agricultural produc.on: assessment of the poten.al use … not in the rearing facility ... the...
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AgriculturalProduc.on:AssessmentofthePoten.aluseofCas9-mediatedGeneDriveSystems
forAgriculturalPestControl
MaxSco(,FredGould,MarcéLorenzen,NathanielGrubbs,Owain
EdwardsandDavidO'Brochta
GeneralConsideraCons
• GenedriveswouldbeusedforpopulaConsuppression
• TargetswouldbeanessenCalgene,orgenerequiredinthegermlineorforfemaledevelopment
• CondiConalexpressionofCas9wouldfacilitatemassrearingofthegenedrivestrain.
• PotenCalforresistancetodrive:considercombiningwithSIT.
cas9expressedinsomaandgermlineandtargetedagainstageneessenCal
infemales
cas9expressedinsomaandgermlineandtargetedagainstagenerequired
forfemaledevelopment
XY XX
XY XX
Cas9condiConalexpression
LHA GFP Cas9 pApAprom
tetOcpI I
vasap
tTA pA U6p
gRNA RHA
Essential gene or gene required for female devlopment
Cas9
NO tet + tet
Or..targetageneessenCalinthefieldbutnotintherearingfacility
Whiteeyeflywithpoorvisione.g.FieldfemalekillingstrainoftheAustraliansheepblowfly.Femaleshadwhiteeyesandmaleshadwildtyperedeyes
CaseStudies
• NewWorldscrewworm• Spo(edwingDrosophila• Diamondbackmoth• Redflourbeetle• Whitefly
Livestock Wildlife
Pets
ScrewwormDamage
HumansImagesprovidedbySteveSkoda,USDA-ARS
SITProgram:Bisexualrelease…bothsterilemalesandfemales
90%deadorsterileoffspring
FERTILE
STERILE
Sterile Fer.le
1991
199419941995
2000
19981999
19751977
EfficacyoftheSITagainstscrewworms
Before Eradication Program Current Distribution
FromVargas-Teránetal.2005
EsCmatedtotalcostoferadicaConprograminNorthAmerica,through2005,is$1,290million
Estimated annual benefits to producers (in US dollars) from eradication of screwworms:
• USA – $896.1 million • Mexico – $328.6 million • Central America - $87.8 million
$1,312.5million
Estimated annual losses to producers (in US dollars) in South America from screwworms:
• Brazil - $1770 million • Argentina - $618 million • Colombia - $264 million • Uruguay - $210 million • Venezuela - $199 million • All others Countries - $445 million
$3,506million
AdvantagesofageneCcallymodified,transgenicmale-onlystrainwithafluorescentproteinmarkergene
• MoreefficientpopulaConsuppressioninthefieldwithmale-onlyrelease
• Considerablesavingsindietcostsiffemalesdiebeforepupalstage
• EssenCallydoublethecapacityoftheplant• IdenCficaConofreleasedsterilizedfliesusingfluorescentmarker
• IfmalesaregeneCcallysterilized,noneedforradiaContreatment.MalesshouldbemorecompeCCveandcostsreduced.
Ifmale-onlySITisefficientwhyconsideragenedrivesystem?
Current Distribution
SITwouldbechallenginginSouthAmerica
GenomemanipulaCon
• Genomeprojectunderway.CompleConendof2016
• TransgenesisisrouCne• CRISPR/Cas9geneediCngappearstobeveryefficientinNewWorldscrewwormandrelatedblowflies
TargetGene:transformerRNAi-mediatedknockdownoftraRNAinposteriorendleadstomaledevelopment
Lietal(2013)PlosOne
Assumingresistancetodrivewillevolve:considercombiningwithSIT?
