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CRISPRinspiresnewtrickstoeditgenes
Themoleculartoolentersanewphaseofcreativeuses
By
TinaHesmanSaey
7:00am,August24,2016

RETOOLEDTweakstothecuttingedgegeneeditorknownasCRISPRenhanceitspowers.
MichaelMorgenstern
Scientistsusuallyshyawayfromusingthewordmiracleunlesstheyretalkingaboutthegeneeditingtool
calledCRISPR/Cas9.YoucandoanythingwithCRISPR,somesay.Othersjustcallitamazing.
CRISPRcanquicklyandefficientlymanipulatevirtuallyanygeneinanyplantoranimal.Inthefouryearssince
CRISPRhasbeenaround,researchershaveusedittofixgeneticdiseasesinanimals,combatviruses,sterilize
mosquitoesandpreparepigorgansforhumantransplants.Mostexpertsthinkthatsjustthebeginning.
CRISPRspowerfulpossibilitieseventhecontroversialnotionsofcreatingdesignerbabiesanderadicating
entirespeciesarestunningandsometimesfrightening.
Doingmore
TraditionalCRISPR/Cas9isonething:atargetedscissors.AguideRNAshepherdstheCas9enzymetoa
specificstretchofDNA.Cas9thencleavestheDNAtodisableorrepairagene.

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E.Otwell

NowresearchersareexpandingwhatCRISPR/Cas9candobyconvertingthecutterintoaplatformforahostof
specializedtools.

E.Otwell

AdisabledordeadCas9canfixasinglebasetypoinDNAsgeneticinstructions.Throughaseriesofsteps,
theenzymecytidinedeaminaseboltedtodeadCas9convertsaCGDNAbasepairintoTAbasepair.

E.Otwell

AttachingfluorescenttagstodeadCas9,researcherscanlocateandobservecertainstretchesofDNA(left)or
RNA(right)inalivingcell.

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E.Otwell

DeadCas9canblockRNApolymerasefromturningonagene,aprocesscalledCRISPRi(forCRISPR
interference).InCRISPRa(CRISPRactivation),aproteinthatturnsongenesisfusedtodeadCas9.

E.Otwell

EpigeneticmodifierproteinsfusedtodeadCas9attachchemicaltagstoDNAortohistones(proteinsthat
packageDNA).Tagsaffectgeneactivitybutdontchangethegenesinstructions.
SofarCRISPRsbiggestimpacthasbeenfeltinbasicbiologylabsaroundtheworld.Theinexpensive,easyto
usegeneeditorhasmadeitpossibleforresearcherstodelveintofundamentalmysteriesoflifeinwaysthat
hadbeendifficultorimpossible.DevelopmentalbiologistRobertReedlikensCRISPRtoacomputermouse.
Youcanjustpointitataplaceinthegenomeandyoucandoanythingyouwantatthatspot.
Anything,thatis,aslongasitinvolvescuttingDNA.CRISPR/Cas9initsoriginalincarnationisahomingdevice
(theCRISPRpart)thatguidesmolecularscissors(theCas9enzyme)toatargetsectionofDNA.Together,they
workasageneticengineeringcruisemissilethatdisablesorrepairsagene,orinsertssomethingnewwhereit
cuts.
EvenwithallthegeneticfeatstheCRISPR/Cas9systemcando,therewereshortcomings.Therewerethings
wewantedtodobetter,saysMITmolecularbiologistFengZhang,oneofthefirstscientiststowieldthe
molecularscissors.Fromhisearliestreportin2013ofusingCRISPR/Cas9tocutgenesinhumanandmouse
cells,Zhanghasdescribedwaystomakethesystemworkmorepreciselyandefficiently.

