Transcript ZFN and mutagenesis in soybean

Precision Genomics in Soybean
Justin Anderson
Advisor: Dr. Robert Stupar
University of Minnesota
Department of Agronomy and Plant
Genetics
Stupar lab
• Natural Variation
– Copy number variation
– Deleterious mutations
• Fast Neutron Induced Mutation
– Evaluating unique and marketable traits such as oil
content, protein content, and plant structure
• Precision Genomics
– Implementation of engineered nuclease technology to
target genes of interest
stuparlab.cfans.umn.edu/
Working with Soybean
• Grown for protein and oil
– National and Global production
– Fixes nitrogen
• Paleopolyploid
– 12 mya and 50 mya
– 60-85% of genes maintain a
paralog from these genome
duplications
• Leads to genetic redundancy
Targeted Mutation
Normal soybean
GOI
ZFN transformed;
Mutates GOI
GOI
Gene Targeting
Endonuclease (Fok1)
Nucleotide Binding (Zinc Finger)
Similar process with other designable nucleases
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Curtin et al. 2012
Zinc Finger Nucleases (ZFN)
Transcription activator-like effector nucleases (TALEN)
CRISPR/Cas9
Meganuclease
Potential of a Double Strand Break
Target Region
ZFN/TALEN
NHEJ
random mutation
Gene Targeting
Donor template
Modify Copy Number
Rhg1
+
ZFN
Rhg1
Rhg1
Rhg1
Rhg1
Designer Nucleases
Zinc Finger Nucleases
TAL Effector Nucleases
PROS
PROS
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They work in soy
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More specificity when
targeting then ZFN
You can design/assemble a
TALEN in 7 Days
CRISPR/ Cas9
PROS
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CONS
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Low specificity compared to
TALENS/CRISPR
Takes 2-3 weeks for assembly
A lot of molecular work
involved
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/lifescience/functional-genomics/zinc-finger-nucleases.
CONS
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They have yet to work in soy
Assembly can be difficult
Very Large
CONS
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http://taleffectors.com/wpcontent/uploads/2011/12/TALENfig1.png
You can design/assemble a
CRISPR in 5 Days
Very easy to design/assemble
Potential for multiplexing
Smaller size than ZFN/TALEN
Has not been tested with
agrobacterium
Potential for off target
mutations
Not as much specificity as
TALENS
http://www.google.com/imgres?imgurl=http://ww
w.pnabio.com/products/image
Technique
• ZFN assembly method published in Legume
Genomics
• TALEN and CRISPR/Cas9 widely available
• Implementation
– Hairy Root (somatic)
– Whole plant (germline)
Curtin SJ, Anderson JE, Starker CG, Baltes NJ, Mani D, Voytas DF, Stupar RM. (2013) Targeted
mutagenesis for functional analysis of gene duplication in legumes. Methods Mol Biol 1069: 25-42.
Hairy roots: Initial testing
• Agrobacterium rhizogenes strain K599 is used
for hairy root transformation
Delivery of nucleases to whole plants
Co-Cultivation with
strain 18r12
(Day 5)
Root Elongation
(Day 90)
Shoot Induction
(Day 19)
Selection Medium
(Day 33)
Planting
(Day 104)
Shoot Elongation
(Day 60)
Screening and Testing
(Day 120)
Contact/Acknowledgments
• Justin Anderson (me) [email protected]
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Advisor: Robert Stupar [email protected]
Dan Voytas
Shaun Curtin
Jean-Michel Michno
Junqi Liu
Plug:
UMN Plant Breeding Symposium
travel funding available
www.plantbreedingsymposium.umn.edu
Gene Targeting in Plants
• ZFNs:
– Shukla et al. 2009
– Townsend et al. 2009
– Cai et al. 2009
• TALENs
– Baltes et al. 2014
• CRISPR/Cas9
Transformation Vector
coding two ZFNs
Inducible
promoter
Left ZFA 1
Right ZFA 1
Left ZFA 1
Right ZFA 2
2
1
Induce
5
R-gene
cluster
4
3
ZFN 1
ZFN 2
1
2
5
R-gene
cluster
4
3
ZFN 1
ZFN 2
Wild Type
5
4
3
2
Deletion
1
1
2
5
R-gene
cluster
4
3
ZFN 1
ZFN 2
Inversion
5
4
ZFN 1
3
2
1
ZFN 2
Inversion
Wild Type
5
4
3
2
1
Inversion
5
4
3
2
1
1
1
2
2
4
5
3
4
R-gene
cluster
5
3
ZFN 1
ZFN 2
Duplication
1
2
4
5
3
1
2
4
5
3
Duplication
Wild Type
5
4
3
2
1
Deletion
Duplication
5
4
3
2
1
5
4
3
2
1