Cas9digesConreducedbymutaConsinregioncomplementarytogRNAandeliminatedbymutaConsinPAM
igtrcn.org
Drosophilasuzukii:Spo(edWingDrosophila(SWD)
D.suzukiibiologyInvasionhistory
2008 2009 2010 2011 2012
DetectedinCAraspberries,misidenCfied
RecordspriortoCAidenCficaCon:DamagetocherriesinJapanin1916(Kanzawa1939),DetectedinHIin1980s
CAsamplesIdenCfiedasD.suzukii
DetectedinOR,WA,and
FL
DetectedinNC,SC,UT,LA,MI,WI,andeasternCanada
DetectedthroughoutNewEnglandandMid
AtlanCc
DetectedthroughouttheMidwest
D.suzukiibiologyLifehistoryandpopula.onbiology
+Male Female
Genera.on.me(adulttoadult):10-15daysAdultscanlive1+monthNoknowndiapause
Pupateonornearfruitoroutsideoffruitinthesoil
D.suzukiibiologyGlobaldetec.on.meline
201120122014
D.suzukiibiologyDetec.onsinUnitedStates
D.suzukiiimpactsCrophostpreference
Whenpresentedwith20goffruitinthelaboratory,femaleflieslaid:
3.04eggs/g2.16eggs/g 1.59
eggs/g
1.16eggs/g
Berrysizepropor6onaltonumberofeggslaidduring4hoursofexposure.(Burracketal.2013)
WhenSWDwerepresentedwithonetypeoffruit(withoutachoice),strawberrieswererankedsecondtoraspberries.Analyseswhichincluded
hostvola.lesandoffspringperformancereachedsimilarconclusions(Bellamey,etal.2013).
D.suzukiiimpactsSelectednoncrophosts
Common name Scientific name Ripe fruit period Honeysuckle
Lonicera spp.
7/1 – 10/7
Common blackberry
Rubus sp.
7/8 – 9/16
Bittersweet nightshade
Solanum dulcamara
7/21 – 10/3
Stiff dogwood
Cornus foemina
8/19 – 10/6
American pokeweed
Phytolacca americana
8/26 – 10/7
Silky dogwood
Cornus amomum
8/29 – 10/7
Spicebush
Lindera benzoin
9/8 – 10/7
Autumn olive
Elaeagnus umbellata
9/8 – 10/6
Leeetal2015.
D.suzukiiimpactsEconomiccosts
Zerotoleranceforinfesta.oninfruitmarketedfruitMeaningonedetectedlarva=rejec7onofanen7reshipment
Croplosspoten6alinthewesternUSover$511millionannually(Bolda,etal2010)
Croplosspoten6alintheeasternUSatleast$207millionannually
IncreaseinpesCcidecostreportedbetween39-262%
(h(ps://swd.ces.ncsu.edu/swd-impacts-2014/)
Increaseinlaborreportedbetween5-48%,dependingoncrop(h(ps://swd.ces.ncsu.edu/swd-impacts-2014/)
Male-onlySIT?• AnecdotalevidencethatpopulaConsofSWDarehighinseasoninsouthernandwesternstates.Wouldbedifficulttoachieveanexcessofreleasedmales
• PopulaConsappeartobelowinNorthernstates,parCcularlyearlyintheseason(e.gMichigan).PotenCalSIT?
• Greenhouses.Confinedspace.Li(leuseofgreenhousesforberryproducConintheUS.
• AlternaCveisapopulaConsuppressiongenedrive
GeneCcManipulaCon
• Highlyqualityreferencegenomesequenceisavailable
• TransgenesisisrouCne• CRISPR/cas9ediCngtoolsdevelopedforD.melanogasterarefuncConalinSWD
Possibledrivetarget:TheSxlgene,essenCalforfemales
CRISPR/Cas9knockoutoftheSxlgeneleadstomasculinizaConoffemales
LiandSco((2016)doi:10.1016/j.bbrc.2015.12.081
Diamondbackmoth(DBM)
Wikipediacommons
www.infonet-biovision.orgwww.canolawatch.org
AnnualReviews
DBMDistribuCon: RED,persistsyear-round BLUE:cannotpersistyear-roundbutcanbeaseasonalpest
DBM• Pestofvegetablecrops(e.g.broccoli,cabbage),canolaandwildmustards
• Annualglobaleconomiccostis$4to$5billion• conCnuousgeneraConsintropics.Cannotoverwinterincoldclimates
• ResistanttomanyinsecCcides• Oxitechasdevelopedamale-onlytransgenicstrain.ApprovalforfieldtesCnginNYhasbeenobtained.