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Heisntalone.Aflurryofpapersinthelastthreeyearshavedetailedimprovementstotheeditor.Goingeven
further,abevyofscientists,includingZhang,havedreamedupwaystomakeCRISPRdoatoolboxsworthof
jobs.
TurningCRISPRintoamultitaskeroftenstartswithdullingthecuttingedgetechnologyscuttingedge.Inmany
ofitsnewadaptations,thedeadCas9scissorscantsnipDNA.Brokenscissorsmaysounduseless,but
scientistshaveupcycledthemintochromosomepainters,typocorrectors,geneactivitystimulatorsand
inhibitorsandgeneralgenometinkerers.
TheoriginalCas9islikeaSwissarmyknifewithonlyoneapplication:Itsaknife,saysGeneYeo,anRNA
biologistattheUniversityofCalifornia,SanDiego.ButYeoandotherresearchershaveboltedotherproteins
andchemicalstothedulledbladesandtransformedtheknifeintoamultifunctionaltool.
ZhangandcolleaguesarealsoexploringtradingtheCas9partofthesystemforotherenzymesthatmight
expandthetypesofmanipulationsscientistscanperformonDNAandothermolecules.Withtheexpanded
toolbox,researchersmayhavethepowertopryopensecretsofcancerandotherdiseasesandanswernew
questionsaboutbiology.
Flexibleguidance
ManyenzymescancutDNAthefirstwerediscoveredinthe1970sandhelpedtolaunchthewholefieldof
geneticengineering.WhatmakesCRISPR/Cas9specialisitsprecision.Scientistscanmakesurgicalslicesin
oneselectedspot,asopposedtothemorescattershotapproachofearlytools.Afewrecentgeneediting
technologies,suchaszincfingernucleasesandTALENs,couldalsolockontoasingletarget.Butthosegene
editorsarehardtoredirect.Ascientistwhowantstosnipanewspotinthegenomehastobuildaneweditor.
Thatslikehavingtoassembleauniqueguidedmissileforeverypossibletargetonamap.WithCRISPR/Cas9,
thatsnotnecessary.
ThesecrettoCRISPRsflexibilityisitsguidancesystem.AshortpieceofRNAshepherdstheCas9cutting
enzymetoitsDNAtarget.TheguideRNAcanhomeinonanyplacearesearcherselectsbychemically
pairingwithDNAsinformationcontainingbuildingblocks,orbases(denotedbythelettersA,T,CandG).
MakinganewguideRNAiseasyresearchersoftensimplyorderoneonlinebytypinginthedesiredsequence
ofbases.
Thatguidancesystemistakinggeneticengineerstoplacestheyveneverbeen.WithCRISPR,literally
overnightwhathadbeenthebiggestfrustrationofmycareerturnedintoanundergraduatesideproject,says
Reed,ofCornellUniversity.Itwasincredible.

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CRISPR/Cas9cangeneticallymanipulatebutterfliescuttingagenecalledDistallesscausesmoreeyespots(right)thanappear
onanormalwing(left).

L.ZhangandR.D.Reed/NatureCommunications2016
Reedstudieshowpatternsarepaintedonbutterflyandmothwings.Colorpatterningisoneofthefundamental
questionsevolutionaryanddevelopmentalbiologistshavebeentryingtoanswerfordecades.In1994,SeanB.
CarrollandcolleaguesdiscoveredthatagenecalledDistallessisturnedoninbutterflywingsinplaceswhere
eyespotslaterform.Thegeneappearedtobeneededforeyespotformation,buttheevidencewasonly
circumstantial.Thatswhereresearchershavebeenstuckfor20years,Reedsays.Theyhadnowayto
manipulategenesinbutterflywingstogetmoredirectproofoftheroleofdifferentgenesinpaintingwing
patterns.
WithCRISPR/Cas9,ReedandCornellcolleagueLinlinZhangcutanddisabledtheDistallessgeneatanearly
stageofwingdevelopmentandgotanunexpectedresult:Ratherthancauseeyespots,Distallesslimitsthem.
WhenCRISPR/Cas9knocksoutDistalless,moreandbiggereyespotsappear,theresearchersreportedin
JuneinNatureCommunications.Reedandcolleagueshavesnippedgenesinnotjustone,butsixdifferent
butterflyspeciesusingCRISPR,hesays.
CRISPRcutsgenesverywell,maybetoowell,saysneuroscientistMarcTessierLavigneofRockefeller
UniversityinNewYorkCity.TheCas9enzymeisjustsoprolific.Itcutsandrecutsandrecuts,hesays.That
constantsnippingcanresultinunwantedmutationsingenesthatresearchersareeditingoringenesthatthey
neverintendedtotouch.TessierLavigneandcolleaguesfiguredouthowtotametheovereagerenzymeand
keepitfromjulienningthegenesofhumanstemcellsgrowninlabdishes.Withbettercontrol,theresearchers
couldmakeoneortwomutationsintwogenesinvolvedinearlyonsetAlzheimersdisease,theyreportedinthe
May5Nature.Growingthemutatedstemcellsintobraincellsshowedthatincreasingthenumberofmutated
copiesofthegenesalsoboostsproductionoftheamyloidbetapeptidethatformsplaquesinAlzheimers
afflictedbrains.Thetechnologycouldmakestemcellsbettermimicsofhumandiseases.
Articlecontinuesbelowgraphic
Controllingtheeditor

