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