Diamondbackmoth-Cornell• OxitecDBM(OX4319L)
– Self-limiCngsystem– Releasesaremale-only– Fieldcagetrialsinsummer2015–haltedearly
– Openfieldtrialsscheduledforsummer2016
GeneCcManipulaCon
• Highlyqualityreferencegenomesequenceisavailable
• Easytorearinthelab• TransgenesisisrouCne• CRISPR/cas9inDBM?
DBMgenedrive• A(racCveintropicalareaswithresource-poorfarmers.
• WithmuchlowerreleaseraCos,apopulaCon-suppressiongenedriveshouldbemorecost-effecCvethanSIT.
• TargetanessenCalgene.• CouldconsidertargeCngagenethatconfersresistancetoachemicalinsecCcide-converCngtosuscepCble
DBMgenedrive
• DBMpopulaConcantriplein6weeksinthefield.Butanefficientgenedrive(>90%)shouldbeeffecCve
• Canmovelargedistances(nohostplant),sotrans-boundaryissueslikelytoarise
• AsforSIT,reproducCvebarriers(e.g.crypCcspecies)couldblockgenedrive
redflourbeetle
TriboliumcastaneumThe1stbeetleandagriculturalpesttobesequenced
Pestofstoredgrains:Lossesof$millionsannually(difficulttoquanCfy) GlobaldistribuCon,tropicalandtemperate
TriboliumLabChow
AdvantagesofTriboliumasaninsectmodel
• Small,easilycultured,rela.velyrapidlifecycle• Sophis.catedgene.cs;chromosomebalancers• Sequencedgenome• RNAinterference• Germlinetransforma.on• CRISPR/Cas9
CRISPR/Cas9
Gillesetal.,2015
Medea:Anaturalgenedrive• Maternal-EffectDominantEmbryonicArrest
– Beemanetal.(1992)
MEDEAMaCngs“Inheritmeordie”
1. Eggs are laid with a toxin 2. Only the eggs which inherit a copy of the MEDEA element can neutralize the toxin
WildTypeMaCngs
Egg
DNA
X
X X
Theorized Mechanism: Toxin/Antidote system:
If the New Gene is linked onto MEDEA, it will spread through the population more effectively
Photo by Alex Wild: myrmecos.net
Bemisiatabaci:silverleafwhitefly
silverleafwhitefly
• OrderHemiptera,atruebug.• OriginallyfromIndia,nowwidespreadtropical,subtropicalregions
• Feedsonhostplantsbypiercinglowerleafsurfaceswithmouthparts,whichcancausedamage
• Feedsonphloemfromover500hosts• Transmitsseveralviraldiseases
GMwhitefly?
Thishasnotyetbeenachieved.Eggsaresmall,0.1mmEggsarea(achedtoaleaf.Appeartoneedtostaya(achedtoaleafduringandaverDNAdeliveryButeasytorearandgeneCccrossessimplifiedasmalesarehaploid
h(p://www.ladybirdplantcare.co.uk/whitefly.html
CRISPR/cas9genedriveforwhitefly?
• DriveanC-viralgene.Similartoapproachesbeingdevelopedformosquitoes.BUT..li(lemolecularunderstandingofvirus-hostinteracConsandwhiteflytransmitsseveralviruses
• PopulaConsuppressiondrive.
Carolina Concha Fang Li Rebecca Linger Ying Yan Esther Belikoff Rebecca Edman Holly Wantuch USDA-ARS Steve Skoda Felix Guerrero
Acknowledgements:Sco(Lab
FundingNIFA,BRAGprogramCOPEGUSDA-ARSNCBiotechCenterNIFA,SCRIprogram