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ResearcherstamedtheCas9cuttersoitwillsliceeitheroneorbothcopiesofagene,thenstop.Cuttingthe
APPandPSEN1genesmademutationsthatmimicthoseinpeoplepronetoearlyonsetAlzheimersdisease.
Neuronsmademoreofanabnormalplaqueformingpeptidewithtwomutations(darkestbars)thanwithone
mutation.

E.Otwell

Source:D.Paquetetal/Nature2016
WhileTessierLavigneandothersareworkingtoimprovetheCRISPR/Cas9system,buildingbetterguideRNAs
andincreasingthespecificityofitscuts,someresearchersareturningawayfromsnippyCas9altogether.
Nuancededits
Cas9isntentirelytoblameforthemesscreatedwhenitcausesadoublestrandedbreakbyslicingthrough
bothrailsoftheDNAladder.Thecellsresponsetodoublestrandedbreaksisthesourceofalotofproblems,
saysDavidLiu,achemicalbiologistatHarvardUniversity.AcellsgotomethodforfixingaDNAbreachisto
gluethecutendsbacktogether.Butoftenafewbasesaremissingorbitsgetstuckwheretheydontbelong.
Theresultismoregenomevandalismthanediting,Liusays,quotingHarvardcolleagueGeorgeChurch.
Liuwantedageneeditorthatwouldntcauseanydestructivebreaches:OnethatcouldA)gotoaspecificsitein
ageneandB)changeaparticularDNAbasethere,allwithoutcuttingDNA.Thetooldidntexist,butinCas9,
Liuandcolleaguessawthemakingsofone,iftheycouldtweakitjustabit.

