Gene Editing and Crop Improvement Using CRISPR-Cas9 ...

CRISPR/Cas9 System DownloadArticle DownloadPDF ReadCube EPUB XML(NLM) totalviews ViewArticleImpact SHAREON ManojK.Sharma SchoolofBiotechnology,JawaharlalNehruUniversity,India KaijunZhao InstituteofCropSciences,ChineseAcademyofAgriculturalSciences,China ElenaKhlestkina InstituteofCytologyandGenetics,RussianAcademyofSciences(RAS),Russia XiaoouDong UniversityofCalifornia,Davis,UnitedStates Theeditorandreviewer'saffiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. Abstract Introduction CRISPR/Cas9System CRISPRSpecificationsinPlants TargetedGenomeModificationinCropPlants ProspectiveApplicationsofCRISPRSystem CRISPR/Cas9OpportunitiesandConcerns VisionaryNotionsofthisTechnology AuthorContributions Funding ConflictofInterestStatement Acknowledgment References Checkforupdates Peoplealsolookedat REVIEWarticle Front.PlantSci.,08November2017Sec.PlantBiotechnology https://doi.org/10.3389/fpls.2017.01932 GeneEditingandCropImprovementUsingCRISPR-Cas9System LeenaAroraandAlkaNarula* DepartmentofBiotechnology,SchoolofChemicalandLifeSciences,JamiaHamdardUniversity,NewDelhi,India AdvancementsinGenomeeditingtechnologieshaverevolutionizedthefieldsoffunctionalgenomicsandcropimprovement.CRISPR/Cas9(clusteredregularlyinterspacedshortpalindromicrepeat)-Cas9isamultipurposetechnologyforgeneticengineeringthatreliesonthecomplementarityoftheguideRNA(gRNA)toaspecificsequenceandtheCas9endonucleaseactivity.Ithasbroadenedtheagriculturalresearcharea,bringinginnewopportunitiestodevelopnovelplantvarietieswithdeletionofdetrimentaltraitsoradditionofsignificantcharacters.ThisRNAguidedgenomeeditingtechnologyisturningouttobeagroundbreakinginnovationindistinctbranchesofplantbiology.CRISPRtechnologyisconstantlyadvancingincludingoptionsforvariousgeneticmanipulationslikegeneratingknockouts;makingprecisemodifications,multiplexgenomeengineering,andactivationandrepressionoftargetgenes.ThereviewhighlightstheprogressionthroughouttheCRISPRlegacy.WehavestudiedtherapidevolutionofCRISPR/Cas9toolswithmyriadfunctionalities,capabilities,andspecializedapplications.Amongvarieddiligences,plantnutritionalimprovement,enhancementofplantdiseaseresistanceandproductionofdroughttolerantplantsarereviewed.ThereviewalsoincludessomeinformationontraditionaldeliverymethodsofCas9-gRNAcomplexesintoplantcellsandincorporatestheadventofCRISPRribonucleoproteins(RNPs)thatcameupasasolutiontovariouslimitationsthatprevailedwithplasmid-basedCRISPRsystem. Introduction Geneticdiversityisakeysourcefortraitimprovementinplants.Creatingvariationsinthegenepoolistheforemostrequirementfordevelopingnovelplantvarieties.Oncethedesiredalterationsareachieved,transgenescanbecrossedoutfromtheimprovedvariety.Cropimprovementhasbeendoneforyearsviatraditionalplantbreedingtechniquesorthroughvariousphysical,chemical(e.g.,gammaradiation,ethylmethanesulfonate)andbiologicalmethods(e.g.,T-DNA,transposoninsertion)leadingtopointmutations,deletions,rearrangements,andgeneduplications.Theadventofsite-specificnucleases(SSNs)highlightedtheimportanceofsitedirectedmutagenesisoverrandommutagenesis(Osakabeetal.,2010;Sikoraetal.,2011).Randommutagenesishasalsoitsownlistofshortcomingstoo.Itproducesmultipleundesirablerearrangementsandmutations,whichareexpensiveandverycomplextoscreen.GeneeditingusesengineeredSSNstodelete,insertorreplaceaDNAsequence.Developmentoftheengineeredendonucleases/mega-nucleases,zincfingernucleases(ZFNs),transcriptionactivator-likeeffectornucleases(TALENs)andtypeIIclusteredregularlyinterspacedshortpalindromicrepeat(CRISPR)/CRISPR-associatedprotein9(Cas9)pavedthewayforsinglenucleotideexcisionmechanismforcropimprovement(Paboetal.,2001;Bochetal.,2009;MoscouandBogdanove,2009)(Figure1).Thesegenome-editingtechnologiesuseprogrammablenucleasestoincreasethespecificityofthetargetlocus. FIGURE1 FIGURE1.Variousgenome-editingtools.(A)Zinc-fingernucleases(ZFNs)actasdimer.EachmonomerconsistsofaDNAbindingdomainandanucleasedomain.EachDNAbindingdomainconsistsofanarrayof3–6zincfingerrepeatswhichrecognizes9–18nucleotides.NucleasedomainconsistsoftypeIIrestrictionendonucleaseFok1.(B)Transcriptionactivator-likenucleases(TALENs):thesearedimericenzymessimilartoZFNs.EachsubunitconsistsofDNAbindingdomain(highlyconserved33–34aminoacidsequencespecificforeachnucleotide)andFok1nucleasedomain.(C)CRISPR/Cas9:Cas9endonucleaseisguidedbysgRNA(singleguideRNA:crRNAandtracrRNA)fortargetspecificcleavage.20nucleotiderecognitionsiteispresentupstreamofprotospaceradjacentmotif(PAM). Genomeeditingmodifiesaspecificgenomeinpreciseandpredictablemanner.Therecouldbevarietiesofgenes,whichcouldbealteredindifferentcelltypesandorganismswiththeaidofnucleasesthatoffertargetedalterations.ZFNsisoneoftheoldestgeneeditingtechnologies,developedinthe1990sandownedbySangamoBioSciences.ZFNsarepremeditatedrestrictionenzymeshavingsequencespecificDNAbindingzincfingermotifsandnon-specificcleavagedomainofFok1endonuclease.Anarrayof4–6bindingmodulescombinestoformasinglezincfingerunit.Eachmodulerecognizesacodon(Paboetal.,2001).ApairofZFNstogetheridentifiesaunique18–24bpDNAsequenceanddoublestrandedbreaks(DSBs)aremadebyFok1dimer.FokInucleasesarenaturallyoccurringtypeIISrestrictionenzymesthatintroducesinglestrandedbreaksinadoublehelicalDNA.HenceFokIfunctionsasadimer,witheachcatalyticmonomer(nickase)cleavingasingleDNAstrandtocreateastaggeredDSBwithoverhangs(Paboetal.,2001).ZFNshavebeensuccessfullyemployedingenomemodificationofvariousplantsincludingtobacco,maize,soybean,etc.