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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
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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.
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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
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http://augureye.blogspot.com/2013_03_01_archive.html
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• 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
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• 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
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• 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
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© 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
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• 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
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© 2013 American Society of Plant Biologists
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
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© 2013 American Society of Plant Biologists
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
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© 2013 American Society of Plant Biologists
Cisgenics can silence genes
RNA silencing
ON
OFF
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
DNA
RNA
Protein
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• Arctic® Apple (PPO enzyme gene silenced)
Arctic® Apple
• Regular Apple (PPO enzyme is active
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• 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/
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• 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/
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• 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)
Innate® Potato Generation 1
http://www.innatepotatoes.com/
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© 2013 American Society of Plant Biologists
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
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Parent A (low performing
variety with disease resistance)
Parent B (elite variety)
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• 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.
Innate® Potato Generation 2
http://www.innatepotatoes.com/
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GMO Answers: Arctic® Apples and Innate® Potatoes
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• 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 in the genome is edited or deleted
Changing technologies
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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
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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)
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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
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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.
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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.
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RNA-guided engineered nucleases
• Easy design, preparation and cost-effective
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You cannot save the world if you do not know how things work at the basic
level
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A YouTube link that will take you to more movies!
• https://www.youtube.com/watch?v=2pp17E4E-O8&feature=share&fb_ref=share
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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)
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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)
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Cibus: Non GMO Roundup Resistant Flax
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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)
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Col-0 bas1 sob7 ben1 ugt73c5 CRISPR: Learning how plants work
Neff Lab: Unpublished
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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?
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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
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Man has been manipulating DNA in plants and animals for millennia
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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.
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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