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TheystartedbydullingCas9scuttingedge,effectivelykillingtheenzyme.ThedeadCas9couldstillgripthe
guideRNAandrideittoitsdestination,butitcouldntslicethroughDNAsdoublestrands.Liuandcolleagues
thenattachedahitchhikingenzyme,whosejobistoinitiateaseriesofstepstochangetheDNAbaseCintoaT,
oraGtoanA.Theresearchershadtotinkerwiththesysteminotherwaystogetthechangetostick.Once
theyworkedoutthekinks,theycouldmakepermanentsinglebasepairchangesin15to75percentoftheDNA
theytargetedwithoutintroducinginsertionsanddeletionsthewaytraditionalCRISPReditingoftendoes.Liu
andcollaboratorsreportedtheaccomplishmentinNatureinMay.Asimilarbaseeditor,reportedinSciencein
AugustbyresearchersinJapan,maybeusefulforeditingDNAinbacteriaandotherorganismsthatcant
toleratehavingtheirDNAcut.
Thereare12possiblecombinationsofDNAbaseswaps.ThehitchhikingenzymethatLiuused,cytidine
deaminase,canmaketwooftheswaps.LiuandothersareworkingtofuseenzymestoCas9thatcandothe10
others.OtherenzymehitchhikersmaymakeitpossibletoeditsingleDNAbasesatwill,Liusays.Suchabase
editorcouldbeusedtofixsinglemutationsthatcausegeneticdiseasessuchascysticfibrosisormuscular
dystrophy.Itmightevencorrectthemutationsthatleadtoinheritedbreastcancer.
Rewritingthescore
DeadCas9isalreadyhelpingresearcherstinkerwithDNAinwaystheycouldntbefore.Variationsonthedull
blademayhelpscientistssolveoneofthegreatmysteriesofbiology:Howdoesthesamesetof20,000genes
giverisetosomanydifferenttypesofcellsinthebody?
Thegenomeislikeapiano,saysJonathanWeissman,abiochemistattheUniversityofCalifornia,San
Francisco.Youcanplayahugevarietyofdifferentmusicwithonly88keysbyhowhardyouhitthekeys,what
keysyoumixupandthetiming.Bydialingdownorturninguptheactivityofcombinationsofgenesatprecise
timesduringdevelopment,cellsarecoaxedintobecominghundredsofdifferenttypesofbodycells.
Forthelast20years,researchershavebeenlearningmoreaboutthatprocessbywatchingwhencertaingenes
turnonandoffindifferentcells.Geneactivityiscontrolledbyadizzyingvarietyofproteinsknownas
transcriptionfactors.Whenandwhereatranscriptionfactoractsisatleastpartlydeterminedbychemicaltags
onDNAandthehistoneproteinsthatpackageit.Thosetagsareknowncollectivelyasepigeneticmarks.They
worksomethinglikethemusicalscoreforanorchestra,tellingthetranscriptionfactormusicianswhichnotes
tohitandhowloudlyorsoftlytoplay.Sofar,scientistshaveonlybeenabletolistentothemusic.Withdead
Cas9,researcherscancreatemoleculesthatwillchangeepigeneticnotesatanyplaceinthescore,Weissman
says,allowingresearcherstoarrangetheirownmusic.
Epigeneticmarksareallegedtobeinvolvedinaddiction,cancer,mentalillness,obesity,diabetesandheart
disease.Scientistshaventbeenabletoprovethatepigeneticmarksarereallybehindtheseandotherailments,
becausetheycouldnevergointoacellandchangejustonemarkononegenetoseeifitreallyproduceda
sournote.
Onesuchepigeneticmark,theattachmentofachemicalcalledanacetylgrouptoaparticularaminoacidina
histoneprotein,isoftenassociatedwithactivegenes.Butnoonecouldsayforsurethatthemarkwas
responsibleformakingthosegenesactive.CharlesGersbachofDukeUniversityandcolleaguesreportedlast
yearinNatureBiotechnologythattheyhadfuseddeadCas9toanenzymethatcouldmakethatepigenetic
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mark.Whentheresearchersplacedtheepigeneticmarkoncertaingenes,activityofthosegenesshotup,
evidencethatthemarkreallydoesboostgeneactivity.WithsuchCRISPRepigeneticeditorsinhand,
researchersmayeventuallybeabletocorrecterrantmarkstorestoreharmonyandhealth.
WeissmanslabgroupwasoneofthefirsttoturndeadCas9intoaconductorofgeneactivity.Parkingdead
Cas9onageneisenoughtonudgedownthevolumeofsomegenesactivitybyblockingtheproteinsthatcopy
DNAintoRNA,theresearchersfound.FusingaproteinthatsilencesgenestodeadCas9ledtoevenbetter
noisedampeningoftargetedgenes.TheresearchersreportedinCellin2014thattheycouldreducegene
activityby90to99percentforsomegenesusingthesilencer(whichWeissmanandcolleaguescallCRISPRi,
forinterference).Asimilartool,createdbyfusingproteinsthatturnon,oractivate,genestodeadCas9(called
CRISPRa,foractivator)letsresearcherscrankupthevolumeofactivityfromcertaingenes.Inaseparatestudy,
publishedinJulyintheProceedingsoftheNationalAcademyofSciences,Weissmanandcolleaguesusedtheir
activationschemetofindnewgenesthatmakecancercellsresistanttochemotherapydrugs.
RNArevolution
New,refittedCas9swontjustmakemanipulatingDNAeasier.TheyalsocouldrevolutionizeRNAbiology.There
arealreadymultiplemoleculartoolsforgrabbingandcuttingRNA,Yeosays.Soforhispurposes,scissors
werentnecessaryorevendesirable.ThehomingabilityofCRISPR/Cas9iswhatYeofoundappealing.
Hestartedsimple,byusingatweakedCRISPR/Cas9totagRNAstoseewheretheygointhecell.Luckily,in
2014,JenniferDoudnaattheUniversityofCalifornia,Berkeleyoneoftheresearcherswhoin2012
introducedCRISPR/Cas9andcolleaguesreportedthatCas9couldlatchontomessengerRNAmolecules,or
mRNAs(copiesoftheproteinbuildinginstructionscontainedinDNA).InastudypublishedinAprilinCell,
Doudna,YeoandcolleaguesstrappedfluorescentproteinstothebackofadeadCas9andpointedittoward
mRNAsfromvariousgenes.