(Curtinetal.,2011;Ainleyetal.,2013;Baltesetal.,2014).Itwastakenbackduetosomedrawbackssuchastime-consumingandexpensiveconstructionoftargetenzymes,lowspecificityandhighoff-targetmutationsthateventuallymadewayforthenewtechnology.TALENsturnedouttobeasubstitutetoZFNsandwereidentifiedasrestrictionenzymesthatcouldbemanipulatedforcuttingspecificDNAsequences.Traditionally,TALENswereconsideredaslongsegmentsoftranscriptionactivator-likeeffector(TALE)sequencesthatoccurrednaturallyandjoinedtheFokldomainwithcarboxylic-terminalendofmanipulatedTALErepeatarrays(Christianetal.,2010).TALENscontainacustomizableDNA-bindingdomainwhichisfusedwithnon-specificFoklnucleasedomain(Christianetal.,2010).TALENscomparedtoZFNs,involvetheinteractionofindividualnucleotiderepeatsofthetargetsiteandaminoacidsequencesofTALeffectorproteins.TheycangenerateoverhangsbyemployingFoklnucleasedomaintopersuadesite-specificDNAcleavage.Ithasbeenwidelyusedtogeneratenon-homologousmutationswithhigherefficienciesindiverseorganisms(JoungandSander,2012). TheemergenceofCRISPRtechnologysupersedesZFNsandTALENsandusedwidelyasanovelapproachfrom“methodsoftheyear”in2011to“breakthroughoftheyear”in2015fortheircaptivatedgenomeediting.Thisprokaryoticsystemispromptlyacceptedforgenomeeditingineukaryotichostcells(Jineketal.,2012;Nakayamaetal.,2013).CRISPRhasanaddedadvantageofgeneknockoutoverRNAi,whichisawell-knowntechniqueforgeneknockdown.CRISPRtargetstheendogenousgenesthatareimpossibletospecificallytargetusingRNAitechnologywithmoreprecisionandsimplicity.RNAigeneregulationisgovernedbytheendogenousmicroRNAs(miRNAs).AnydisplacementofthesemiRNAsfromtheexogenousmiRNAscanleadtohypomorphicmutationsandoff-targetphenotypes(Khanetal.,2009).CRISPR/Cas9targetsspecificgenomiclociwiththehelpof∼100nucleotide(nt)guideRNA(gRNA)sequence.sgRNAbindstotheprotospaceradjacentmotif(PAM)ontargetedDNAviaWatsonandCrickbasepairingthrough17–20ntatthegRNA5′-endandguideCas9forspecificcleavage(Tsaietal.,2015).Cas9stimulatestheDNArepairmechanismbyintroducingDSBsinthetargetDNA.Repairmechanisminvolveserrorpronenon-homologousendjoining(NHEJ)orhomologousrecombination(HR)toproducegenomicalterations,geneknockoutsandgeneinsertions(Figure2).NHEJbyfaristhemostcommonDSBrepairmechanisminsomaticplantcells(Puchta,2005).RandominsertionsordeletionsbyNHEJinthecodingregionleadtoframeshiftmutations,hencecreatinggeneknockouts.CRISPRtechnologyholdspotentialforloss-of-function,gain-of-function,andgeneexpressionanalysis.CRISPRhasversatileapplicationsinplantbiologyandisreadilyappliedtoproducehighqualityagriculturallysustainableproducts(Table1).TherearemanyplantswhichareintheprocessofgettingalteredthroughCRISPR/Cas9.TheCRISPReditedtomatoeswillbeexpectedtohaveenhancedflavor,sugarcontentandaromaascomparedtomoderncommercialvarieties;cornismaderesistanttodroughtwithhighyieldperhectare;wheatiseditedagainstpowderymildewdisease,andmushroomsaretargetedtoreducethemelanincontent(Wangetal.,2014;Waltz,2016;Shietal.,2017;Tiemanetal.,2017). FIGURE2 FIGURE2.Genomeeditingwithsite-specificnucleases(SSNs).Thedoublestrandedbreaks(DSBs)introducedbyCRISPR/Cas9complexcanberepairedbynon-homologousendjoining(NHEJ)andhomologousrecombination(HR).(A)NHEJrepaircanproduceheterozygousmutations,biallelicmutations(twodifferentmutationsateachchromosome)andhomozygousmutations(twoindependentidenticalmutations)leadingtogeneinsertionorgenedeletion.(B)InthepresenceofdonorDNAdigestedwiththesameendonucleaseleavingbehindsimilaroverhangs,HRcanbeachievedleadingtogenemodificationandinsertion. TABLE1 TABLE1.ListoftargetedgenesviaCRISPR/Cas9systemindifferentplantspecies. CRISPR/Cas9System CRISPRprogressintoday’sworldasgenomeeditingtoolcanbetracedbacktoitsorigininthelate1980s(Ishinoetal.,1987)andadecadeofextensiveexperimentationsince2005(Figure3).CRISPR/Cas9microbialadaptiveimmunesystemanditsprogresstilldateistheoutcomeoftheworkofnumerousresearchersaroundtheglobe.Aseriesofcomprehensivereviews(BortesiandFischer,2015;AmitaiandSorek,2016;Puchta,2016)givesthedetailedinformationofeachaspectofCRISPR/Castechnology. FIGURE3 FIGURE3.KeydiscoveriesandadvancesinCRISPR/Cas9technology. DecipheringtheroleofCRISPR/Cassysteminbacteriaandarchaeaelucidatedthepowerofthissystemasagenome-editingtool.AseriesofexperimentsinvolvingbioinformatictoolsunveiledvariousCRISPR/Cascomponentsandtheirfunctioninprovidingadaptiveimmunitytobacterialcells.ACRISPRlocusconsistsofclustersofCRISPR-associated(Cas)genesandCRISPRarrayswhereallimmunologicalmemoriesareengraved(Barrangouetal.,2007).CRISPRarrayisagenomiclocushavingseriesof21–40bprepeatsequences(directrepeats)interspacedby25–40bpvariablesequences(spacers)(Jansenetal.,2002;Tangetal.,2002).In2005,threeindependentresearchgroups(Bolotinetal.,2005;Mojicaetal.,2005;Pourceletal.,2005)hypothesizedtheroleofspacerelementsastracesofpastinvasionsofforeignDNAthatprovideimmunityagainstphageinfection.Theyalsonotedthatspacersshareacommonendsequence,nowknownasPAM.Barrangouetal.(2007)experimentallydemonstratedtheinvolvementofCRISPRarraysinresistancetobacteriophagesinassociationwithCasgenes.Ateveryinfection,newphageDNAgetsincorporatedintotheCRISPRarraybuildingpotentialtofighttheupcominginfection.StudiesfromBrounsetal.(2008)unveilthetranscriptionofphagespacersequencesintosmallRNAs(crRNAs)thatguideCasproteinstothetargetDNA.ThemechanismofinterferencebasedonRNA-mediatedDNAtargetingandtheroleofCas9inintroducingDSBsatapreciseposition,threenucleotidesupstreamofPAMwasalsodemonstrated(MarraffiniandSontheimer,2008;Garneauetal.,2010).Furtheratrans-activatingCRISPRRNA(tracrRNA)formsaduplexwithcrRNAandguidesCas9toitstarget(Deltchevaetal.,2011).FusionofthecrRNAandtracrRNAtoformasingle,syntheticguideRNAfurthersimplifiedthesystem(Jineketal.,2012).Finally,Congetal.(2013)reportedtheabilityofCas9tofacilitatehomologydirectedrepairwithminimummutagenicactivity. ClassificationofCRISPR/Cas9System ThefirstattempttoclassifyCRISPR/CassystemwasdonebyHaftetal.(2005).Hedefined45CRISPR-associated(Cas)proteinfamiliesthatarecategorizedintocoreproteins(Cas1,Cas2,Cas3,Cas4,Cas5,Cas6),8CRISPR/CassubtypesandRAMP(repairassociatedmysteriousprotein)moduleinprokaryoticgenomes.Makarovaetal.