PROCEEDINGS 2017 Crop Pest Management Short Course & Minnesota Crop Production Retailers Association Trade Show

Transcription

1 PROCEEDINGS 2017 Crop Pest Management Short Course & Minnesota Crop Production Retailers Association Trade Show Institute for Ag Professionals Do not reproduce or redistribute without the written consent of author(s).

2 Using CRISPRs for soybean genetic improvement: moving from promise to application Bob Stupar Department of Agronomy & Plant Genetics University of Minnesota

3 Univ. of Minnesota Saint Paul campus

4 Univ. of Minnesota Saint Paul campus

5 Soybean domestication Domesticated about 3,000-5,000 years ago in central China Glycine soja Glycine max earch/2010/100426masoybean.html Slide courtesy of Ben Campbell

6 Approaches to genetic improvement of crops Conventional breeding Mutation breeding GMOs Gene editing

7 Approaches to genetic improvement of crops Conventional breeding Make crosses, look for best progeny Mutation breeding Induce new mutations, then introgress traits GMOs Add new DNA to the genome to confer a specific trait Gene editing A genetic Swiss army knife; target changes in the DNA of specific genes for specific traits; outcomes can be analogous to either mutation breeding or GMOs

8 Does anyone recognize this cover? What is CRISPR? Why is it on the cover of Time? What does this have to do with agriculture? CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats CRISPR is the newest and most powerful tool used for gene editing (i.e., it can be used to rewrite sections of genetic code)

9 Why use CRISPR? CRISPR opens doors to new possibilities: Identification of gene functions Development of novel traits Information for regulators A promising strategy for crop improvement Academics will use the technology and how" will be determined by regulation

10 Who is using CRISPR? Academics Medical researchers (therapeutic tool) Pharma (drug discovery) Agricultural companies Big and small

11 What types of traits can be made using CRISPR?

12 CRISPR/Cas overview Mutate genes Eliminate function Change genes Modify function Insert genes Add new functions; simplify gene stacking

13 CRISPR/Cas overview Charpentier & Doudna (2013) Nature 495:50 51.

14 CRISPR/Cas overview MIT video: Bozeman Science video:

15 What are some desired outcomes? Bortesi & Fischer (2015) Biotechnology Advances 33:41 52.

16 What are some desired outcomes? Application Most common desired outcomes Similar to Targeted mutagenesis Targeted deletion, duplication, inversion, translocation Loss of function to native gene Loss of function to native gene(s); change of dosage to native gene Irradiation or chemical mutagenesis Irradiation mutagenesis Targeted DNA substitution Change of function to native gene Chemical mutagenesis Targeted small DNA insertion Targeted gene insertion Change of function to native gene Introduce new function with novel function Irradiation or chemical mutagenesis Genetic transformation

17 What are some desired outcomes? Application Most common desired outcomes Similar to Targeted mutagenesis Targeted deletion, duplication, inversion, translocation Loss of function to native gene Loss of function to native gene(s); change of dosage to native gene Irradiation or chemical mutagenesis Irradiation mutagenesis Targeted DNA substitution Change of function to native gene Chemical mutagenesis Targeted small DNA insertion Targeted gene insertion Change of function to native gene Introduce new function with novel function Irradiation or chemical mutagenesis Genetic transformation

18 What are some desired outcomes? Application Most common desired outcomes Similar to Targeted mutagenesis Targeted deletion, duplication, inversion, translocation Loss of function to native gene Loss of function to native gene(s); change of dosage to native gene Irradiation or chemical mutagenesis Irradiation mutagenesis Targeted DNA substitution Change of function to native gene Chemical mutagenesis Targeted small DNA insertion Targeted gene insertion Change of function to native gene Introduce new function with novel function Irradiation or chemical mutagenesis Genetic transformation

19 What are some desired outcomes? Application Most common desired outcomes Similar to Targeted mutagenesis Targeted deletion, duplication, inversion, translocation Loss of function to native gene Loss of function to native gene(s); change of dosage to native gene Irradiation or chemical mutagenesis Irradiation mutagenesis Targeted DNA substitution Change of function to native gene Chemical mutagenesis Targeted small DNA insertion Targeted gene insertion Change of function to native gene Introduce new function with novel function Irradiation or chemical mutagenesis Genetic transformation

20 What are some desired outcomes? Application Most common desired outcomes Similar to Targeted mutagenesis Targeted deletion, duplication, inversion, translocation Loss of function to native gene Loss of function to native gene(s); change of dosage to native gene Irradiation or chemical mutagenesis Irradiation mutagenesis Targeted DNA substitution Change of function to native gene Chemical mutagenesis Targeted small DNA insertion Targeted gene insertion Change of function to native gene Introduce new function with novel function Irradiation or chemical mutagenesis Genetic transformation

21 Systems of engineered nucleases TAL Effector Nucleases (TALENs) Zinc Finger Nucleases (ZFNs) Slide courtesy of Nick Baltes Homing Endonucleases (HEs) CRISPR/Cas9

22 Advantages of CRISPR/Cas compared to other genome editing platforms CRISPR platforms are faster, cheaper and easier to develop High success rate Provide the ability to target multiple targets in a single construct (aka. multiplexing) Question: Does anyone still use ZFNs or TALENs? If so, why would they?

