Step aside CRISPR, RNA editing is taking off - Nature

RNA editing, by contrast, could allow clinicians to make temporary fixes that eliminate mutations in proteins, halt their production or ... Skiptomaincontent Thankyouforvisitingnature.com.YouareusingabrowserversionwithlimitedsupportforCSS.Toobtain thebestexperience,werecommendyouuseamoreuptodatebrowser(orturnoffcompatibilitymodein InternetExplorer).Inthemeantime,toensurecontinuedsupport,wearedisplayingthesitewithoutstyles andJavaScript. Advertisement nature newsfeature article StepasideCRISPR,RNAeditingistakingoff DownloadPDF IllustrationbyJoannaGębal DownloadPDF ThorstenStafforstfoundhisbigbreakattheworstpossibletime.In2012,histeamattheUniversityofTübingeninGermanydiscoveredthatbylinkingenzymestoengineeredstrandsofRNA,theycouldchangethesequencesofmessengerRNAmoleculesincells.Inessence,theycouldrewritethegenome’sinstructionsenroutetomakingproteins.Theprocesscouldtheoreticallyservetotreatnumerousdiseases,bothoneswithgeneticunderpinningsandthosethatwouldbenefitfromachangeintheamountortypeofaproteinbeingproduced.ButStafforsthadalotoftroublegettingthediscoverypublished—itwassimplynotinterestinganymore.Hisfinding1wasovershadowedbythediscoveryafewmonthsearlierthattheDNA-editingtoolCRISPR–Cas9couldbeusedtopermanentlyalterthegenome.Sincethen,CRISPRhasbecomeafixtureinthelaboratoryandhasspawnedanumberofcompaniesaimedatusingthetechnologytodevelopdrugsandtreatments.WithCRISPRsuckingupalltheattention,Stafforstsays,peoplereactedtohispaperwithindifference.Theyasked,“Whydoweneedthiswhenthere’sDNAediting?”ButCRISPRediting—atleastasatherapeutictechniqueinpeople—hasturnedouttobemoredifficultthaninitiallythought.ResearchershavedocumentedwaysthatCas9,oneoftheenzymesusedinCRISPRgeneediting,couldtriggerimmuneresponses,orcauseaccidentalchangestothegenomethatwouldbepermanent.RNAediting,bycontrast,couldallowclinicianstomaketemporaryfixesthateliminatemutationsinproteins,halttheirproductionorchangethewaythattheyworkinspecificorgansandtissues.BecausecellsquicklydegradeunusedRNAs,anyerrorsintroducedbyatherapywouldbewashedout,ratherthanstayingwithapersonforever.ExcitementoverRNAeditingisfinallycatchingon.In2019,researcherspublishedmorethan400papersonthetopic,accordingtodatafromScopus,anabstractandcitationdatabase.Ahandfulofstart-upcompaniesarebeginningtouseRNA-editingsystemstodeveloppotentialtreatmentsforeverythingfromgeneticdiseasessuchasmusculardystrophytotemporarymaladiessuchasacutepain.AndalthoughRNA-baseddrugshavehaddifficultyreachingthemarketowingtochallengesindeliveryandtolerance,someregulatoryapprovalsinthepastfewyearsmighthelptopavethewayforRNA-editingtherapies.Severalhurdlesremain:currenttechnologiescanalterRNAsequencesinonlyafewlimitedways,andgettingthesystemtoworkasintendedinthehumanbodywillprovechallenging.Still,researchershopethatnewtechnologies,suchasproteinengineering,andimprovedmethodsfordeliveringRNAtocellscanhelptoovercometheselimitations.“Itreallyopensaworldwehaven’tseenbefore,”Stafforstsays.AroleforRNAAfoundationaltenetinmoleculargenetics—itscentraldogma—wasthatcellularmachineryfaithfullytranscribesgeneticinformationfromadouble-strandedDNAtemplateintoasingle-strandedRNAmessenger,whichisthentranslatedintoaprotein.Butinthe1980s,ahandfuloflabsnoticedthatsomemRNAtranscriptscontainedalteredorextralettersthatwerenotencodedintheDNA.ThefindingswerecontroversialuntilscientistsuncoveredafamilyofenzymescalledadenosinedeaminasesactingonRNA(ADARs).TheseproteinsbindtoRNAsandaltertheirsequencebychangingafamiliarbaseknownasadenosineintoamoleculecalledinosine.AlthoughnotoneofthecanonicalRNAbases,inosineisreadbythecell’sprotein-translationmachineryasthefamiliarguanosine.AhandfulofotherRNA-editingenzymessurfacedaroundthesametime.ScientistshavestruggledoverthepastthreedecadestounderstandwhatexactlyRNAeditingaccomplishes.Theeditorsworkonlyondouble-strandedRNAs,whichsometimesshowupinthecellasregulatoryelements—orasviruses.SomehavespeculatedthattheADARproteinsevolvedasadefenceagainstviruses,butmanyviruseswithdouble-strandedRNAareunaffectedbytheenzymes.Theeditingmightservearegulatoryfunction,butmostadulttissuesdon’tproducethehighlevelsoftheproteinsrequiredfortheeditingtooccur. Thekill-switchforCRISPRthatcouldmakegene-editingsafer BrendaBass,abiochemistattheUniversityofUtahinSaltLakeCity,wasamongthefirsttoidentifyADARsinfrogembryos2.Shesaysthatnoonehasfoundaspecificroleforthechangesmadetonon-protein-codingRNAs,whichaccountforthemajorityofeditedmolecules.Theeditingcouldservetoprotectdouble-strandedRNAsfromimmuneattack.BasssuspectsthatADARseditthedouble-strandedtranscripts,addinginosinesasawayoftellingthebodytoleavethemalone.Theenzymesalsoseemtohavearoleinembryonicdevelopment:micethatlackADARgenesdiebeforebirthordon’tlivelongafter.Theeditorsalsoseemtohavesomefunctioninselecttissuesofadultorganisms—suchasthenervoussystemofcephalopods.ItwasthisactivitythatdrewmarinebiologistJoshuaRosenthaltoRNAeditingintheearly2000s.Itseemsthathighlyintelligentcephalopods,suchassquid,cuttlefishandoctopuses,useRNAeditingextensivelytoadjustgenesinvolvedinnerve-celldevelopmentandsignaltransmission.NootheranimalsareknowntouseRNAeditinginthisway.Inspiredbytheseobservations,Rosenthalwonderedwhetheritwaspossibletousethesystemtocorrectthemessagesproducedbydysfunctionalgenesinatherapeuticsetting.In2013,hisgroupattheUniversityofPuertoRicoinSanJuanre-engineeredADARenzymesandattachedthemtoguideRNAsthatwouldbindtoaspecificpointinanmRNA—creatingadoublestrand.Withthese,theywereabletoedittranscriptsinfrogembryos,andeveninhumancellsinculture3.SimilartoStafforst,Rosenthal,nowattheMarineBiologicalLaboratoryinWoodsHole,Massachusetts,sawhispublicationmostlyignored.Asimilarfate,helearnt,hadbefallentheworkofresearchersatacompanycalledRibozyme,whoin1995proposed‘therapeuticediting’ofmutatedRNAsequencesbyinsertingcomplementarysequencesintofrogembryosandallowingADARstoedittheresultingdouble-strandedmoleculeandcorrectthemutation4.Butinthepastseveralyears,multiplefactorshaveconvergedtobringRosenthal’sandStafforst’sfindingstothefore.PeterBeal,achemistattheUniversityofCalifornia,Davis,saysthatthe2016publication5ofthemolecularstructureofADARboundtodouble-strandedRNAmadethesystemmoreunderstandableandenabledscientiststobetterengineertheenzymetoenhanceitsdeliveryormakeitmoreefficient.Andin2018,theUSFoodandDrugAdministration(FDA)approvedthefirsttherapyusingRNAinterference(RNAi):atechniqueinwhichasmallpieceofRNAisinsertedintoacellinwhichitbindstonativemRNAsandhastenstheirdegradation.TheapprovalhasopenedthedoorforothertherapiesthatinvolvemRNAinteractions,saysGerardPlatenburg,chiefinnovationofficerofProQRTherapeuticsinLeiden,theNetherlands,whichispursuingvariousRNA-basedtherapies.