(2011)classifiedCRISPR/Cassystemsintothreetypes:typeI,typeII,andtypeIIIdependingonthepresenceofsignatureCas3,Cas9andCas10proteins,respectively(Table2).Thissystemwasdividedinto10subtypesdependingonthepresenceofadditionalsignatureproteins.Thisthree-typeclassificationsystemisfurthermodifiedintotwoclass-fivetypeclassificationsystemsdependingonthetypeofsignatureproteinsandCRISPRloci(Makarovaetal.,2015).MajordifferencesbetweenCRISPRclassesarebasedonthecompositionofcrRNPcomplexes.Class1CRISPRshavemultiplesubuniteffectorcomplexeswhileclass2CRISPRsconcentratesmostoftheirfunctionswithsingleproteineffectors.Class1CRISPRsystem,forexample,havedifferentnucleasesforpre-crRNAprocessing,spacersequenceloading,andtargetedcleavageprocessing.Inclass2,asingleproteinperformsallofthesefunctions.TypeIVandtypeVbelongstoclassIandclassIIsystemsrespectively.TwosubtypesoftypeVsystemandVItypeisalsorecognized,elaboratingtheclassificationtotwo-class–six-type–19-subtypesystem(Shmakovetal.,2015;Table2).Cas1andCas2genesareubiquitousinallCRISPR/Castypes(Makarovaetal.,2011). TABLE2 TABLE2.ClassificationofCRISPR/Cas9system. CRISPR-Cpf1(ClassII,TypeVCRISPRfromPrevoltellaandFrancisella1)isanadvancedtoolthatusesasingleCpf1proteinforcrRNAprocessing,targetsiterecognition,andDNAcleavage.Cpf1isfunctionallyconservedtoCas9proteinbutdifferssubstantiallyinmanyaspects.Thedifferencesareasfollows:itisaribonucleasethatprocessesprecursorcrRNA;itrecognizesathyminerich(like5′-TTTN-3′)PAMsites(Zetscheetal.,2015a).PAMsequenceislocatedupstreamoftheprotospacersequenceandtracrRNAisnotrequiredforguidingCas9tothetargetsite.ThemostimportantcharacteristicofCpf1isthegenerationof4bpoverhangsincontracttobluntendsproducedbyCas9(Zetscheetal.,2015a).Thesestickyendswouldprovidemoreefficientgenomicinsertionsduetosequencecomplementarityintoagenome.AmongseveralproteinsintheCpf1family,LbCpf1fromLachnospiraceaebacteriumND2006andAsCpf1fromAcidaminococcussp.BV3L6actmoreeffectivelyinhumancellscomparedwithotherorthologs(Kimetal.,2016).Class2typeVIischaracterizedbyaneffectorproteinC2c2(Class2,candidate2).C2c2containstwonucleotidebinding(HEPN)conserveddomains,whichlackshomologytoanyknownDNAnuclease(Abudayyehetal.,2016).HEPNdomainsfunctionasRNases,henceitisvisualizedasanewRNAtargetingtoolguidedbyasinglecrRNAwhichcanbeengineeredtocleavessRNAcarryingcomplementaryprotospacers.Hence,C2c2doesnottargetDNA(Abudayyehetal.,2016).C2c2issimilartotypeIII-AandIII-BsystemsinhavingHEPNdomainsthatarebiochemicallycharacterizedasssRNAspecificendoribonucleasesbutthereisasignificantlineofdifferencebetweenthesetwotypes.Cas10-CsmintypeIIIAandCsxintypeIIIBhavelesstargetspecificityandhavetodimerizetoformactivesites.C2c2,incontrast,containstwoHEPNdomainsandfunctionasmonomericendoribonuclease(Abudayyehetal.,2016).dCas9analogsofC2c2,dC2c2canbeproducedbyalaninesubstitutionofanyofthefourpredictedHEPNdomain.FurtherexaminationisrequiredtoclarifythemechanismoftheC2c2systemandtheclassofpathogensagainstwhichitcanprotectbacteria.Currently,typeVIsystemisfoundinCarnobacteriumgallinarum,Leptotrichiabuccalis,L.shahii,L.wadei,Listerianewyorkensis,L.seeligeri,L.weihenstephanensis,Paludibacterpropionicigenes,andRhodobactercapsulatus(ChoiandLee,2016). CRISPR/Cas9Mechanism TheadaptiveimmunityofCRISPR/Cas9systemconsistsofthreephases:adaptation,expression,andinterference(Figure4).AdaptationinvolvestheinvadingDNAfromvirusorplasmidsthatarecleavedintosmallfragmentsandincorporatedintoCRISPRlocus.CRISPRlociaretranscribedandprocessedtogeneratesmallRNA(crRNA),whichguidetheeffectorendonucleasestotargettheviralmaterialbybasecomplementarity(Barrangouetal.,2007;Yosefetal.,2012).DNAinterferenceinTypeIICRISPR/CassystemrequiresasingleCas9protein(Haleetal.,2009;Zetscheetal.,2015b).Cas9isahugeproteinpossessingmultipledomains(RuvCdomainattheaminoterminusandtheHNHnucleasedomainpositionedinmiddle)andtwosmallRNAsnamelycrRNAandtracrRNA.Cas9assistsadaptation,participatesinpre-crRNAprocessingtocrRNAandintroducetargetedDSBsguidedbytracrRNAanddoublestrandedRNAspecificRNaseIII(Jacksonetal.,2014;Mulepatietal.,2014).AscomparedtotypeIICRISPR,theuniquefeaturesoftypeIIICRISPRarethecleavageofbothDNAandRNA,anditsassociationwiththecleavageproteinCas10.Thecleavageisatranscription-dependentDNAsequencemodificationthatalsocontainsatranscriptionallyactivepromoter(Samaietal.,2015).Cas10systemenablesbacteriatoacquireviralspacerelementsenablingatypeofresistanceagainstforeignDNAunderspecialconditions.Thisresistancetoforeign/viralDNApreventsactivationofthelyticpathway,whichisdetrimentaltothehostcell.Thesesequencescouldalsoalterthephysicalcharacteristicsofthecell,potentiallyprovidingasurvivaladvantageforthehostcell(Samaietal.,2015). FIGURE4 FIGURE4.MechanismofCRISPR/Cas9action:intheacquisitionphaseforeignDNAgetsincorporatedintotheCRISPRlociofbacterialgenome.CRISPRlociisthentranscribedintoprimarytranscriptandprocessedintocrRNAwiththehelpoftracrRNAduringcrRNAbiogenesis.Duringinterference,Cas9endonucleasecomplexedwithacrRNAandcleavesforeignDNAnearPAMregion. MultiplexgenomeengineeringusingmultipleguideRNAstotargetvariousgenomicsitessimultaneouslywasalsodemonstrated.CRISPRwasfirstappliedinplantsinAugust2013(Fengetal.,2013;Lietal.,2013;XieandYang,2013).Fengetal.(2013)targetedvariousendogenousgenesandtransgenesbyprotoplasttransfection,agroinfiltrationandgeneratedstabletransgenicplantsbybothNHEJandHRmechanisms.VariousgenesleadingtophenotypicvariationsweretargetedlikeBrassinosteroidInsensitive1(BRI1),Jasmonate-Zim-DomainProtein1(JAZ1)andGibberellicacidinsensitive(GAI)inArabidopsisandRiceOutermostCell-specificgene5(ROC5),StromalProcessingPeptidase(SPP),andYoungSeedlingAlbino(YSA)inriceandobtainedpositiveresults.Similarly,XieandYang(2013)introducedthreeguideRNAsatdistinctricegenomiclociandanalyzedthemutationefficiencyof3–8%.Offtargetmutationswerealsonoticedbutwithminimumgenomeeditingefficiencythanthematchedsite. Studiesonmaize(Liangetal.,2014),wheat(Wangetal.,2014),andsorghum(Jiangetal.,2013)providedanexcellentfoundationfortheuseofCRISPRingeneediting.Theseinvestigationspostulatedthefirstcomprehensivedataonparameterssuchasmutationefficiency,cleavagespecificity,largechromosomaldeletionsandresolutionoflocusstructure.Jiangetal.