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Professor Steve StraussCoop Director

GREAT TREESA proposal for Cooperative Research in Gene Editing & Genetic Transformationof Eucalypts at Oregon State University

2Links to documents here

Oregon State Forest Biotech Coop: Nearly 25 years of success

• Coop will complete its 25th year in June 2019• Current proposal is for a sixth 5-year phase:

July 2019 – June 2024• Proposal builds upon many Coop successes

– Eucalypt transformation– Flowering modification– Field trials– Genomics– Gene editing– Publications and presentations– Millions in leveraged grants

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What is genetic engineering (GE)

• Direct modification of DNA– vs. indirect modification

in breeding• Asexually modified in

somatic cells– Then regenerated into

whole organisms, usually starting in Petri dishes

• Regeneration of modified cells into organisms is the main obstacle

Genetic modification of forest trees can have a variety of proven, stable benefits based on field

studies – we would like to use it

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Gene editing is a powerful new form of genetic modification

Plant gene editing may have large impacts – avoid some GMO problems?

“CRISPR/Cas9 is a game-changing technology that is poised to revolutionize basic research and plant

breeding.”

The capacity for transformation/editing of eucalypts, and most other forest trees,

greatly limits commercial potential

• The great variation in response among genotypes and species in real breeding and plantation programs stymie most real-world applications

• The genetic engineering method is powerful and reliable, but does not scale beyond research

• Years of research with conventional approaches have not yielded breakthroughs

• We need a new tool, a new approach, that might provide a quantum leap in capacity 8

Concept for innovation in transformation and gene editing

• New focus area: Innovation in tree transformation and gene editing in eucalypts– Urgent need for improved methods if gene editing and

genetic engineering to be applied to the diverse genotypes and species important to forestry

• Approach: Use of regeneration and transformation stimulating genes, in vitro or in vivo – Promotes regeneration of the modified cells – often a

critical problem in eucalypts– Avoid or reduce use of antibiotics? A major stressor for

eucalypt tissue culture 9

Concept for innovation in transformation and gene editing

• Goals: To promote transformation rate, reduce genotype variability, reduce costs

• Why now?: Great success in application of genes as transformation reagents in monocots and model species, through to complete transformation system

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DuPont Pioneer breakthrough advances

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Pioneer construct types that promote regeneration of transformed cells than are removed

Regeneration inducing genes

Excision inducing genes

Genes stimulate regeneration and/or transformation, and reporter genes mark

the process

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Pioneer system continues to advance, further inspiring confidence in success

• Posters/talks at Society for In Vitro Biology meeting in Raleigh, NC

• More rapid• Less genotype specific• Excision not essential to obtain fertile plants• Workable in many species• Workable in leaf as well as seed tissues• Pioneer now working to adapt the system to

dicots14

Growing interest and success with approachLiterature review revealed 120 relevant peer-reviewed publications

from 1988-2018, with spike in 2017

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Many gene options to explore forwhat works best in eucalypts

Examples of genes whose modified expression enhances regeneration of transgenic callus, shoots, roots, or embryos in some plant species

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Gene or gene family Relevant organsLEAFY COTYLEDON Shoot/embryo

BABY BOOM Shoot/embryoRESPONSE REGULATOR shoot

ENHANCER SHOOT REGENERATION (ESR/EBB1) shootWUSCHEL shoot, root, embryo

WUSCHEL-ASSOCIATED HOMEOBOX shoot, root, embryoMITOGEN-ASSOCIATED PROTEIN KINASE callus

TARGETING PROTEIN FOR XKLP2 callus

Examples of success include poplar

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• Overexpression of BBM in Populus tomentosa led to development of somatic embryos from callus (Deng et al., Plant Science, 2009)– Somatic embryos regenerable, developed into whole plants

• Can use either embryogenesis or organogenesis pathway?– In monocots, BBM overexpression can lead to a mix of

organogenic and embryogenic structures (Lowe Plant Cell 2016)

EBB1 gene discovered in Strauss lab by activation tagging, promotes organogenesis

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EBB1 (ESR) overexpression promotes shoot organogenesis in poplar and Arabidopsis

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Preliminary studies suggest success in cottonwood

Michael Nagle PhD work, eucalypts also included in ongoing work

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57%

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10%

20%

30%

40%

50%

60%

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CTR

N=3

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EBB1

N=6

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LEC1

N=5

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WU

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Expl

ants

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vis

ible

GFP

Nisqually-1Replicate 1

0%

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18%

3%

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5%

10%

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20%

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CTR

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EBB1

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LEC1

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Nisqually-2Replicate 2

Comparison of cottonwood with and without developmental gene overexpression

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Stem explants with control vectors (including GFP)

