESS TOLERANCE gene
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Examples of Traits Developed by CRISPR-Cas Gene Editing
Corn:
▪Reduced phytate
▪Waxy starch
▪ Improved oil quality
▪ Delayed flowering
Powdery mildew
resistance
▪ Yield components
improvement
▪ Fragrant
▪ High amylose
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Virus resistance
▪ Partenocarpic (seedless)
▪ Powdery mildew
resistant
RICE
WHEAT
Canker resistance
CAMELINA
Improved oil quality
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• Mutagenesis is fundamental to evolution and crop breeding
• Mutational products have a long history of safe use. Over 3,200* cultivars have been used commercially.
• Gene editing technologies continue the history of improving crop development through modern targeted mutational applications
• Gene editing allows, for the first time, mutations to be targeted to a specific, desired location in the plant genome
Gene Editing is Precision Mutagenesis
* https://mvd.iaea.org16
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➢ Global regulatory policies for gene edited products are in development in many countries
➢ Challenge: what is the appropriate policy framework to facilitate innovation while protecting human health and the environment?
Regulatory Policies for Genome Editing
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Appropriate, science-based regulatory oversight for plants developed with CRISPR-Cas advanced plant breeding, consistent with plants developed through other plant breeding methods
➢ Protecting health and the environment while promoting innovation
➢ Science-based, accommodate new evidence and learning
➢ Cost effective and commensurate with the risk
➢ Transparent, consistently applied and enforced
➢ Globally harmonized
✓ Gene edited plants are indistinguishable from plants that could be produced with conventional breeding tools and have a long history of safe use
✓ It is the characteristics of the plant, not the production method, that determines its safety
Considerations for Regulatory Policies
Public Acceptance
Engage Full Range of Stakeholders
➢ Recognize that all new technologies require a “social license”
➢ Engage traditional and non-traditional stakeholders about their hopes and questions related to CRISPR-Cas and how to balance the two
➢ Using insights as we develop our plans and as we work with others in agriculture and with those applying CRISPR-Cas across industries
➢ On-going discussion
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CIMMYT & DuPont Pioneer Public-Private Collaboration
Sugarcane Mosaic Virus (SCMV)
Maize chlorotic mottle viruses (MCMV)
new to Africa, more severe then SCMVMaize Lethal
Necrosis
Range of symptoms:
• Vascular discoloration
Even “clean” plants may
show:
• Sterile tassels
• No ears
• High cob rot/
predisposed to other
challenges
➢First observed in Kenya in 2011, spread to neighboring countries in less than five years
➢Average reduction in maize production: 3% in drylands; 32% in moist environments; yield reduction at individual farms can be as high as 90%*
➢ In Kenya, MLN affects nearly a quarter of total maize production; yearly losses ~$US110 million**
*de Groot et al., CIMMYT
**Biosciences for Farming In Africa, 2016
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21Thank You!
http://crisprcas.pioneer.com/
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Disclaimer
This document and material is for use only by the entity to which it is submitted by
DuPont Pioneer, and only with the explicit consent of DuPont Pioneer. Except in
accordance with law, any other use of this material, without prior written consent of
DuPont Pioneer, is strictly prohibited. By submitting this document, Pioneer does not
grant any party or entity not authorized by Pioneer any right or license to the
information or intellectual property described in this document.
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