Precision Plant Breeding With CRISPR Genome Editing .
Precision Plant Breeding With CRISPRGenome Editing: Opportunities andChallengesAlfred HuoMid-Florida Research and Education CenterUniversity of Florida
Current Challenges7.7 B 8.5 B by 2030 9.7B by 2050Urban sprawl encroaches rapidlyon farmlandHunger
Current ChallengesExtreme Weather/ Climatic ChangeFloodDroughta home is surrounded by floodwatersfrom Tropical Storm Harvey in Spring,TexasA Texas State Park police officer walked across the lakebed of O.C. Fisher Lake in San Angelo, Texas. 2012
Current ChallengesExtreme Weather/ Climatic ChangeHeatHeat Wave, July 2018Tornado
Current ChallengesExtreme Weather/ Climatic ChangeIce covers the Lake Michigan shoreline on January30 in Chicago 2019Temperature increase at North Pole
Current ChallengesExtreme Weather/ Climatic ChangeHurricaneDevastated buildings damaged by theHurricane Irma, 2017Wild FireWildfire on the hillside in Burbank, Calif. 2018
Objectives of Plant Breeding Primary objective is to increase crop yield and improve quality of crop produceWeed ControlIntegrated Pest ManagementHerbicide resistantSoybeanInsect resistant corn anding-genetic-basisdrought-tolerant-soybeans
Objectives of Plant Breeding Primary objective is to increase crop yield and improve quality of crop produceFertilizer Use Efficiency (N, P, K)Water Use EfficiencyDrought Sensitive Solution for limiting resources (e.g. P) Reduce energy and cost Reduce environmental impactDrought TolerantSoybean plants subjected to drought tanding-genetic-basisdrought-tolerant-soybeans
2016 CRISPR/WAX Corn
Plant Domesitcation Plant domestication: selection by nature powerDesired traits: Larger grain, high yield, less bitterness
Plant Domestication?
Plant Domestication?
Hybridization Breeding: Basic ConceptsSkin color gene
Hybridization BreedingHybridization breeding Depends on genetic variation
Crossing Different Parents to Create New Variations
Hybridization Breeding The pug and beagle have been bred to produce the ‘puggle’,a mixed breed with both pug and beagle traits. Introduce desirable traits from one parent to another Selection based on morphological characteres With aritifical intervention
Hybridizations Breeding: Backcrossing to Clean Up Genome
18Hybridizations Breeding: Backcrossing to Clean Up GenomeF1100% 3.38%:96.62%Tolerant todroughtLowerquality plantSensitiveto droughtGOAL100% high quality plant drought toleranceHigherqualityplant100% B
Linkage Drag: Nightmare for Traditional Plant BreedersGood FlavorDisease SusceptibilityNever hasprogenies withgood flavor andbetterpostharvestquality, diseaseresistant tomatoGood FlavorPoor postharvestqualityHigh Yield
Pros and Cons of Hybridization BreedingPros: No requirement for knowing genetic and genomic background Not regulated by USDA, ecological, environmental friendly Straightforward phenotypic selection, technically easyCons: Heavily relies on genetic variation, may not exist in nature Time and labor consuming compatibility, embryo lethality Quantitative traits are hard for selection Linkage drag
Mutation Breeding Spontaneous mutations: continuous source of natural geneticvariation Traditional mutagenesis: inducing mutations by radiation or chemicalmutagens-Large population of mutagenized plants needed (5.000-10.000)
Traditional MutagenesisPopulation of plants withgenome-wide mutations(e.g. 17,000x)Select plantswith mutationsin targetedgeneFurther crossing toremove undesirablemutations and toobtain optimalvarieties
Examples of Mutation BreedingBad mutationsSeedless orangeSalt Tolerant RiceDiverse colorful flowersBad mutationsDiverse colorful flowersFoliage with different leaf pattern
