advances in crop biotechnology- cisgenics and genome editing · • genes can be edited or replaced...

Advances in Crop Biotechnology- Cisgenics and Genome Editing

Michael M. Neff Ph.D.

[email protected] Washington State University

Department of Crop and Soil Sciences Molecular Plant Sciences Graduate Program

Thoughts from previous talk   Many examples of GMO bacteria in medicine (e.g. insulin,

taxol) and food (vitamins, chymosin for making cheese)

  Some people fear GMOs- photoshop, misinformation, lack of trust

  Correlation does not equal causation

  GM food production is increasing

  Monsanto is one of the smallest of the large agribusiness companies, about the same size as Whole Foods.

  No credible, reproducible, scientific evidence that the GMO process is dangerous for us. Need case-by-case analysis.

  Some GMOs could benefit organic agriculture.

Examples of GMO crops Crop Trait Process Organic?

Papaya (and summer squash)

Virus Resistance “Vaccination” by expressing a piece of the virus DNA. Silences Virus

Could benefit organic: High use in Hawaii has lowered virus levels to allow organic production. “Herd immunity”

Corn, soybean, cotton

BT-mediated insect resistance

Expressing a bacterial (BT) toxin that is specific to particular insect pest.

Could benefit organic: Toxin and bacteria sprayed in organic production settings

Corn, soybean, cotton, sugar beets, alfalfa, canola

Herbicide resistance Depends on mode of action for herbicide

Not for organic but useful in no-till farming

http://augureye.blogspot.com/2013_03_01_archive.html

•   Frustrated with organizations that systematically attack GMO technology without learning about the science •   Cherry picking data •   False statements •   Use of social media

•   Use internet, often with industry support, to address public concerns about GMOs. Educate public: •   GMO Answers (www.gmoanswers.com) •   Genetic Literacy Project

(www.geneticliteracyproject.org) •   Cornell Alliance for Science

(www.allianceforscience.cornell.edu) •   Industry involvement still causes skepticism •   Developing new technologies that alleviate some concerns.

Scientists are trying to help

•   Old technology: Transgenics •   Introducing DNA from one non-closely related species to

another •   Herbicide tolerance and BT-insect resistance (both

bacterial genes expressed in plants) •   New technology: Cisgenics •   Introducing DNA from the same species or a closely

related species •   Arctic® Apple and Innate® Potato •   Using a process called RNAi to silence a gene •   Also can introduce or over-express a gene

•   New technology: CRISPR/Cas9-based gene/genome editing •   Sometimes call Subgenic •   A gene is edited or deleted

Changing technologies

© 2013 American Society of Plant Biologists

Emerging technologies circumvent some concerns about transgenics

In the conventional approach to transgenic plant production, a large piece of DNA, derived from several sources, is inserted randomly into the genome

Open Reading Frame 5′ UTR 3′ UTR Promoter

Plant genome Transgene

cassette

The promoter, 5′ and 3′ untranslated regions (UTRs) and protein coding region may all come from different sources

All the cells carry the transgene





•   New technology: CRISPR/Cas9-based gene/genome editing •   Sometimes call Subgenic •   A gene is edited or deleted



Cisgenics: Genes from the same or closely-related species

Schouten, H.J., Krens, F.A. and Jacobsen, E. (2006). Cisgenic plants are similar to traditionally bred plants. EMBO Rep. 7: 750-753.

Apple gene

Apple gene Apple gene

That might mean that little foreign DNA is introduced (“cisgenic”)

Some traits can be modified by the introduction of a cisgene – a gene from the same or closely-related species

Or, bacterial and viral DNA may be included, but no protein-coding regions from other organisms (“intragenic”)

Small amounts of DNA from T-DNA borders may be incorporated


Cisgenics can add or silence genes

RNA silencing

ON

OFF

Resistance gene

One application of cisgenics is to add resistance genes

Advantages: Avoids lengthy backcrossing process Particularly useful for plants propagated vegetatively, such as potato or apple

Disadvantages: Gene must exist in gene pool

Gene silencing can be induced by introduction of antisense or hairpin RNA, or overexpression

of an endogenous gene

Silencing construct

The gene that causes cut apples to turn brown can be silenced

See for example Arctic Apple


Cisgenics can silence genes

RNA silencing

ON

OFF



Silencing construct



DNA

RNA

Protein

•   Arctic® Apple (PPO enzyme gene silenced)

Arctic® Apple

•   Regular Apple (PPO enzyme is active

•   Arctic® Apple (PPO enzyme gene silenced) •   Developed by Okanagan Specialty Fruits Inc. as their

flagship product •   Arctic® Golden and Arctic® Granny (Gala and Fuji next)