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CRISPRattachesfluorescenttagstochromosomeendcaps,calledtelomeres,inacellsnucleus(top,green)aswellasto
centromeres,wherechromosomesarepinchedtogether(center,red).Combiningimagesshowswherethestructuresarein
relationtoeachother.

S.Wangetal/ScientificReports2016
WiththeglowingCas9,theresearcherstrackedmRNAsproducedfromseveraldifferentgenesinlivingcells.
(PreviousmethodsforpinpointingRNAslocationinacellkilledthecell.)InMay,ZhangofMITandcolleagues
describedatwocolorRNAtrackingsysteminScientificReports.Yetanothergroupofresearchersdescribeda
CRISPRrainbowforgivingDNAamulticoloredglow,alsoinlivingcells.Thatglowallowedtheteamtopinpoint
thelocationsofuptosixgenesandseehowthethreedimensionalstructureofchromosomesinthenucleus
changesovertime,theresearchersreportedintheMayNatureBiotechnology.AteamfromUCSanFrancisco

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reportedinJanuaryinNucleicAcidsResearchthatithadtrackedmultiplegenesusingcombinationsoftwo
colortags.
ButYeowantstodomorethanwatchRNAmovearound.Heenvisionsboltingavarietyofdifferentproteinsto
Cas9tomanipulateandstudythemanystepsanmRNAgoesthroughbetweenbeingcopiedfromDNAand
havingitsinstructionsreadtomakeaprotein.LearningmoreaboutthatmultistepprocessandwhatotherRNAs
doinacellcouldhelpresearchersunderstandwhatgoeswronginsomediseases,andmaybelearnhowtofix
theproblems.
ZhangwantstoimproveCas9,buthewouldalsolikeotherversatiletools.Heandcolleaguesarelookingfor
suchtoolsinbacteria.
CRISPR/Cas9wasfirstdiscoveredinbacteriaasarudimentaryimmunesystemforfightingoffviruses(SN:
12/12/15,p.16).ItzeroesinonandthenshredstheviralDNA.ResearchersmostoftenusetheCas9cutting
enzymefromStreptococcuspyogenesbacteria.
ButalmosthalfofallbacteriahaveCRISPRimmunesystems,scientistsnowknow,andmanyuseenzymes
otherthanCas9.InthebacteriumFrancisellanovicidaU112,Zhangandcolleaguesfoundageneediting
enzyme,Cpf1,whichdoesthingsalittledifferentlythanCas9does.Ithasadifferentcutheresignalthatcould
makeitmoresuitablethanCas9forcuttingDNAinsomecases,theteamreportedlastOctoberinCell.Cpf1
canalsochoponelongguideRNAintomultipleguides,soresearchersmaybeabletoeditseveralgenesat
once.AndCpf1cutsDNAsothatonestrandoftheDNAisslightlylongerthantheother.Thatcouldmakeit
easiertoinsertnewgenesintoDNA.
ZhangmorerecentlyfoundanenzymeinthebacteriumLeptotrichiashahiithatcouldtinkerwithRNA.The
RNAcuttingenzymeiscalledC2c2,heandcolleaguesreportedAugust5inScience.LikeCas9,C2c2usesa
guideRNAtoleadtheway,butinsteadofslicingDNA,itchopsRNA.
ZhangsteamisexploringotherCRISPR/Cas9styleenzymesthatcouldhelpthemeditormodulateorinteract
withagenomemoreefficientlyormoreeffectively,hesays.Oursearchisnotdoneyet.
TheexplosionofnewwaystouseCRISPRhasntended.Thefieldisadvancingsorapidly,saysZhang.Just
lookingathowfarwehavecomeinthelastthreeandahalfyears,Ithinkwhatwellseecominginthenextfew
yearswilljustbeamazing.
ThisarticleappearsintheSeptember3,2016,issueofScienceNewswiththeheadline,"CRISPRgetsa
makeover:Thegeneeditingtooldoesonethingwell,buthat'sjustthebeginning."
Citations
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eyespotcolorpatterns.NatureCommunications.Vol.7,June15,2016,p.11769.doi:10.1038/ncomms11769.
H.Yinetal.GenomeeditingwithCas9inadultmicecorrectsadiseasemutationandphenotype.Nature
Biotechnology.Vol.32,June2014,publishedonlineMarch30,2014,p.551.doi:10.1038/nbt.2884.