Pros and Cons of Mutation BreedingPros: Induction of desirable mutant that is not present in natural plant materials Not regulated by USDA, ecologically, environmentally friendly Straightforward phenotypic selection, technically easyCons: generally random and unpredictable “good” mutations come with “bad” mutations Need large mutant pool to identify “good” one Costly and Slow
Molecular Marker Assisted Selection Breeding Marker assisted selection refers tothe use of DNA markers that aretightly-linked to target loci Assumption: DNA markers canreliably predict phenotype
Pros and Cons of MAS BreedingPros: Similar to traditional breeding, not regulated by USDA Accelerating breeding process Easier for stacking multiple traits within the same cultivarCons: Must know genomic and genetic background Very costly False markers
Genetic Engineering
Are these crops GMOs?riceSoybeanSafflowerChicoryFlax seedsCanolaAlfalfaCreep BentgrassTobaccoPolarPlumEucalyptus
Are these crops GMOs?riceSoybeanSafflowerChicoryFlax seedsCanolaAlfalfaCreep BentgrassTobaccoPolarPlumEucalyptus
How many approved GMOs in U.S.?525rice233Soybean33Chicory222308203 approvedVarieties for 21crop 2species4313Flax seedsAlfalfa1Canola1Creep BentgrassTobacco111Plum43
What Traits Were Modified?TraitsCropsAltered lignin productionAlfalfaNon-browningAppleModified fatty acidCanolaHerbicide ToleranceAlfalfa, Canola, Chicory, cotton, creeping bentgrass, flax, corn, rice, soybean, sugar beet,wheatMale sterilityAlfalfa, Chicory, corninsect-resistanceCotton, corn, potato, rice, soybean, sugar cane, tomatoNutritionCanola, corn, potato, rice, soybeanDrought toleranceCornDelayed ripeningMelon, tomatodisease resistancePapaya, potato, squashModified flower colorroseYieldsoybeanNicotine reductiontobacco
GMO ExamplesDeveloped by AustraliaApproved in Australia, Japan, European UnionModified color ( genes from petunia)Herbicide resistance (mutated gene from tobacco)
GMO ExamplesRingspot Virus-resistantPapayaSusceptible plantsRingspot virusresistant papaya
GMO ExamplesStopping Citrus Greening
Development Of Glowing Ornamental PlantsLED(365nm) UV lightWTeGFPeYGFPuvScientific Reports (2018) 8:16556
How Are GMOs Created?Insectresistanttomato
Pros and Cons of Genetic EngineeringPros: Fast way to verify gene function precisely modify crop productivity and qualityCons: Necessary to know gene function Very costly, complicated procedures Heavily regulated by USDA
40The Latest Innovation: CRISPR/Cas9 Genome EditingCRISPR:Clustered Regularly Interspaced Short Palindromic RepeatsCRISPR Loci induce acquired immunity in Bacteria against the virus infection orplasmid transferHorvath and Barrangou, 2010, Science: 167-170
Features of CRISPR/Cas9 Genome Editing High precision, High efficiency, Broad application Procedures are identical to genetic modification Final products are similar to traditional breeding
High Precision: no or less non-targeted mutationsTraditional Mutagenesis Vs Targeted MutagenesisPopulation of plants with genome-wide mutations(e.g. 17,000x)Traditional mutagenesisA few plants with targeted mutationsTargeted mutagenesis
How CRISPR/CAS9 Can Create Precise Mutation?Cas9: one proteinCan recognize the unique locationGuided sequencesTurn offTurn on
Turn Off or Turn OnDisease susceptible cultivarGenesDisease sensitive geneDisease resistant cultivarGenesDisease resistant geneGenesDisease susceptible cultivarGenesDisease resistant cultivar
