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Precision Genomics in Soybean
Justin Anderson
Advisor: Dr. Robert StuparUniversity 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
GOI
GOI
Normal soybean
ZFN transformed;Mutates GOI
Targeted Mutation
Curtin et al. 2012
Gene Targeting
Similar process with other designable nucleases• Zinc Finger Nucleases (ZFN)• Transcription activator-like effector nucleases (TALEN)• CRISPR/Cas9• Meganuclease
Nucleotide Binding (Zinc Finger)Endonuclease (Fok1)
Target Region
ZFN/TALEN
NHEJrandom mutation
Donor template
Gene Targeting
Potential of a Double Strand Break
Modify Copy Number
Rhg1
+Rhg1
ZFN Rhg1
Rhg1
Rhg1
Rhg1 Rhg1
Rhg1
PROS• They work in soy
Designer NucleasesZinc Finger Nucleases CRISPR/ Cas9TAL Effector Nucleases
CONS Low specificity compared to
TALENS/CRISPR Takes 2-3 weeks for assembly A lot of molecular work
involved
PROS More specificity when
targeting then ZFN You can design/assemble a
TALEN in 7 Days
CONS They have yet to work in soy Assembly can be difficult Very Large
PROS You can design/assemble a
CRISPR in 5 Days Very easy to design/assemble Potential for multiplexing Smaller size than ZFN/TALEN
CONS Has not been tested with
agrobacterium Potential for off target
mutations Not as much specificity as
TALENS http://taleffectors.com/wp-content/uploads/2011/12/TALENfig1.png
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/life-science/functional-genomics/zinc-finger-nucleases.
http://www.google.com/imgres?imgurl=http://www.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)
Shoot Induction(Day 19)
Selection Medium (Day 33)
Shoot Elongation (Day 60)
Root Elongation(Day 90) Planting
(Day 104)Screening and Testing
(Day 120)
Contact/Acknowledgments
• Justin Anderson (me) [email protected]
• Advisor: Robert Stupar [email protected]• Dan Voytas• Shaun Curtin• Jean-Michel Michno• Junqi Liu
Plug:UMN Plant Breeding Symposium
travel funding availablewww.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
Inducible promoter
Left ZFA 1 Right ZFA 1 Left ZFA 1 Right ZFA 2
Transformation Vector coding two ZFNs
R-gene cluster
12
34
5
Induce
ZFN 1
ZFN 2
12
34
5
ZFN 1
ZFN 2
R-gene cluster
Deletion
12345
Wild Type
12
34
5
ZFN 1
ZFN 2
R-gene cluster
Inversion
ZFN 1
ZFN 2
Inversion
12
34
5
Inversion
12345
Wild Type
1 2 3 4 5
12
34
5
ZFN 1
ZFN 2
R-gene cluster
12
34
5
Duplication
12
34
5
12
34
5
Duplication
12
34
5
12
34
5
12345 12345
Deletion
Duplication
12345
Wild Type