(2013)alsodemonstratedtheexpressionofgRNAsunderthecontrolofmultiplepromoters.Fauseretal.(2014)emphasizedtheuseofbothCRISPR/CasbasednucleasesandnickaseswiththeirstudiesconductedonArabidopsisthaliana.NucleasesareefficienttoolsforNHEJmediatedmutagenesisandthecombinedactionoftwonickasescanenhancerecombinationbetweentandemlyarrangeddirectrepeats,geneconversionguidedbyinvertedrepeatsandcanregulatemechanismsinvolvingHR(Fauseretal.,2014).SinglechimericgRNAarefoundtobemoreefficientthanindividualcrRNAandtracrRNAcomponents(Miaoetal.,2013;Zhouetal.,2014).Interestingly,fourindependentgroups(Shanetal.,2013;Brooksetal.,2014;Zhangetal.,2014;Zhouetal.,2014)havedemonstratedtheintroductionofbiallelicorhomozygousmutationsinT1generationofriceandtomatoindicatingthehighefficiencyofthissystem.Thegeneticchangesaresegregatednormallyinsubsequentgenerationswithoutfurthermodifications(Zhouetal.,2014).SomeexamplesoftheCRISPR/Cas9applicationsinplantsarecitedinTable1. CRISPR/Cas9systemiscontinuouslybeingupgradedforbetterefficiencyandspecificityofgenetargeting.TheneedforrepurposingCRISPR/Cas9systemtoaltereukaryoticgenomehasnecessitatedtheadditionofnuclearlocalizationsignalsatoneorbothendsoftheprotein.TheintroductionoforthogonalCRISPR/Cas9systemshasbroadenedtheapplicationofthistechnologymanifold.TheseorthologsincludeRNAguidedendonucleasesfromStreptococcusthermophilus(St),Neisseriameningitidis(Nm),Campylobacterjejuni(Cj),andStaphytococcusaureus(Sa).EachorthogonalCas9systemhasuniquespecificationsincludingvariationsinCas9proteins,PAMsitesandgRNAscaffoldsfortargetrecognition(Table3).Houetal.(2013)demonstratedefficienttargetingofendogenousgenesinhumanpluripotentstemcellsviaNmCas9.TheyarethepioneersinthedevelopmentofNmCas9thatuses24-nucleotide(nt)protospacertotargetDNAover20ntprotospacerrequirementsofSpCas9andStCas9.ExtendedPAMsequence(5′-NNNNGATT-3′)ascomparedtoNGGsequencemayfurtherenhancethespecificity. TABLE3 TABLE3.Cas9variantswiththeiroriginandspecifications. CRISPRSpecificationsinPlants EfficientCRISPR/Cas9genomeseditinginplantsrequiresuitablevectorsystem(codonoptimizedCas9geneandpromotersforCas9andsgRNA),efficienttargetsitesandtransformationmethodusedinappropriateplantspecies.CRISPReditingrequiresthedeliveryandexpressionofsingleguideRNA(sgRNA)andcas9proteininthetargetcell.Specificexpressionvectorsaredesignedtoachievethisgoal.sgRNAisgenerallyregulatedbytissuespecificRNApolymeraseIIIpromoterssuchasAtU6,TaU6etc.thatdrivestheexpressionofsmallRNAsintheirrespectivespecies.Similarly,Cas9isplaceddownstreamofRNApolymeraseIIpromoterslikeubiquitinpromotersthatguidetheexpressionoflongerRNAs.Cas9isgenerallytaggedwithnuclearlocalizationsequence(NLS)totargetnuclearDNA.Thechoiceofthevectorslargelydependsuponthetypeoftheexpressionsystemtobeworkedon,typeofrestrictionsitespresenttoinsertsgRNAandthetypeofCas9system.BothsgRNAandCas9canbeco-expressedinasingleplasmidex.pFGC-pcoCas9,pRGEB32,pHSE401.Differenttypesofplasmidscanbestudiedfromhttps://www.addgene.org/crispr/plant/.TheuseoftheseplasmidsislimiteddependinguponthetypeofCas9(cut,nick,activate,interfere)present(Table4). TABLE4 TABLE4.DifferentplasmidswithspecificCas9activity. IndependentsgRNAplasmidsarealsodesignedwhereCas9isnotco-expressedbutcanbepairedalongenablingusageofthewidevarietyofCas9types.pICSL01009::AtU6pandpICH86966::AtU6p::sgRNA_PDSwhichencodesanArabidopsisU6promoterandexpressessgRNAtargetingPDSinNicotianabenthamiana.ThechoiceoftheoptimalpromoterstodrivetheexpressionofsgRNAorCas9andcodonoptimizedversionofCas9isimportantforefficientgenomeediting.MostoftheworkineukaryoticcellsisdoneusingcodonoptimizedversionsofSpCas9.Resultshavebeenobtainedusinghumancodonoptimized(Lietal.,2013;Miaoetal.,2013)orplantcodonoptimizedversionsofCas9(Fengetal.,2013;Nekrasovetal.,2013;XieandYang,2013).Themutationsinducedcanbeheterozygous,biallelic(twodistinctallelicmutations),homozygousorrarelychimeric.AnumberofreportsconfirmedthestableinheritanceofCRISPR/Cas9inducedmutationsinmodelandcropplants.EfficientCRISPR/Cas9genomeeditingandinheritanceofmodifiedgenesintheT3andT2generationswasreportedforthefirsttimeinArabidopsis(Jiangetal.,2014).Achangeinnon-functionalGFPgenewasobservedinT1generation.AllGFP-positivetransgenicplantswereidentifiedwithmutagenizedGFPgenes.Outof42transgenicsdeveloped,50%haveinheritedasingleT-DNAinsert. ThegeneralmethodologyforimplementingtargetedmutagenesisusingCRISPR/Cas9technologyisoutlinedinFigure5.ItstartswiththeselectionofspecifictargetsitehavingashortPAMsequenceat3′end.Targetsiteshouldbeselectedconsideringminimumornooff-targeteffects(preventingcutsatunintendedsitesinthegenome).ManybioinformaticstoolshelpindesigningsgRNAwithhighspecificityanddetectionofoff-targetssuchasCOSMID(CRISPROff-targetSiteswithMismatches,Insertions,andDeletions).Off-targetsaremoreprevalentinbacterialandculturedmammaliancellsthaninplantcells.Manystudieshaveshownthepotentialoff-targetsofcas9suchas,insoybean,theoff-targetfrequencywasfoundtobe13%(Jacobsetal.,2015).Nodetectableoff-targetsarefoundinA.thaliana,wheat,riceandsweetorange.Cas9nickasehasalsoemergedasanalternativetoreduceofftargeteffects.NickaseisguidedbythesgRNAattwoadjacentpositionsatthetargetsiteproducingasinglestrandedbreakoneachofthetwoDNAstrands. FIGURE5 FIGURE5.SimplifiedflowchartrepresentingCRISPR/Cas9mediatedplantgenomeediting.Aftertheselectionofthetargetsite,sgRNAsaredesignedusingvariousbioinformaticsoftwaresandpackedintospecificvectorsalongwithcodonoptimizedCas9.Afterdeliveryintoplantcells,putativetransformantscanbescreenedbymultipleassaysandusedforfurtheranalysis. CRISPR-PLANTisanewlydesignedwebportalsupportedbyPennStateandArizonaGenomicsInstitute(AGI)establishedtohelpresearcherstousetheCRISPR-Cas9systemforgenomeediting.ItestimatesthehighlyspecificsgRNAbyavoidingoff-targetsequences(Xieetal.,2014).Afterthetargetsiteconfirmation,targetspecificoligonucleotides(20nt)aredesignedwhichfurtherfuseswithtracrRNAsequencetoformsgRNA.sgRNAisfurtherplacedinavectoreitheralongwithCas9sequence(abinaryvector)orindividuallyunderasuitablepromoterforanoptimalexpression.Theconstructsarethentransformedusingasuitablemethod.Thedeliverysystemsvarybasedonplantspecies,researchpurpose,andrequirements.FgRNA-Cas9mediatededitingcanbedetectedbyarestrictionenzymedigestionsuppressedPCR(RE-PCR)method,whichinvestigatestheNHEJ-introducedmutations(XieandYang,2013).RE-qPCRcanalsobeperformedformoreaccurateestimationofgenome-editingefficiency.Finally,wholegenomesequencingisdonetoreduceoff-targetmodifications. TargetedGenomeModificationinCropPlants