Advances in Crop Biotechnology- Cisgenics and Genome Editing Michael M. Neff Ph.D. 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 mmneff@wsu.edu Washington State University Department of Crop and Soil Sciences Molecular Plant Sciences Graduate Program 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 Scientists are trying to help 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. Changing technologies 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 1
Changing technologies 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 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 Transgene cassette Plant genome Promoter 5 UTR 3 UTR Open Reading Frame The promoter, 5 and 3 untranslated regions (UTRs) and protein coding region may all come from different sources All the cells carry the transgene Changing technologies 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 Cisgenics: Genes from the same or closely-related species 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 proteincoding regions from other organisms ( intragenic ) Small amounts of DNA from T-DNA borders may be incorporated Apple gene Schouten, H.J., Krens, F.A. and Jacobsen, E. (2006). Cisgenic plants are similar to traditionally bred plants. EMBO Rep. 7: 750-753. Cisgenics can add or silence genes Resistance gene Gene silencing can be induced by introduction of antisense or hairpin RNA, or overexpression of an endogenous gene Cisgenics can silence genes DNA Gene silencing can be induced by introduction of antisense or hairpin RNA, or overexpression of an endogenous gene One application of cisgenics is to add resistance genes Silencing construct ON RNA silencing OFF RNA Silencing construct ON RNA silencing OFF Advantages: Avoids lengthy backcrossing process Particularly useful for plants propagated vegetatively, such as potato or apple Disadvantages: Gene must exist in gene pool The gene that causes cut apples to turn brown can be silenced Protein The gene that causes cut apples to turn brown can be silenced See for example Arctic Apple See for example Arctic Apple 2
Arctic Apple Arctic Apple (PPO enzyme gene silenced) Regular Apple (PPO enzyme is active Arctic Apple 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) http://www.okspecialtyfruits.com/o ur-science/apple-browning/ Innate Potato Generation 1 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 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) http://www.innatepotatoes.com/ http://www.innatepotatoes.com/ Cisgenics can add or silence genes 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 ON RNA silencing OFF The gene that causes cut apples to turn brown can be silenced Conventional Plant Breeding Parent A (low performing Parent B variety with disease (elite variety) resistance) vs Genetic Engineering Transgene See for example Arctic Apple 3
Innate Potato Generation 2 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 http://www.innatepotatoes.com/ Changing technologies 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 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) 4
Targetable nucleases/cleavage reagents Key requirement: protein nuclease (an enzyme that cuts DNA) + targeting mechanism ZFNs = DNA-binding modules from transcription factors 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 TALENs = DNA binding modules from bacteria CRISPR = RNA-guide for nuclease 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 RNA-guided engineered nucleases Easy design, preparation and cost-effective Kim and Kim, 2014. Nature Reviews. 5
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) 6
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) CRISPR: Learning how plants work Col-0 bas1 sob7 ben1 ugt73c5 Neff Lab: Unpublished Genome editing controversy Man has been manipulating DNA in plants and animals for millennia Is this just another form of GMO? Is it ethical to alter a plant genome? Is it ethical to alter other genomes? All due to mutations and genomic alterations. All required human intervention for breeding and/or selection 7
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 mmneff@wsu.edu 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 8