“Learningfromthepast,andwiththenumberofapprovalspickingup,thefieldhasmaturedalot,”saysPlatenburg.ManyseeRNAeditingasanimportantalternativetoDNAeditingusingtechniquessuchasCRISPR.CRISPRtechnologyisimproving,butDNAeditingcancauseunwantedmutationsinotherpartsofthegenome—‘off-targeteffects’—whichmightcreatenewproblems. Super-precisenewCRISPRtoolcouldtackleaplethoraofgeneticdiseases Rosenthalexpects,moreover,thatRNAeditingwillproveusefulfordiseaseswithoutageneticorigin.HeiscurrentlyusingADARstoeditthemRNAforageneencodingthesodiumchannelNav1.7,whichcontrolshowpainsignalsaretransmittedtothebrain.PermanentlychangingtheNav1.7genethroughDNAeditingcouldeliminatetheabilitytofeelpainanddisruptothernecessaryfunctionsoftheproteininthenervoussystem,buttuningitdownthroughRNAeditinginselecttissuesforalimitedamountoftimecouldhelptoalleviatepainwithouttheriskofdependencyoraddictionassociatedwithconventionalpainkillers.Similarly,RNAeditingcouldallowresearcherstomimicgeneticvariantsthatprovideahealthadvantage.Forexample,peoplewithcertainmutationsinthegenePCSK9,whichregulatescholesterolinthebloodstream,tendtohavelowercholesterollevels,andmodifyingPCSK9mRNAcouldconferasimilaradvantagewithoutpermanentlydisruptingtheprotein’sotherfunctions.ImmunologistNinaPapavasiliouoftheGermanCancerResearchCenterinHeidelbergsaysthatRNAeditingcouldbeusedtofighttumours.Somecancershijackimportantcell-signallingpathways,suchasthoseinvolvedincelldeathorproliferation.IfRNAeditorscouldbeconscriptedtoturnoffkeysignallingmoleculestemporarily,shesays,“wecouldseethetumourdie”.Then,thepatientcouldstopthetherapy,allowingthepathwaytoresumeitsnormalfunctions.Asatreatment,RNAeditingmightbelesslikelytocauseapotentiallydangerousimmunereactionthanareCRISPR-basedapproaches.UnliketheDNA-editingenzymeCas9,whichcomesfrombacteria,ADARsarehumanproteinsthatdon’ttriggeranattackfromtheimmunesystem.“Youreallydon’tneedheavymachinerytotargetRNA,”saysPrashantMali,abioengineerattheUniversityofCalifornia,SanDiego.Inapaperpublishedlastyear6,MaliandhiscolleaguesinjectedguideRNAsintomicebornwithageneticmutationthatcausesmusculardystrophy.TheguideRNAsweredesignedtotriggerproductionofamissingproteincalleddystrophin.AlthoughthesystemeditedonlyasmallamountoftheRNAencodingdystrophin,itrestoredtheproteintoabout5%ofitsnormallevelintheanimals’muscletissue,anamountthathasshowntherapeuticpotential. IllustrationbyJoannaGębal Inotherdiseasesthatresultfromamissingordysfunctionalprotein,suchassometypesofhaemophilia,“itmakesahugedifferencetogofromnothingtosomething”,Stafforstsays,anditmightnotbenecessarytoeditRNAineverycellinthebody.RNAeditingmightperformbetterthanformsofgenetherapythatwouldinvolveinjectinganewgene.MaliandotherssaythatdirectingnativeADARstooperateonthecell’sownmRNAmightprovideamorenaturalresponsethanintroducinganexternal,engineeredgene.RNA-editingtechnologyisfarfromperfect,however,evenwhenitcomestolaboratoryapplications.“Itisearlydays,”Basssays.“There’slotsofquestions.”BecauseADARsaremuchlessefficientthanCRISPR,theycouldbelessusefulformakinggeneticallymodifiedplantsandanimals.“Asaresearchtool,it’sverylimiting,”saysJinBillyLi,ageneticistatStanfordUniversityinCalifornia.AnothermajordisadvantageisthatADARscanmakeonlyafewkindsofchangetoRNA.CRISPRsystemsactasscissorsbycuttingDNAatadesignatedspotandremovingorinsertinganewsequence;ADARsaremorelikeanoverwritefunctionthatchangesletterschemically,withoutbreakingtheRNAmolecule’s‘backbone’.Althoughthisprocessislesslikelytocauseunintendedmutations,itlimitstheenzymestomakingspecificchanges—adenosinetoinosineinthecaseofADARs,andcytosinetouridinebyasetofenzymescalledAPOBECs(see‘TheRNAcorrections’).Thereareafewotherpossibilities.Grapeplants,forinstance,canchangecytidinestouridines,andsometumourscanchangeguanosinestoadenosines.“Biodiversityisgivingustonsofanswerstothesethings,”Rosenthalsays.