Overtheyearsthebiotic(bacteria,fungi,insects,andviruses)andabiotic(salinity,drought,flooding,heavymetaltoxicity,hightemperature)stresseshaveadverselyaffectedcropplantation.Oneofthecurrentresearchesinplantbiologyfocusesongeneratingcropstotolerateharshagroclimaticconditionsandtomeettheneedsoftheever-growinghumanrace. GenomeModificationforNutritionImprovement CRISPR/Cas9systemcangeneratestableandheritablemutationswithoutaffectingtheexistingvaluabletraits.Thisresultsinthedevelopmentofhomozygousmodifiedtransgenefreeplantsinonlyonegenerationandit’sstabletransmissiontosuccessivegenerations(Fengetal.,2014;Panetal.,2016).Cas9continuedtobeabettertoolwithrelativelyhighcleavageefficiencywhencomparedtoTALENsandZFNs(Gajetal.,2013;Johnsonetal.,2015).ResearchesdoneonvariouscropssincetheadventofCRISPRtechnologyintheplantworldishighlightedinTable1.Classicworksarebeingdoneforproducingacrylamidefreepotatoes(Haltermanetal.,2015),non-browningapples,mushroomsandpotatoesbymutatingPolyphenoloxidase(PPO)genes(Haltermanetal.,2015;Nishitanietal.,2016;Waltz,2016)andlowphyticacidinmaize(Liangetal.,2014). Wangetal.(2014)pioneeredtheworkoftargetedgenomeeditinginsweetorangeusingCas9/sgRNA.Geneticimprovementofcitrusislimitedduetoitsslowgrowth,pollenincompatibility,polyembryony,andparthenocarpy.Xcc(Xanthomonascitrisubsp.citri)-facilitatedagroinfiltrationwasemployedtodeliverCas9andCsPDSgenespecificsgRNAintosweetorange.DNAsequencingconfirmedthemutatedCsPDSgeneatthetargetsitewithamutationrateof3.2to3.9%.Nooff-targetmutagenesiswasreported.Lawrensonetal.(2015)targetedmulticopygenesinHordeumvulgareinvestigatingtheuseandtargetspecificityrequirementsofCas9editing.HvPM19geneencodinganABA-inducibleplasmamembraneproteinwastargetedtostudythecharacteristicsofdormancy.T0werephenotypicallyidentifiedwithexpecteddwarfphenotypeassociatedwithaknockoutofthetargetgene.Liangetal.(2014)discussedthepresenceofantinutritionalcompoundPhyticacid(PA),inositol1,2,3,4,5,6-hexakisphosphateinmaize.PAispoorlydigestedinhumansandpossesathreattotheenvironment,thus,PAcontentofmaizeseedswasreducedbydesigningtwogRNAstargetingtheZmIPK(InositolPhosphateKinase)genethatcatalyzesakeystepinPAbiosyntheticpathway. BioticandAbioticStressResistanceviaCRISPR/Cas9 MultiplediseaseresistanceplantshavebeenobtainedusingCRISPR/Cas9technology(Table5).SomehighlightsinvolvetheresistanceagainstriceblastdiseasebytargetingOsERF922geneinrice(Wangetal.,2016).TransgenefreemutantlinesfromT1andT2generationswereselectedbysegregationandfurtherexamined.Transgeniclinesshowedasignificantreductionblastlesionsformedduetopathogeninfection.Wangetal.(2014)introducedmutationsusingsite-specificendonucleasesinhomeoallelesencodingMildew-resistancelocus(MLO)proteinsofhexaploidbreadwheat.Pengetal.(2017)targetedcitruscankercausedbyXanthomonascitrisubsp.XccinCitrussinensis.CRISPR/Cas9targetedmodificationofthesusceptibilitygeneLateralorganboundaries1(CsLOB1)promoterenhancesdiseaseresistance.DeletionoftheentireEBEPthA4sequencefrombothCsLOB1allelesconferredthehighestlevelofresistancetoWanjinchengorange.AlltransformedplantsweremorphologicallysimilartowildtypeindicatingthatCsLOB1promotermodificationdoesnotdisruptplantdevelopment.42%ofthemutantplantsharboreddesiredmutationsand23.5%ofthemutantsshowedresistancetocitruscanker.ThestackingupofmultiplenucleasesasonetransgenebyCRISPR/Cas9systemalsoleadstothetargetedcleavageofmultipleinfectionsbyviruses(Iqbaletal.,2016). TABLE5 TABLE5.ListofsomecropsthataremaderesistanttodiseasesviaCRISPR/Cas9system. CRISPRSysteminMetabolicEngineering FurtherapplicationsofCRISPR/Cas9includeextensiveresearchinthefieldofmetabolicengineeringwhereplantcellsaretargetedforproductionofspecificmetabolites.Alagozetal.(2016)manipulatedthebiosynthesisofbenzylisoquinolinealkaloids(BIAs)fornextgenerationmetabolicengineeringinPapaversomniferumbyknockingout3′OMT2geneviaNHEJDNArepairCRISPR/Cas9mechanism.4′OMT2(4′-O-methyltransferase)isaregulatorygeneinvolvedinthebiosynthesisofcodeine,noscapine,papaverine,andmorphineviadifferentBIApathways.Suchstrategiescanbeemployedtoconvertvaluablemedicinalplantsintobiofactoriesformassproductionofspecificmetabolitessimplybyintroducingbreaksinrelatedgenesequencing.Lietal.(2017)targetedditerpenesynthasegene(SmCPS1),involvedintanshinonebiosynthesisinSalviamiltiorrhiza,Chineseherbwell-knownforvasorelaxationandantiarrhythmiceffects.SmCPS1istheentryenzymethatusesGGPP(geranylgeranyldiphosphate)asitssubstrateforgeneratingtanshinones.GGPPalsoactsasaprecursorfortaxolbiosynthesis,therefore;SmCPS1knockout(post-GGPPsynthesisstep)blocksthemetabolicfluxthroughGGPPtotanshinone,switchingGGPPtotaxolsynthesis.AgrobacteriumrhizogenesmediatedtransformationusingCRISPR/Cas9generatedthreehomozygousandeightchimericmutantsfrom26independenttransgenichairyrootlinesofSalvia.Metabolomicsanalysisrevealedzerotanshinoneaccumulationinhomozygousmutantsanddecreasedpercentageinchimericmutants. ProspectiveApplicationsofCRISPRSystem CRISPR/Cas9technologyisadvancingatanunprecedentedpace.MostoftheresearchdonesofarincludegeneknockoutorgenesilencingmechanismsviaNHEJ,whichisnotpreciseandmostprevailingmechanism.Geneknock-inorgenereplacementstrategiesthatfollowtargetedmutagenesisviaHDRevidencedpromisingresultsinmammalianandplantcells.Homologydrivenrepairwasadifficulttaskearlierinplantsbecauseoflowefficiencyandinefficientdeliveryofhomologousdonorsequencesintotransfectedplantcells(PuchtaandFauser,2014;Steinertetal.,2016).Multipleapproachesareusedforefficienthomologydirectedrepairmechanismandsuccessfulresultshavebeenreported(Collonnieretal.,2017;Humanesetal.,2017).Genomicstudiesinwoodyplantsarechallengingbecauseofthelongvegetativeperiods,lowgenetictransformationefficiencyandlimitedmutants.Fanetal.