PrecisionALS2 gene editing for herbicide resistant corn Ability to target ALS2, but not sister ALS1, for herbicide resistance in cornALS1ALS2Herbicide-resistant
High Efficiency: Multi-TargetingSimultaneous targeting of multiple genesIn rice, more than 8targets weresuccessfully targeted
High Efficiency: Time
High Efficiency: Time Significantly reduce the number of crossing cycles Significantly reduce plant number for each generation
High Efficiency: Time
Broad applicability: Turn Off a Disease GeneMLO1 gene, controlling powder mildewsensitivity, has been identified in wheat,barley, rice, tomato, petunia, tobacco,eggplant, cucumber, squash, melon,strawberry, lettuce, orange, and more.Use CRISPR to knock out MLO1gene to create powdery mildewpathogen resistant tomato
Broad applicability: Multiple Plant Species
Broad applicability: Multiple Plant SpeciesIn collaboration withDr. Deng in Gulf CoastRec, SWEET gene hasbeen edited for testingcitrus canker diseaseresistance.Gene editing of OsSWEET14 to createBlight(Xanthomonas)- resistant riceSimilar strategy can beemployed to otherplant species
Broad applicability: polyploidy plantsSimultaneous targeting of multiple copies of samegenes in polyploidy cropsWT and tadep1-aabbddmutant (high yield)
High Gene Editing Efficiency in Allotetraploid Tobacco and Octoploid StrawberryAllotetraploid TobaccoOctoploid StrawberryIn collaboration with UF/IFAS Gulf Coast Rec
Non-GMO: NO Foreign DNAGMO approachNon-GMO approach
Breeding Non-GMO Lettuce with CRISPR-Genome EditingUse your eyes to find Non-GMO plantsLBNPT-GFP::pCassavaMosaic VirusControlpUBI::HypaCas9ProAtU6::crRNA-2 x BsaI-crRNAGene EditedLettuce Seeds are inhibited byFlorida high temperature, butgene edited lettuce can germinatewellF2 Segregated SeedsRB
Complimentary To Traditional Breeding : Solution to Linkage DragNever hasprogenieswith goodflavor Good FlavorDisease SusceptibilityGood FlavorPoor postharvestqualityHigh Yield
Complimentary To Traditional Breeding : Solution to Linkage DragGood FlavorCIRSPR EditingDisease SusceptibilityGene editingcan removelinkage dragto createbetter plantsGood FlavorPoor postharvestqualityHigh Yield
How Different Is Between Conventional Breeding AndCRISPR Genome Editing?RadiationmutagenesisConventional BreedingGMOGenome Editinggenetic modificationYES, Artificial (e.g.radiation)Yes, by nature (e.g. reshuffling ofDNA fragment or recombinationYes, TransgeneYes, transgeneTurn gene offyesyesyesyesTurn gene onNo(extremely rare)yesyesyesForeign DNANoNoyesNo (case by case)Distinguished fromnoconventional breeding-yesnoEfficiencylowlowhighVery High (mainlyLowfor dealing withregulation)Difficulties ofprocedureseasyeasycomplicatedcomplicated
Rise of Private Sector in Application of GenomeEditing Techniques for Crop ImprovementCompanyLocationBenson HillBiosystemsSt. Louis,MOYearSelected Tools/ServicesFocus CropsEstablished2012CROP-OS software;Row crops edited for higher yield, stressgene editing usingresistance, and herbicide toleranceCRISPR-Cpf1 and -Cms1CortevaWilmington,2018(DowDuPont) DECRISPR-Cas9Waxy corn modified for altered starchcompositionPairwiseDurham, NC 2018Row crops such as corn and soybeans withCRISPR-Cas9 with baseincreased productivity, disease resistance;editingmore-convenient fruits and Corn, soy, wheat, tomato, sunflower, modifiedto increase yieldTropicBiosciencesNorwich, UK 2016CRISPR and othertechniquesDisease-resistant bananas, decaffeinated coffeeYield10BioscienceWoburn, MA 2015CRISPR-Cas9Camelina engineered for higher oil content
Rise of Public Sector in Application of Genome EditingTechniques for Crop ImprovementNovember 20, 2018.
USDA-Regulated?
