Download A New Plant Breeding Technique: Gene Editing

A New Plant Breeding Technique: Gene Editing Dr. Shveta Bagga
DuPont Pioneer
Contents
 Background on New Breeding Techniques (NBTs)
 Gene Editing methods
 Features and examples
 Gene Editing as precision mutagenesis
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New Breeding Techniques
Additional Tools in the Breeder’s Toolbox
 Makes plant breeding more precise and efficient
 Essential to address world’s agricultural challenges  Many techniques result in products that do not contain foreign DNA
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New Breeding Techniques
Additional Tools in the Breeder’s Toolbox
 Makes plant breeding more precise and efficient
 Essential to address world’s agricultural challenges  Many techniques result in products that do not contain foreign DNA
Gene editing techniques
•Oligo Directed Mutagenesis
• Site‐directed nucleases:
•Zinc‐finger nucleases
• CRISPR‐Cas
• TALENs
• Meganucleases
Recombinant DNA is transient and absent in the final product
•Reverse breeding
•RNA‐dependent DNA methylation
•Virus‐induced gene silencing
•Grafting of on GM rootstock
Recombinant DNA is from the same* species
•Cisgenesis
•Intragenesis
•*or sexually compatible 4
Gene Editing: Evolution of Precision Breeding
Gene Induced
variability
Natural
variability
Marker assisted selection
editing
(chemical, X‐ray mutagenesis)
Towards efficient precision breeding to meet agricultural and food demands
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Endonuclease‐Based Gene Editing Platforms
Meganuclease
Zinc Fingers
TALEns
CRISPR-Cas9
Precision Biosciences
Illustration: Bertoni 2014 with modifications
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Precise Targeting of Native Plant Genes
DNA Bind
Cut Repair template
Gene disruption
OFF
Gene correction
EDIT
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Generating Gene Edited Plants
Confirm:
 Intended mutation in the target
gene
 No unintended integration of the construct components in the final plant line
Target gene
identification and experiment design
Plant transformation and regeneration
Molecular analysis
FINAL PLANT LINE
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Technology Features




Precision
Efficiency Broad applicability
Similarity to mutagenesis
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Precision: Why Is It Possible?
Advancements in genome sequencing
• Lowering cost of sequencing technologies
• Availability of complete genome sequences
• Ability to identify unique target sites in the gene of interest
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Precision: Native Gene Editing
ALS2 gene editing and chlorsulfuron‐tolerant maize
• Two acetolactate synthase (ALS) genes in maize, sharing 94% homology
• Single amino acid change confers tolerance to chlorsulfuron • Ability to target ALS2, but not ALS1
4‐week old plants sprayed with chlorsulfuron
Svitashev et al (2015) Plant Physiology 169: 931‐945
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Efficiency: Multiplexing
Simultaneous targeting of three maize genes
• Liguleless gene region (Lig34)
• Male fertility gene Ms26
• Male fertility gene Ms45
Svitashev et al (2015) Plant Physiology 169: 931‐945
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Efficiency: Time Maize transformation example
Selection and plant regeneration
2‐3 months
Growth to maturity and seed set in controlled environment
3‐4 months
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Broad Applicability: Across Crops
and Traits
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Technology Opportunities
Traits by editing plant genes instead of adding new genes 16
How Similar to Conventional Breeding?
 Can similar changes be obtained by conventional
breeding (natural diversity, mutagenesis)?
 Is DNA from a different organism present in the final product?
 Can we distinguish the final plant lines from those that could be obtained through conventional breeding? 17
Native Gene Editing is Precision Mutagenesis • 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 and are globally adopted.
• 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
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 Resulting products are indistinguishable
from products of natural variability or
mutagenesis
Knowledge
Specificity
Efficiency
Time
Expense
Development
 Gene editing is achieved through a precise, yet flexible, set of tools allowing breeders to make specific, targeted improvements to plant genomes
Breeding
Summary
 Both public and private sector have significant opportunities
to employ gene editing in their breeding programs; however, technology adoption will be highly dependent on the imposed regulatory regime
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Acknowledgements
Mark Cigan and DuPont Pioneer Genome Modification Group:
Josh Young
Sergei Svitashev
Huirong Gao
Lijuan Wang
Zhan‐Bin Liu
Chris Scelonge
Dave Petersen
Lanie Feigenbutz
Myeong‐Je Cho
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