(2015)reportedthedisruptionofsite-specificendogenousphytoenedesaturasegene(PtoPDS)inPopulustomentosaCarr.via.Homoallelicandheteroallelicpdsmutantsweredetectedinfirstgeneration.CRISPR/Cas9hasalsobeenappliedtolowermembersofkingdomPlantaelikealgae,bryophytes,pteridophytes,etc.Liverwortsemergeasmodelspeciesforstudyinglandplantevolution.MoleculargeneticsofMarchantiapolymorphaL.isstudiedbytheapplicationofCRISPR/cas9targetedmutagenesis(Suganoetal.,2014).Beyondgenomeediting,CRISPR/Cas9technologyiswidelydevelopingandusedforvariousotherpurposestounderstandfunctionalgenomicsandmolecularbiology.Thecurrentfocusisonloss-of-functionandgain-of-functionanalysisofindividualgenesandidentificationofgenemodulesandgeneticexpression.Figure6representstheexpandingfootprintsofCRISPR/Cas9systemofwhichmanyareyettobetestedinplants. FIGURE6 FIGURE6.VariousapplicationsofCRISPR/Cas9systemmanyofwhichareyettobetestedinplants. CRISPRhasreplacedtheRNAinterference(RNAi)genesilencingtechnologyforefficientandprecisegeneknockdown.IthasovercomevariouslimitationsofRNAitechnology,suchasincompleteloss-of-functionanalysisandextensiveoff-targetactivities.WiththedevelopmentofsimultaneousexpressionofmultipleguideRNAs(sgRNAs),CRISPR/Cas9systemallows,“multiplexgenomeediting.”Multiplexgenome-editingactsasapowerfultoolforreducinggeneticredundancyinparalogoussequencesbycreatingmultiplexgeneknockouts.IthasalsobeenusedtocreatechromosomaldeletionsfrommultipleDNAbasepairsinArabidopsis,Nicotianabenthamianaetc. GeneExpressionRegulation Manipulatingthegenomeofthetargetcellsisanotherwell-knownCRISPR/Cas9application.RepurposingCRISPR/Cas9geneeditingtogeneexpressionregulationisknownasCRISPRinterference(CRISPRi).CRISPRinterferenceinvolveseitheractivationorrepressionofthegeneexpression(BikardandMarraffini,2013).TheestablishmentofCRISPR/Cas9asgeneregulatorymachinerycameupmajorlyfromexperimentalstudiesonintracellularpathogenFrancisellanovicida.FTN-0757geneexpressesthevirulencefactorthatrepressestheproductionofabacteriallipoprotein(BLP).FTN-0757isfurtherexaminedasatypeIICas9proteinthatinassociationwithtracrRNAinactivateBLPexpressioninFrancisellanovicida.tracrRNAhasanimperfectcomplementaritytoBLPmessenger,whichrequiresCas9andasmallCRISPR-Cas-associatedRNA(scaRNA)forBLPmRNAdegradation(BikardandMarraffini,2013).AnumberofexcellentreviewsgivethedetailedinformationonprinciplesofgeneregulationbyCRISPR/Cassystemincluding(Larsonetal.,2013;Qietal.,2013;Xuetal.,2014;Leeetal.,2016). Targetedregulationofgeneexpressionprovidesinterestinginsightsintotheplantgenomeaswell(PetolinoandDavies,2013).Theectopicgeneexpressionregulationprovidesimportantinformationforgenefunctioningandcanalsobeappliedtodevelopregulatorycircuitsforsyntheticbiologyapplications(Puchta,2016).Precisemanipulationofthedesiredgeneexpressionbyrepressionoractivationcanelucidatethefunctionofindividualgenesandtheirroleincomplexdevelopmentalprocesses(Dominguezetal.,2016).Geneexpressionregulationdependsonthetypeofinducibleorrepressiblepromotersandthechemicalorphysicaltreatmentsforpromoteractivationandrepression.SimultaneousmultigenerepressioninplantswasevaluatedbyLowderetal.(2015).Asyntheticrepressorsystem(pCo-dCas9-3X-SRDX)wasdesignedandtestedonArabidopsiscleavagestimulatingfactor64(AtCSTF64)geneandonnon-proteincodinggenes(redundantmicroRNAs-miR159AandmiR159B).ThemultigenegRNAdesignedagainstthesegeneswereconstructedintoaT-DNAcassetteharboringpCo-dCas9-3X(SRDX)pUBQ10control.Thetranscriptlevelswerereducedapproximatelyby60%ascomparedtocontrolamongthethreeindependenttransgeniclines.Similarly,thetranscriptlevelswerereducedto50%andmoreintransgeniclinesexpressingmiR159AandmiR159Btargetingconstruct. LiveCellImaging Plantchromosomesarehighlyorganizedandcompactstructures.ThespatiotemporalorganizationofplantgenomedeterminestheregulatorycharacteristicsofvariouscellfunctionssuchasDNAreplicationandrepair,transcriptionandcelldeath.Studiesanalyzingsubcellularlocalizationofgenesandchangeinchromosomalstructuresprovideinsightsintogenomeregulationandthesystemicregulationofcodingandnon-codinggenesduringdevelopment.InvivovisualizationofthedefinedDNAsequencesisdonepriorbyfluorescentinsituhybridization(FISH)butCRISPRimaginghasovercomevariousissuesrelatedtoFISHsuchasitsinabilitytovisualizedynamicprocessesandtherequirementoffixedtissuesamples.FISHalsorequiresthecellfixationandDNAdenaturationstepwhichmayalterthechromatinstructure(Boettigeretal.,2016).CRISPR/Cas9technologyiscustomizedwiththeintroductionofCas9variantknownas“deadCas9”(dCas9).dCas9isacatalyticallyinactiveformofthenuclease(pointmutationineitherofthetwocatalyticdomains,HNHandRuvC)thatfuseswithgeneraltranscriptionfactorstoitsC-termini(Figure7).dCas9hastheabilitytobindtospecifictargetDNAguidedbysgRNAandallowsdirectimagingandmanipulationoftranscriptionwithoutalteringtheDNAsequence(Dominguezetal.,2016).Puchta(2017)developedaCRISPR-dCas9basedcellimagingtechniquebasedonsitedirectedmutagenesisoftwoCas9orthologsderivedfromStreptococcuspyrogens(Sp-dCas9)andStaphylococcusaureus(Sa-dCas9)followedbyfusionofmultiplecopiesoffluorescenceproteinstotheC-terminalendofeachdCas9variant.TheuseofdCas9toinhibitgeneexpressionisreferredasCRISPRinterference(CRISPRi).Itisalsousedtodeliverspecificcargosandeffectorproteinstotargetedgenomiclocifortranscriptionalgeneregulation.dCas9hasalsobeingutilizedtorecruittranscriptionalactivatorstothetargetpromoter(Bikardetal.,2013).GeneactivationandrepressioninplantsisstilladvancingwithpositiveresultsreportedinNicotianabenthamiana(Piateketal.,2015)andA.thaliana(Lowderetal.,2015).ThisnewCas9basedsystemcanfurtherbeemployedtocontrolthespatiotemporalpatternsofgeneexpressioninplantsandmodulatinglifecyclesofvariouseconomicallyusefulcrops(Yang,2015). FIGURE7 