USDA-Regulated?Not regulated as long as they are not plant pests or developed using plant pests. Noxious weedThis would include plant varieties with the following changes: Deletions Single base pair substitutions Insertions from compatible plant relatives (foreign DNA from bacteria, insects,virus etc. will be still regulated) Off-springs of a genetically engineered plant that does not retain the change ofits parent
USDA Authorizations: Products Of Genome EditingYearNumber ofPermits 9012462Total354Dupont/Pioneer aim to launch the firstCRISPR-enabled waxy corn around 2020.Consulted by FDA
Food And Drug Administration (FDA) -Regulated? All food is regulated, regardless of how plant varieties are bred No unique requirements exist for food developed with biotechnology All food must meet universal regulatory requirements-All food must be safe-GE food labelling? Yes-Genome-Edited food labeling? probablyGE soybeanoil
Global Regulatory StatusEurope: YES
Challenges Availability of Genomic Sequence Efficient plant transformation pipe line DNA-free genetic transformation method forperennial plant species
IP Licensing Agreements LandscapeSimplotCalyxtFree for academicresearchers
Social Acceptance And Ethical Concerns Consumer acceptance Ecological concern Health Concern Ethical concern
Summary Genome editing requires similar procedures used for GeneticEngineering (GMOs), yet creates precise mutation in plant genomescontaining non-foreign DNA Resulting products are indistinguishable from products of naturalvariability or mutagenesis, yet genome edited plants are regulated byUSDA in a manner of case-by-case Limitation of genome editing application are plant transformationpipeline and genome availability Both public and private sector have significant opportunities to applygenome editing in their breeding programs
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Pros and Cons of Mutation Breeding Pros: Induction of desirable mutant that is not present in natural plant materials Not regulated by USDA, ecologically, environmentally friendly Straightforward phenotypic selection, technically easy Cons: generally random and unpredictable “good” mutations come with “bad” mutations
Adding CRISPR to your Bio ARROW Protocol Page 2 of 6. Work Covered by this Guidance Document: This guidance document covers how to add the use of CRISPR systems (e.g., CRISPR/Cas9, CRISPR/Cpf1) – whether for genome editing or other purposes (e.g., CRISPR-mediated
CRISPR ethics and vice versa. Third, we assess several key ethical considerations. Notably, while some of these concerns are specific to CRISPR technology, many, such as research on human embryos, have been debated long before the CRISPR revolution [15]. Moreover, since CRISPR is still a maturing technology, novel applications in
CRISPR-EZ: CRISPR- RNP Electroporation of Zygotes Chen et al., JBC, 2016 CRISPR-EZ Advantages 100% Cas9 RNP delivery Highly efficient NHEJ and HDR editing indel, point mutation, deletion, insertion 3x increase in embryo viability Easy, economic and high-throughput CRISPR-EZ Challenges Larg
industrial applications. Finally, the efforts of researchers paid off with the development of CRISPR, a robust molecular tool that can edit DNA at virtually any locus. CRISPR technology is igniting a revolution across the life sciences and is quickly becoming a standard tool in many labs. Given its ease-of-use and versatility, CRISPR is already .
nents comprising CRISPR genome editing, each of which is considered next. Components of CRISPR Genome Editing Component 1: Cas9 Endonuclease The most common endonuclease used in CRISPR genome editing is the class II effector protein, Cas9, from S pyogenes (
Medicinal and aromatic crops 36 2.2.7. Fruits 37 2.2.8. Vegetables 39 2.2.9. Forecast data for the production of basic cereals and oilseeds from the 2020 harvest 44 2.3. Livestock Breeding Production Results 45 2.3.1. Cattle Breeding and Buffalo Breeding 50 2.3.2. Sheep Breeding and Goat Breeding 53 2.3.3. Pig Farming 56. 2.3.4. Horse Breeding 59
the production of eective compounds [11]. e applica-tion of the CRISPR-Cas system to gene functional studies and metabolic networks regulation of medicinal plants is essential and meaningful, presenting a promising method for improving quality and breeding ideal germplasms in medicinal plants. Here, we review CRISPR-based tools and briey intro-
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