“Ithinkdowntheline,thingslikethesquidaregoingtoteachusalot.”Buthesaysthefieldisunderstudied—researchersdon’tunderstandtheprocessthatdrivesthisediting.Anditremainstobeseenwhetheraplantenzyme,forinstance,couldfunctioninhumancells. ScientistsarealreadylookingforwaystoengineernewenzymesthatcouldexpandRNA-editingcapabilities.“It’squiteaprocesswhereyoudon’tknowwhatyou’llfind,”saysOmarAbudayyeh,abiologicalengineerattheMassachusettsInstituteofTechnology(MIT)inCambridge.WorkingwithFengZhang,aCRISPRpioneeratMIT,AbudayyehandhiscolleagueslinkedanADARenzymetoCas137.AbacterialenzymesimilartotheCRISPR-associatedproteinCas9,Cas13cutsRNAinsteadofDNA.TheresearchersalteredthesequenceoftheADARuntilitcouldconvertcytidinestouridines.TheythenusedthenewsysteminhumancellstochangebasesinmRNAsencodedbyseveralgenes,includingAPOE.OnenaturallyoccurringgeneticvariantofthisgeneisassociatedwithAlzheimer’sdisease,andeditingitcouldswitchthevarianttotheharmlessform.AbudayyehandhisMITcollaborator,biologicalengineerJonathanGootenberg,admititispossiblethatchangingtheADARproteincouldcausetheimmunesystemtostoprecognizingitasanaturalhumanproteinandattackcellsthatcontainit.Buttheysaythatbecausetheseeditsaresmall,thisriskpalesnexttoknownconcernsabouttheimmunesystemattackingCas13orthevirususedtodelivertheeditingtoolsintocells.Researchersseepromiseinanaturalprocesscalledpseudouridylation,inwhichasetofproteinandRNAenzymeschemicallymodifythestructureofuridinesinmRNA.UnlikeADARmodifications,pseudouridylationdoesn’tchangethesequenceofthemRNAorprotein.Instead,forreasonsthatarenotentirelyclear,theprocessstabilizestheRNAmoleculeandcausesthetranslationmachinerytoignoresignalsinstructingittostopmakingprotein.Theabilitytoturnthesemolecularredlightsintogreenlightscouldbepowerful.Yi-TaoYu,abiochemistattheUniversityofRochesterinNewYork,saysthathundredsofgeneticdiseasesarecausedbyDNAmutationsthatcreateincorrectstopsignalsinmRNAs,resultinginashortenedproteinthatdoesn’tfunctionnormallyinthebody.“Thelistisverylong,”Yusays,andincludescysticfibrosis,theeyediseaseHurler’ssyndromeandnumerouscancers.Despiteitsearlystage,researchers—andbiotechinvestors—areexcitedaboutthewidepotentialofRNAediting.“Igotintoitwaybeforeitbecamecool,”saysPapavasiliou,whoistryingtomapwherenaturalADARsworkinthebody.“Formanyyearsthiswasabackwater,andallofasuddenthere’sacompanypoppingupeverytwoweeks.”Numerousstart-upsandestablishedDNA-editingfirmshaveannouncedtheirintentiontomoveintoRNA.TheyincludeBeamTherapeuticsinBoston,Massachusetts,whichwasco-foundedbyZhangandLiuandhasbeendevelopingCRISPRDNAeditingasatherapyforseveralblooddiseases.Locana,basedinSanDiego,isalsopursuingCRISPR-basedRNAeditingthatithopescouldtreatconditionsincludingmotor-neurondiseaseandHuntington’sdisease. CRISPRbabies:whenwilltheworldbeready? ThechallengeforindustryistoworkoutthebestwaytogettheguideRNAsintothecellwithouttriggeringanimmunereactionorcausingthecelltodegradethem.BealsaysthatthiscouldincludemakingstrategicchemicalmodificationstotheengineeredRNAsthatstabilizethem,orembeddingtheminananoparticleorvirusthatcansneakintocells.AndalthoughADARsarealreadyinhumancells,thehumanbodymakesonlysmallamountsoftheminmosttissues,meaningthatanytherapymightneedtoaddADARsorotherenzymestoboostcells’editingcapabilities.PackingviruseswiththegenesthatencodeallthemachineryneededforRNAeditingmightnotbeefficient.Manyhopethatitwon’tbenecessary.PlatenburghopestoaddRNAsandrelyonthenaturallyoccurringADARstohelptocorrecttheletteringofmRNAsthatcontributetoretinaldisorders.