FIGURE7.SchematicrepresentationofCas9nucleaseactivityanditsmodifications.SpCas9endonucleasescreateDSBsintargetDNAthroughtheactivityofRuvCandHNHnucleasedomains.SpCas9nucleasescanbeconvertedintoDNAnickasebysubstitutionofitskeyaminoacidsD10AandH840Athatproducessinglestrandedbreaks.SitedirectedmutagenesisinD10AproducesCas9nD10AandmutationinHNHdomainproducesCas9n(H840A).MutationsinbothcatalyticresiduesmodifyCas9toaninactivedeadCas9(dCas9). GenerationofMutantLibraries Agenomiclibraryisanindispensabletooltoidentifygenefunctionbyassessingthecellularphenotypesoflossoffunctionmutants.Progressionofgeneticmutantlibrarieshassimplifiedthegenomicexplicationofgenefunctioninmultipleorganisms(SchaefferandNakata,2016).Geneticperturbationscanbeachievedviaconventionalapproacheslikealteringthecopynumberofthegene,mRNAorprotein;useofchemicalmutagens;irradiation(AhloowaliaandMaluszynski,2001);orrandomintegrationofforeignDNAs(Tadegeetal.,2008).cDNAlibrariesforgain-of-functionmutationsandshortinterfering(si)RNAlibrariesforloss-of-functionmutationsareconsideredashigh-throughputscreeningapproachesbuthavevariousdrawbackslikelackofcontrolofoverexpressionlevelsandobstinatedownstreamanalysisduetomutationatmultipleloci(AgrotisandKetteler,2015).NowCRISPR/Cas9isrepurposedtoenablehighthroughputsequencescreening.Functionalscreeningisgenerallydoneintwoformats-arrayedandpooled(Shalemetal.,2015).VariouspublicationsillustratetheroleofCRISPR/Cas9technologyinscreening.Thearrayedformatisaonegeneperwell-analyzingtool.Individualreagentsarearrangedinmultiwellplateswithasinglereagentperwell(Shalemetal.,2015).Sinceeachreagentispreparedseparately,thismethodisexpensiveandtime-consumingbutallowsinvestigationofawiderrangeofcellularphenotypes.Pooledlibrariesaresinglepreparationsofmanydifferentplasmids.Thesescreensarelessexpensiveandlaborintensive(Shalemetal.,2015). DNAFreeModificationsofPlantGenome Cas9editedcropsareassumedtocrossmanyhurdlesandissuestobeclassifiedasgeneticallymodifiedcrops.Generally,CRISPR/Cas9DNAconstructsaredeliveredintoplantcellsbyAgrobacterium-mediatedinfiltration(Lietal.,2013),particlebombardment(Miaoetal.,2013)andprotoplasttransfection(Shanetal.,2013).TheAgrobacterium-mediatedmethodismorepopularbecauseithasapropensitytoinsertsingleoralowcopynumberoftransgenesanddoesnotrequireanexpensiveparticlegunapparatus(Charetal.,2016).However,theextraDNAdeliveredalongthegRNA,Cas9andselectablemarkergenesfrequentlyintegrateintotheplantgenomeandmaycausesideeffectslikegenedisruption,plantmosaicismandofftargetdisruptions.ForeignDNAmoleculescanfurtherintegrateintothetargetedDSBsites,lessenstheefficiencyofgeneeditingandgeneinsertion. ToalleviatethedisadvantagesofplasmidbasedexpressionofCas9/gRNA;efficientDNA-freegenomeeditingisadoptedwhichusesCas9ribonucleoproteins(RNPs).Cas9RNPsareinvitropre-integratedCas9nucleasesandgRNAthataredeliveredintoplantcellsasRNAmolecules(Figure8).Cas9RNPsareequallyefficienttoplasmidbasedexpressionsystemsforgeneknockoutsandgeneediting.Theseribonucleoproteinscanbedeliveredinmammaliancellsvialipid-mediatedelectroporationortransfectiontechniques(Liangetal.,2015).However,inplantsthepresenceofcellwallhindersthesetechniques.Therefore,RNPsaredeliveredinisolatedplantprotoplastsandsuccessfulresultshavebeenobtainedinavarietyofplantssuchastobacco,Arabidopsis,lettuce,rice,Petunia,andwheat(Wooetal.,2015;Subburajetal.,2016;Zhangetal.,2017). FIGURE8 FIGURE8.ProposedworkflowforDNAfreegenomeediting.Cas9isexpressedpurifiedfromE.coli.InvitrotranscriptionofsingleguideRNA(sgRNA)andtranscribedinvitroandRNPcomplexformation.RNPsandDNAprecipitationonto0.6μmgoldparticlesfollowedbyParticlebombardmentintargetedcells.PlantsregenerationwithoutanyselectiveagentfrombombardedcellsandscreenedformutationsviaPCR/restrictionenzymeassayanddeepsequencing. Similarly,Malnoyetal.(2016)havetargetedtheMLO-7geneingrapesfordevelopingresistanceagainstpowderymildewdiseaseandDIPM-1,DIPM-2,andDIPM-4genesinappleforresistanceagainstfireblightdiseaseusingCRISPR/Cas9ribonucleoproteins.CommerciallyavailablerecombinantCas9protein(160kDa)wasusedandsgRNAwasdesignedviaCRISPRRGENtoolswebsitefortargetspecificsiteshavinghigheroutofframescorestoachievemaximumknockoutefficiency.DirectdeliveryofCRISPRRNPsinplantprotoplastandefficienttargetedmutagenesiswith0.1%and0.5–0.69%indels(insertionordeletion)intargetedsitesofMLO-7andDIPM-1,2,and4wasreportedrespectively.Butplantregenerationfromprotoplastischallengingformostofthecerealcrops,mainlymonocots.Therefore,DNA-freeefficientgenomeeditinghasbeendoneinmultiplecropslikerice,maize,wheatusingCRISPR/Cas9ribonucleoproteincomplexesviaparticlebombardmentinembryocells.Svitashevetal.(2016)arethepioneerstoreportbiolisticdeliveryofCas9-gRNARNPintoimmaturemaizeembryo.Liguleless1(LIG)gene,Malefertilitygenes(MS26andMS45)andAcetolactatesynthasegene(ALS2)wastargetedandmutationfrequenciesofCas9/gRNAplasmidbasedsystemandCas9RNPswereevaluated.MutationfrequenciesofplasmidbasedCas9system-0.004,0.020,0.004,and0.002%respectivelyforLIG,ALS2,MS26,andMS45wasremarkablylowwhencomparedtoRNPdeliverywherethefrequencieswere0.57,0.45,0.21,and0.69respectively.Finally,efficientdeliveryandhighcleavageactivityofRNPswasdemonstrated(Svitashevetal.,2016). CRISPR/Cas9OpportunitiesandConcerns