Stem explants with LEC1 (left), EBB1 (right) experimental vectors (including GFP)

Experimental elements

• Develop model eucalypt genotypes and transformation system (easy to difficult) – possible collaboration with U. Pretoria/Myburg

• Identify genes and promoters in literature, bioinformatics databases, and by our own experiments– Mutants, transcriptomics, GWAS results

• Test system control elements (promoters, recombinases, insulators, etc)

• Integrate system elements to produce platform constructs– Transformation and gene editing versions

• Test in diverse genotypes/species• Apply to develop an efficient gene editing and removal system

– Demonstrate effectiveness in different genotypes/species• Test genes in high throughput system using a new phenomics

platform developed as part of major NSF GWAS grant (4 million)22

Elements of new phenomics platform

• High throughput in vitro imaging system• Custom product by Middleton Spectral,

Wisconsin– Visible light (RGB)– Hyperspectral, fluorescent camera

• Image decomposition and quantification– Tissue/organ development– Transformation rate as reported by fluorescent

reporter genes like GFP• Machine vision systems (Fuxin Li, Computer

Science, OSU)23

Automated imaging system overview (Middleton)

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Automated imaging system detail (Middleton)

Hyperspectral component decomposition (Middleton)

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Machine vision analysis of tissue development (Li)

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Timeline: 5-year agenda to study enhanced regeneration and transformation

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Objectives Years

Develop non-proprietary eucalypt model transformation systems Examine in vitro and possibly in vivo transformation platforms Transform plants with variety of promoters/genes likely to be

positive regulators of organogenesis or embryogenesis Assay callus induction, shoot regeneration, adventitious rooting and

transformation efficiency in transgenic plants using reporter genes

1-3

Build transformation constructs with transgene excision systems Compare efficiency of excision when induced by heat, drought or

meristematic promoters using reporters3-4

Study additive and synergistic effects of effective transgenes, and variations in promoters to drive them

Test in wider variety of genotypes and species under diverse conditions

Integrate knowledge from tissue culture optimization to develop comprehensive transformation and gene editing system for trees

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Not a simple or easy path - transformation

• Need to develop eucalypt system for study, and system to adapt/match in vitro and/or in vivo systems to genotypes

• Application to eucalypts that do not easily undergo embryogenesis may require different genes/approaches vs. the Pioneer monocot system

• Constructs are large and complex to assemble• Transformation and regeneration experiments are

slow, difficult to replicate, and require many weeks to months for completion 29

Not a simple or easy path – gene editing

• Adaption to produce an efficient gene editing system will require significant research– Rapid technological evolution in this area

• Need to assess or develop means to avoid chimerism with excision system to remove CRISPR locus– Footprint from excision left in genome – regulatory

status unknown

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Applied transformation difficult to fund and difficult to share progress -- Coop a sensible

means to advance and share technology?

• Scope outside of most granting agency programs• Problem of common interest to many companies• However, methods often highly proprietary – thus

much duplication, blind alleys, high costs• Thus need for multi-year, industry cooperative to

make progress on new approaches, create a platform to share relevant results

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Memorandum of Agreement (MOA) and budget

• MOA similar to last Coop phase– MOA emailed to all potential Coop members in March

2018• Expressions of interest from several companies• University has authorized much reduced

administrative costs rate: 17.5% (vs. 48.5%) !!!• Companies can take up to three membership

equivalents – relevant to merger-derived and other mega-companies?

• Historic model: Grant leveraging with Coop– Example: Recent USDA grant for gene editing in

eucalypts and poplar (1/2 million)32

Summary budget options –12 member equivalents

• Transformation technician, 1.0 FTE (full-time equivalent proportion)

• Program Manager, 0.1 FTE• Postdoc, 1.0 FTE• Undergrads for in vitro/greenhouse, ~1.0 FTE• Supplies/pubs/travel, minor eqpt, ~43K/year• Calculated cost = 30K/member per year

(average over 5 years)

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Summary budget composition –5 member equivalents

• Transformation technician, 0.3 FTE (full-time equivalent proportion)

• Program Manager, 0.1 FTE• Postdoc, 0.2 FTE• Undergrads for in vitro/greenhouse, ~1.0 FTE• Supplies/travel/pubs/minor eqpt, ~34K/year• Calculated cost = 32K/member per year

(average over 5 years)

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Questions

• What science or other concerns do you have? • What membership costs do you consider

possible for your company? • What is your time frame for a decision?

– Fall 2018? – First billing planned in February 2019 for

July 1, 2019 –June 30, 2020 fiscal year• How can I further support your company in

making a decision?

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For contact

• Steve.Strauss@OregonState.Edu• (1) 541-760-7357 (cell) or

(1) 541-737-6578 (office)

• Thanks much for your consideration of this proposal!

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