Arctic® Apple

http://www.okspecialtyfruits.com/our-science/apple-browning/

•   Innate® Potato Generation 1 (PPO enzyme gene silenced) •   Developed by Simplot Plant Science (J.R. Simplot

Company) •   Russet Burbank Potatoes with reduced browning (44%)

Innate® Potato Generation 1

http://www.innatepotatoes.com/

•   Innate® Potato Generation 1 (PPO enzyme gene silenced) •   Russet Burbank Potatoes with reduced browning (44%) •   Also reduces acrylamide (52 to 78%) when baked, fried or

roasted at high temps (a neurotoxin and carcinogen)




Cisgenics can add or silence genes

RNA silencing

ON

OFF

Resistance gene

One application of cisgenics is to add resistance genes

Advantages: Avoids lengthy backcrossing process Particularly useful for plants propagated vegetatively, such as potato or apple

Disadvantages: Gene must exist in gene pool



Silencing construct



Parent A (low performing

variety with disease resistance)

Parent B (elite variety)

•   Innate® Potato Generation 2 (PPO enzyme gene silenced and late blight resistance gene from wild relative added)

•   Late blight is caused by Phytophthora infestans and is the disease that led to the Great Irish Famine in 1840s.



GMO Answers: Arctic® Apples and Innate® Potatoes





•   New technology: CRISPR/Cas9-based gene/genome editing •   Sometimes call Subgenic •   A gene in the genome is edited or deleted


DNA editing techniques Possible to carry out controlled genome modifications to create desirable mutations

Techniques are efficient and specific

Via targetable DNA cleavage…

…that uses the cellular DNA repair pathways

Targetable nucleases/cleavage reagents

•   Four major classes •   Zinc-finger nucleases (ZFNs) •   Transcription activator-like effector nucleases

(TALENs) •   Meganucleases •   Clustered regularly interspaced short

palindromic repeats (CRISPRs)

Targetable nucleases/cleavage reagents

•   Key requirement: protein nuclease (an enzyme that cuts DNA) + targeting mechanism

•   ZFNs = DNA-binding modules from

transcription factors

•   TALENs = DNA binding modules from

bacteria

•   CRISPR = RNA-guide for nuclease

Zinc Finger Nucleases •   Consists of ZFP domain and a nuclease domain

(FokI) = ZFN •   Zn Fingers provide specifity •   Zn Fingers with desired specifities can be

constructed

Kim and Kim, 2014. Nature Reviews.

TALENs •   Consists of TALE domain and a nuclease domain

(FokI). TALE domain from Xanthomonas spp bacteria proteins

•   Domains can be engineered to bind predetermined DNA sequences

Kim and Kim, 2014. Nature Reviews.

RNA-guided engineered nucleases

•   Easy design, preparation and cost-effective

You cannot save the world if you do not know how things work at the basic

level

A YouTube link that will take you to more movies!

•   https://www.youtube.com/watch?v=2pp17E4E-O8&feature=share&fb_ref=share

What can we do with CRISPR/Cas9? •   Genes can be deleted (knocked out) •   Genes can be edited or replaced with altered

sequences •   Genes can be silenced (like RNAi but by a different

mechanism) •   Genes can be over-expressed •   Multiple genes can be targeted at one time •   We can both learn how plants work (fundamental

knowledge) and use that information to change crops (knowledge applications)

Cibus: Non GMO Roundup Resistant Flax

What can we do with CRISPR/Cas9? •   Genes can be deleted (knocked out) •   Genes can be edited or replaced with altered

sequences •   Genes can be silenced (like RNAi but by a different

mechanism) •   Genes can be over-expressed •   Multiple genes can be targeted at one time •   We can both learn how plants work (fundamental

knowledge) and use that information to change crops (knowledge applications)

Col-0 bas1 sob7 ben1 ugt73c5 CRISPR: Learning how plants work

Neff Lab: Unpublished

Genome editing controversy

•   Is this just another form of GMO? •   Is it ethical to alter a plant genome? •   Is it ethical to alter other genomes?

Man has been manipulating DNA in plants and animals for millennia

All due to mutations and genomic alterations. All required human intervention for breeding and/or selection

Man has been manipulating DNA in plants and animals for millennia

Looking to the Future: •  Organic definition includes GMO-free •  Can GMO approaches help organic farmers? •  Can GMO approaches facilitate sustainable farming and sustainable living? •  Can GMO approaches help remove dependence on fossil fuels? •  Can an “open source” approach be used with GMOs?

•  All of this should be open to discussion.

Some final talking points ��[email protected]

•   Empower through education but don’t expect attitude changes

•   Be passionate and stay committed •   Be credible and listen •   Adapt to your audience •   Be aware of your effects

•   Use all channels •   Collaborate

advances in crop biotechnology- cisgenics and genome editing · • genes can be edited or replaced...

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