23 Soybean traits of interest: Seed composition Soybean producers need new varieties and germplasm to stay competitive in world markets Slide courtesy of Austin Dobbels

24 Dobbels et al. (2016) G3 7: Fast neutron mutation shows elevated sucrose over five years

25 Dobbels et al. (2016) G3 7: Homozygous Wild-type Homozygous Translocation Heterozygous Translocation

26 The genetic pipeline Soybean plant transformation

27 Typical The soybean genetic transformation pipeline Normal soybean Transformed soybean Gene of interest

28 Typical The soybean genetic transformation pipeline Normal soybean Transformed soybean Gene of interest

29 Transformation/segregation with The genetic pipeline genome editing Normal soybean KASI CRISPR/Cas9 transformed; KASI

30 Transformation/segregation with The genetic pipeline genome editing Normal soybean KASI CRISPR/Cas9 transformed; Mutates KASI KASI

31 Transformation/segregation with The genetic pipeline genome editing Normal soybean KASI CRISPR/Cas9 transformed; Mutates KASI KASI

32 Transformation/segregation with The genetic pipeline genome editing Normal soybean KASI CRISPR/Cas9 transformed; Mutates KASI KASI kas1 KAS1 kas1

33 Transformation/segregation with The genetic pipeline genome editing Normal soybean KASI CRISPR/Cas9 transformed; Mutates KASI KASI kas1 KAS1 kas1

34 Gene editing results for KasI Target site 1 Target site 2 Target site1 WPT# Sequenced clones 677-3_shoot _shoot indels

35 Plant Biotechnology Journal Volume 12, Issue 7, pages , 23 MAY 2014 DOI: /pbi The genetic pipeline An excellent example in soybean Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family

36 The genetic pipeline An excellent example in soybean High oleic is an interesting case study because it has been modified using all four methods: Conventional breeding Mutation breeding GMO breeding (RNAi) Gene editing

37 Conclusions and long-term outlook No tidy conclusions, but products emerging There are many ways to develop traits and the methods are continuously evolving CRISPR offers a powerful tool in the toolbox Knowing the function of genes underlies this power; reminds us about the importance of basic research When, how, and who develops these traits/products will largely be determined by how the technology is regulated Domestic and international markets

38 Thank you!

39 Extra slides

40 Acknowledgments Genetics and Informatics Ben Campbell, Austin Dobbels, Jean-Michel Michno, Tom Kono, Fengli Fu, Suma Sreekanta, Fernanda Rodriguez, Anna Hofstad, Jeff Roessler Transgene/CRISPR group Shaun Curtin, Jun Liu, Kamaldeep Virdi, Adrian Stec, Yer Xiong, Xiaobo Wang, Nicole Mihelich, Daniele Peglow, Samatha Gunapati Collaboratoring labs Dan Voytas, Gary Muehlbauer, Carroll Vance, Seth Naeve, Peter Morrell, Wayne Parrott, Scott Jackson, Gary and Bing Stacey, Tom Clemente, Jamie O Rourke, Francois Belzile, many FN users

41 Resequencing of biotech types Project participants Peter Morrell Bob Stupar Wayne Parrott Scott Jackson

42 Resequencing of biotech types

43 Lps1 candidate gene PF03140: Plant protein of unknown function (DUF247) (IPR004158) Expressed: pod, stem, flowers VIGS silencing Average petiole lengths of the top five leaves (cm) Plants with leaflets removed Slide courtesy of Ben Campbell and Jamie O Rourke 0.0 LPSH_EV Empty Vector LPSH Construct Empty Vector Construct

44 Lps1 candidate gene PF03140: Plant protein of unknown function (DUF247) (IPR004158) High expression in pod, stem, and flowers VIGS silencing shows short petiole Extra Lysine added at position 480 Slide courtesy of Ben Campbell Non-cytoplasmic Domain Transmembrane Domain Cytoplasmic Domain

45 Calyxt traits (Dec 2017) Soybean High Oleic Reduced Trans Fat in Oil Wheat High Fiber Herbicide Tolerant Gluten Reduced Canola Lower Saturated Fat Potato Low Acrylamide