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

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/

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

Targeted Mutation Normal soybean GOI ZFN transformed; Mutates GOI GOI

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

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

Modify Copy Number Rhg1 + Rhg1 ZFN Rhg1 Rhg1 Rhg1 Rhg1

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 http://www.sigmaaldrich.com/content/dam/sigma-aldrich/life-science/functional-genomics/zinc-finger-nucleases. http://taleffectors.com/wp-content/uploads/2011/12/TALENfig1.png 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) ande9112@umn.edu Advisor: Robert Stupar stup0004@umn.edu 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

Induce 1 2 ZFN 1 3 R-gene 4 cluster 5 ZFN 2 Lee et al 2012 saw 230 kb deletion in human cell lines Frequencies from 10 ^-1 to 10 ^ -4 depending on length and ZFN activity ZFN 2

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

1 2 3 4 5 Wild Type Deletion

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

1 2 3 4 5 ZFN 1 ZFN 2 Inversion

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.

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

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 Duplication

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