1. Precision Genomics in Soybean
Justin Anderson
Advisor: Dr. Robert Stupar
University of Minnesota
Department of Agronomy and Plant Genetics

2. Stupar lab Natural Variation Fast Neutron Induced Mutation
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/

3. Working with Soybean Grown for protein and oil Paleopolyploid
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

4. Targeted Mutation
Normal soybean
GOI
ZFN transformed;
Mutates GOI
GOI

5. Gene Targeting Similar process with other designable nucleases
Nucleotide Binding (Zinc Finger)
Endonuclease (Fok1)
Similar process with other designable nucleases
Zinc Finger Nucleases (ZFN)
Transcription activator-like effector nucleases (TALEN)
CRISPR/Cas9
Meganuclease
Curtin et al. 2012

6. Potential of a Double Strand Break
Target Region
ZFN/TALEN
NHEJ
random mutation
Gene Targeting
Donor template

7. Modify Copy Number
Rhg1 +
Rhg1
ZFN
Rhg1
Rhg1
Rhg1
Rhg1

8. Designer Nucleases Zinc Finger Nucleases TAL Effector Nucleases
CRISPR/ Cas9
PROS
They work in soy
PROS
More specificity when targeting then ZFN
You can design/assemble a TALEN in 7 Days
PROS
You can design/assemble a CRISPR in 5 Days
Very easy to design/assemble
Potential for multiplexing
Smaller size than ZFN/TALEN
CONS
Low specificity compared to TALENS/CRISPR
Takes 2-3 weeks for assembly
A lot of molecular work involved
CONS
They have yet to work in soy
Assembly can be difficult
Very Large
CONS
Has not been tested with agrobacterium
Potential for off target mutations
Not as much specificity as TALENS

9. 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:

10. Hairy roots: Initial testing
Agrobacterium rhizogenes strain K599 is used for hairy root transformation

11. 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)

12. Contact/Acknowledgments
Justin Anderson (me)
Advisor: Robert Stupar
Dan Voytas
Shaun Curtin
Jean-Michel Michno
Junqi Liu
Plug:
UMN Plant Breeding Symposium
travel funding available

13. Gene Targeting in Plants
ZFNs:
Shukla et al. 2009
Townsend et al. 2009
Cai et al. 2009
TALENs
Baltes et al. 2014
CRISPR/Cas9

14. Transformation Vector coding two ZFNs
Inducible promoter
Left ZFA 1
Right ZFA 1
Left ZFA 1
Right ZFA 2

15. Induce 1 2 ZFN 1 3 R-gene 4 cluster 5 ZFN 2
Lee et al saw 230 kb deletion in human cell lines
Frequencies from 10 ^-1 to 10 ^ -4 depending on length and ZFN activity
ZFN 2

16. 1 2
ZFN 1 3
R-gene
cluster 4 5
ZFN 2

17. 1 2 3 4 5
Wild Type
Deletion

18. 1 2
ZFN 1 3
R-gene
cluster 4 5
ZFN 2
Inversion

19. 1 2 3 4 5
ZFN 1
ZFN 2
Inversion

20. 1 2 3 4 5
Wild Type 1 2 3 4 5
Inversion
Lee et al 2012 claim inversions and duplications up to 1 Mb.

21. 1 1 2 2
ZFN 1 3 3
R-gene
cluster 4 4 5 5
ZFN 2
Duplication

22. 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
Duplication

23. 1 2 3 4 5 Wild Type Deletion 1 2 3 4 5 1 2 3 4 5 Duplication
Lee et al 2012 saw 230 kbp duplicaion