Customizablesequencespecificnucleasesareapowerfultoolforplantgenomeediting.Historically,meganucleases,ZFNs,andTALENshavebeenSSNsofchoicebuttheintroductionofCRISPR/Cas9systemhasrevolutionizedthegenomeeditingtechnologies.Theimportanceofthissystemliesinitsrelativeeaseofuse,highprecision,andlowstart-upcost.ThemostdistinctfeatureofCRISPRtechnology,i.e.,DNAcleavagerecognitionthroughWatsonandCrickbasepairingdrasticallysimplifiestheDNAtargeting.TheemergenceoftwoRNAcomponents(CRISPRRNAandtransactivatingCRISPRRNA)intosgRNAhasfurthersimplifiedtheCRISPR/Cassystemandenhancedreagentdelivery(Jineketal.,2012;Congetal.,2013).CRISPR/Cassystemallowssimultaneoustargetingofmultiplegenomiclociduetothesimplifiedengineeringoftargetspecificity(Zhouetal.,2014).Moreover,CRISPR/CassystemcanreadilybeengineeredtoCas9nickases,introducingsinglestrandedbreaks.Comparedtozincfingernickasesandtranscriptionactivator-likeeffectornickases,Cas9nickaseshavenoresidualnucleaseactivityandgreatlyalleviatetheriskofoff-targetactivity. AdvancementsandcharacterizationofnewCRISPReffectorproteinshavebroadenedtherangeofbiotechnologicalapplicationsviaCRISPR/Cassystem.Forexample,dormancyinanycellssuchascancercellscanbeachievedusingtypeVIC2c2effectorproteins.C2c2caninhibitcellgrowthinvivowhenprimedwithcognateRNA(Abudayyehetal.,2016).ThepotentialofaninactiveprogrammableRNA-bindingprotein(dC2c2)canbeusedtotrackandvisualizespecificRNAsandtomodulatethefunctionofeffectormodulesthatcanbeusedfortheconstructionofsyntheticregulatorycircuitsandlarge-scalescreening(Abudayyehetal.,2016).Thecontinuousdevelopmentandvalidationofnewfunctionaltoolkitsprovideimmenseopportunitiestoactivateanimprintedgeneandgeneexpression.Lowderetal.(2015)havedevelopedamultifacetedtoolkitconsistingofGoldenGateandGatewaycompatiblevectors.TheydemonstratedthelessexploredmultiplexingbyexpressingthreeindependentgRNAssimultaneouslyintobacco,rice,andArabidopsisandsuccessfullytriggeredorsuppressedtheexpressionofproteincodingandnon-codinggenes.Kleinstiveretal.(2015)proposedasolutiontogRNAmismatchandoff-targeteditingbyfeaturingtheinteractionbetweenfourdifferentdomainsofCas9.ThesedomainsincreasethebindingenergyofCas9totargetedsequencesuptomismatches,thusweakeningtheseinteractionswouldprovidebetterresultstoimproveoff-targetinteractions.MajorlimitationsoftheCRISPR/Cas9systemincludeinefficientHDRtoNHEJratioandveryfewsimultaneouschangespercell.Thefrequentoccurrenceofnon-targeteffectsfurtherhamperstheuseofthistechnology.OneofthemajordrawbacksofCas9editingismismatchedcleavagewhenthegRNAmismatchesafewbases.ManyreportsindicatedtheinfrequencywithwhichCRISPRcutsthenon-targetedsequences(Fuetal.,2013;Hsuetal.,2013). VisionaryNotionsofthisTechnology ResearchinvestigationinthepastquadrennialhastranscendgenomeeditingtoolsrangingfromtargetedgenemodificationstodesigningeIF4Eresistancealleleswhichisakeyplayerinvirusresistance(Bastetetal.,2017)toaltergenestocreatemultipleattributesliketolerancetoabioticandbioticstressinplantsviz.droughttolerance,virusanddiseaseresistance,enhancednutritional,highyieldcropandenhanceshelf-lifeoftheplants.CRISPR-Cas9technologywitnessesthefutureofversatilegenomeeditingwithrobustandefficaciousconsequences.TheforteofgeneeditinginplantsincludingcropshasbeenradicallychangedbyCRISPR-Cas9technology.Exploringthefundamentalbiologyofplantdevelopmentandstressresponsewillfacilitateindesigningeliteandsuperiorcrops.TheCRISPR-Cas9holdsaverypromisingfutureinmakingdesignerplantsbytakingonlythegeneofinterestfromawildtypespeciesandthegeneisthendirectlyinterpolatedatapreciselocation,whichinturnopensmanyavenuesforplantbreedersformakingdesignerplants.Variousapproachesaregoingtodesignplantsinsuchamanner,whichcouldwithstandwithallpossibleharshchallenges.ThenewlyemergedCRISPR/Cas9RNPsystemevadedtheneedtorelayontargetcellpotentialforCas9translationanditsplausiblemeetingwithgRNA. CRISPR/Cas9sequencespecificnucleaseeditingisaneffectiveapproachtocombatriceblastdisease(Wangetal.,2016).OsERF922geneinricewastargetedand21CRISPR-ERF922inducedmutantswereidentifiedfrom50T0transgenicplants(Wangetal.,2016).Furthermore,thehighthroughputcanbeobtainedbycoalescenceofcytidinedeaminaseenzymewithCas9,whichpermitshigh-efficiencyemendationoftargetcodonsinrice(LiL.etal.,2016).dCas9fusionwithcytidinedeaminaseallowsdirectconversionofcytidinetouridineleadingtoapointmutationfromC/GbptoT/Abpduringreplicationinoneofthedaughtercells(Puchta,2016).Researchesintheadvancementoflegendarytechnologyaredeliberatelygoingonbutonestubbornandconstantlyfollowingpitfallrelatedtooff-targetsinplants,whichcouldbeexecutedbydoingwholegenomesequencing.Manycompaniesarealsoengagedinusingthistechnologyfortheproductionofelitefoodandfeedcrops.Theproducts,whichareobtainedbyeditingthroughCRISPR-Cas9,havenoexogenousDNAandfurthermoreeditingcanbedoneinsuchaway,whichabidesbyalltherulesandregulationsthatarecomplaisanttowithstandagainstGeneticallyModifiedissuesandcangetaneasyapprovalbytheDepartmentofAgriculture(USDA).Inconclusion,CRISPR-Cas9technologyboastsofapromisingfutureinmakingthedesiredmutationinplantsbecauseithastransformedandmetamorphosedourpotentialtomodifyandregulateprokaryoticandeukaryoticgenomes.Theprevalentuseofthistechnologywillsurelyexpediteitspace. AuthorContributions LAhaswrittenthemanuscriptunderthesupervisionanddraftingofAN.ThereviewwasfinallyeditedandsubmittedbyAN. Funding ThearticleforpublicationissupportedbyFrontiersinPlantScience. ConflictofInterestStatement Theauthorsdeclarethattheresearchwasconductedintheabsenceofanycommercialorfinancialrelationshipsthatcouldbeconstruedasapotentialconflictofinterest. Acknowledgment Theauthorsarethankfultofrontiersforthefinancialassistance. 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Editedby: ManojK.Sharma,JawaharlalNehruUniversity,India Reviewedby: ElenaKhlestkina,InstituteofCytologyandGenetics(RAS),Russia KaijunZhao,ChineseAcademyofAgriculturalSciences,China XiaoouDong,UniversityofCalifornia,Davis,UnitedStates Copyright©2017AroraandNarula.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(CCBY).Theuse,distributionorreproductioninotherforumsispermitted,providedtheoriginalauthor(s)orlicensorarecreditedandthattheoriginalpublicationinthisjournaliscited,inaccordancewithacceptedacademicpractice.Nouse,distributionorreproductionispermittedwhichdoesnotcomplywiththeseterms. *Correspondence:AlkaNarula,[email protected] Peoplealsolookedat Download