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C.Longetal.Postnatalgenomeeditingpartiallyrestoresdystrophinexpressioninamousemodelofmuscular
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M.Tabebordbaretal.Invivogeneeditingindystrophicmousemuscleandmusclestemcells.Science.Vol.
351,January22,2016,publishedonlineDecember31,2015,p.407.doi:10.1126/science.aad5177.
D.Paquetetal.EfficientintroductionofspecifichomozygousandheterozygousmutationsusingCRISPR/Cas9.
Nature.Vol.533,May5,2016,publishedonlineApril27,2016,p.125.doi:10.1038/nature17664.
D.A.Nellesetal.ProgrammableRNAtrackinginlivecellswithCRISPR/Cas9.Cell.Vol.165,April7,2016,p.
488.doi:10.1016/j.cell.2016.02.054.
S.Wangetal.AnRNAaptamerbasedtwocolorCRISPRlabelingsystem.ScientificReports.Vol.6,May27,
2016,p.26857.doi:10.1038/srep26857.
I.B.Hiltonetal.EpigenomeeditingbyaCRISPRCas9basedacetyltransferaseactivatesgenesfrom
promotersandenhancers.NatureBiotechnology.Vol.33,May2015,publishedonlineApril6,2015,p.510.
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C.J.Braunetal.VersatileinvivoregulationoftumorphenotypesbydCas9mediatedtranscriptional
perturbation.ProceedingsoftheNationalAcademyofSciences.Vol.113,July5,2016,p.E3892,published
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A.C.Komoretal.ProgrammableeditingofatargetbaseingenomicDNAwithoutdoublestrandedDNA
cleavage.Nature.Vol.533,May19,2016,publishedonlineApril20,2016,p.420.doi:10.1038/nature17946.
H.Maetal.MultiplexedlabelingofgenomiclociwithdCas9andengineeredsgRNAsusingCRISPRainbow.
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B.Zetscheetal.Cpf1isasingleRNAguidedendonucleaseofaclass2CRISPRCassystem.Cell.Vol.163,
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FurtherReading
T.H.Saey.Breakthroughgeneeditorsparksethicsdebate.ScienceNews.Vol.188,December26,2015,p.18.

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T.H.Saey.Genedrivesspreadtheirwings.ScienceNews.Vol.188,December12,2015,p.16.
T.H.Saey.Geneeditingmakespigssaferforhumantransplants.ScienceNews.Vol.188,November14,2015,
p.6.

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