“Weusethesystemgiventousbynatureandharnessit,”hesays.ResearchersincludingStafforstareengineeringguideRNAswithchemicalmodificationsthatattractADARsinthecelltotheeditingsite.ButsomeresearchersworrythatconscriptingthenaturalADARsintoeditingspecificmRNAscouldpullthemawayfromtheirnormaltasksandcauseotherhealthproblems.Alteringgeneexpressioninonepartofthebodycouldaffectotherpartsinunforeseenways.InMali’smuscular-dystrophystudy,forinstance,micedevelopedliverproblemsforunknownreasons.“It’satoolindevelopmentstill,”hesays.“ADARevolvedtoallowthebodytomodifybasesinaverytargetedfashion,”saysNessanBermingham,chiefexecutiveandaco-founderwithRosenthalandothersofbiotechnologycompanyKorroBioinCambridge,Massachusetts.BerminghamisoptimisticabouttheprospectsofRNAediting,butcautiousnottogetaheadofthebiology.“Wehavealotofworktodoaswestarttomaturethesetechniques,”hesays.“We’renotleavinganythingoffthetable,butwehavetorecognizecertainlimitations.” Nature578,24-27(2020) doi:https://doi.org/10.1038/d41586-020-00272-5 ReferencesStafforst,T.&Schneider,M.F.Angew.Chem.Int.Ed.Engl.51,11166–11169(2012).PubMed  Article  GoogleScholar  Bass,B.L.&Weintraub,H.Cell55,1089–1098(1988).PubMed  Article  GoogleScholar  Montiel-Gonzalez,M.F.,Vallecillo-Viejo,I.,Yudowski,G.A.&Rosenthal,J.J.C.Proc.NatlAcad.Sci.USA110,18285–18290(2013).PubMed  Article  GoogleScholar  Woolf,T.M.,Chase,J.M.&Stinchcomb,D.T.Proc.NatlAcad.Sci.USA92,8298–8302(1995).PubMed  Article  GoogleScholar  Matthews,M.M.etal.NatureStruct.Mol.Biol.23,426–433(2016).PubMed  Article  GoogleScholar  Katrekar,D.etal.NatureMethods16,239–242(2019).PubMed  Article  GoogleScholar  Abudayyeh,O.O.etal.Science365,382–386(2019).PubMed  Article  GoogleScholar  Downloadreferences RelatedArticles Thekill-switchforCRISPRthatcouldmakegene-editingsafer Super-precisenewCRISPRtoolcouldtackleaplethoraofgeneticdiseases CRISPRbabies:whenwilltheworldbeready? CRISPRgene-editingsystemunleashedonRNA Subjects Biologicaltechniques Genetics Molecularbiology Lateston: Biologicaltechniques Howtomakespatialmapsofgeneactivity—downtothecellularlevel TechnologyFeature27JUN22 Synonymousmutationsinrepresentativeyeastgenesaremostlystronglynon-neutral Article08JUN22 Mechanismofmitoribosomalsmallsubunitbiogenesisandpreinitiation Article08JUN22 Genetics ‘Ghost’DNAfromtheworld’srarestwolveslingersincoyotes ResearchHighlight01JUL22 StructuralinsightsintodsRNAprocessingbyDrosophilaDicer-2–Loqs-PD Article29JUN22 Howtomakespatialmapsofgeneactivity—downtothecellularlevel TechnologyFeature27JUN22 Molecularbiology GTSF1acceleratestargetRNAcleavagebyPIWI-cladeArgonauteproteins Article30JUN22 StructuralinsightsintodsRNAprocessingbyDrosophilaDicer-2–Loqs-PD Article29JUN22 StructureoftheDicer-2–R2D2heterodimerboundtoasmallRNAduplex Article29JUN22 Jobs Postdoc(gn)-SingleCellTranscriptomics UniversityHospitalofMuenster(UKM),WWU Münster,Germany 68763:BachelorStudentAerospaceengineeringorsimilar(f/m/x)-LeachingExperimentsoflunarDustSimulantsinaqueousSolutions GermanAerospaceCenter(DLR) Bremen,Germany PhDPosition-InterfaceCatalysisbyDesignforScalableElectrosynthesisofValue-addedOrganicCompounds JülichResearchCentre(FZJ) Erlangen-Nürnberg,Germany Technicianmeteorologicalobservatories(m/f/d) AlfredWegenerInstitute-HelmholtzCentreforPolarandMarineResearch(AWI) Bremerhaven,Germany DownloadPDF RelatedArticles Thekill-switchforCRISPRthatcouldmakegene-editingsafer Super-precisenewCRISPRtoolcouldtackleaplethoraofgeneticdiseases CRISPRbabies:whenwilltheworldbeready? 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