2016 - Cell - Biology and Applications of CRISPR Systems -

LeadingEdge

Review

BiologyandApplicationsofCRISPRSystems:

HarnessingNature’sToolboxforGenomeEngineering

?ez,1andJenniferA.Doudna1,2,3,4,5,6,*AddisonV.Wright,1JamesK.Nun

ofMolecularandCellBiology,UniversityofCalifornia,Berkeley,Berkeley,CA94720,USA

HughesMedicalInstituteHHMI,UniversityofCalifornia,Berkeley,Berkeley,CA94720,USA

3DepartmentofChemistry,UniversityofCalifornia,Berkeley,Berkeley,CA94720,USA

4CenterforRNASystemsBiology,UniversityofCalifornia,Berkeley,Berkeley,CA94720,USA5InnovativeGenomicsInitiative,UniversityofCalifornia,Berkeley,Berkeley,CA94720,USA

6PhysicalBiosciencesDivision,LawrenceBerkeleyNationalLaboratory,Berkeley,Berkeley,CA94720,USA*Correspondence:doudna@berkeley.eduhttp://dx.doi.org/10.1016/j.cell.2015.12.035

2Howard1Department

Bacteriaandarchaeapossessarangeofdefensemechanismstocombatplasmidsandviralinfec-tions.UniqueamongthesearetheCRISPR-Cas(clusteredregularlyinterspacedshortpalindromicrepeats-CRISPRassociated)systems,whichprovideadaptiveimmunityagainstforeignnucleicacids.CRISPRsystemsfunctionbyacquiringgeneticrecordsofinvaderstofacilitaterobustinter-ferenceuponreinfection.InthisReview,wediscussrecentadvancesinunderstandingthediversemechanismsbywhichCasproteinsrespondtoforeignnucleicacidsandhowthesesystemshavebeenharnessedforprecisiongenomemanipulationinawidearrayoforganisms.

CRISPR-Cas(clusteredregularlyinterspacedshortpalindromicrepeats-CRISPRassociated)adaptiveimmunesystemsarefoundinroughly50%ofbacteriaand90%ofarchaea(Makarovaetal.,2015).Thesesystemsfunctionalongsiderestriction-modi-?cationsystems,abortiveinfections,andadsorptionblockstodefendprokaryoticpopulationsagainstphageinfection(Labrieetal.,2010).Unlikeothermechanismsofcellulardefense,whichprovidegeneralizedprotectionagainstanyinvadersnotpos-sessingcountermeasures,CRISPRimmunityfunctionsanalo-gouslytovertebrateadaptiveimmunitybygeneratingrecordsofpreviousinfectionstoelicitarapidandrobustresponseuponreinfection.

CRISPR-Cassystemsaregenerallyde?nedbyagenomiclo-cuscalledtheCRISPRarray,aseriesof??20–50base-pair(bp)directrepeatsseparatedbyunique‘‘spacers’’ofsimilarlengthandprecededbyanAT-rich‘‘leader’’sequence(Jansenetal.,2002;Kuninetal.,2007).NearlytwodecadesafterCRISPRlociwere?rstidenti?edinEscherichiacoli,spacerswerefoundtoderivefromviralgenomesandconjugativeplasmids,servingasrecordsofpreviousinfection(Bolotinetal.,2005;Ishinoetal.,1987;Mojicaetal.,2005;Pourceletal.,2005).SequencesinforeignDNAmatchingspacersarereferredtoas‘‘proto-spacers.’’In2007,itwasshownthataspacermatchingaphagegenomeimmunizesthehostmicrobeagainstthecorrespondingphageandthatinfectionbyanovelphageleadstotheexpansionoftheCRISPRarraybyadditionofnewspacersoriginatingfromthephagegenome(Barrangouetal.,2007).

CRISPRimmunityisdividedintothreestages:spacerac-quisition,CRISPRRNA(crRNA)biogenesis,andinterference(Figure1A)(Makarovaetal.,2011b;vanderOostetal.,2009).Duringspaceracquisition,alsoknownasadaptation,foreignDNAisidenti?ed,processed,andintegratedintotheCRISPRlocusasanewspacer.ThecrRNAbiogenesisorexpression

stageinvolvesCRISPRlocustranscription,oftenasasinglepre-crRNA,anditssubsequentprocessingintomaturecrRNAsthateachcontainasinglespacer.Intheinterferencestage,aneffectorcomplexusesthecrRNAtoidentifyanddestroyanyphageorplasmidbearingsequencecomplementaritytothespacersequenceofthecrRNA.

ThesestepsarecarriedoutprimarilybyCasproteins,whichareencodedbycasgenes?ankingtheCRISPRarrays.Thespe-ci?ccomplementofcasgenesvarieswidely.CRISPR-Cassys-temscanbeclassi?edbasedonthepresenceof‘‘signaturegenes’’intosixtypes,whichareadditionallygroupedintotwoclasses(Figure1B)(Makarovaetal.,2011b;Makarovaetal.,2015;Shmakovetal.,2015).TypesI–IIIarethebeststudied,whileTypesIV–VIhaveonlyrecentlybeenidenti?ed(MakarovaandKoonin,2015;Makarovaetal.,2015;Shmakovetal.,2015).ThesignatureproteinofTypeIsystemsisCas3,aproteinwithnucleaseandhelicasedomainsthatfunctionsinforeignDNAdegradationtocleaveDNAthatisrecognizedbythemulti-protein-crRNAcomplexCascade(CRISPR-associatedcomplexforantiviraldefense).InTypeIIsystems,thesignaturecas9geneencodesthesoleproteinnecessaryforinterference.TypeIIIsystemsaresigni?edbyCas10,whichassemblesintoaCascade-likeinterferencecomplexfortargetsearchanddestruction.TypeIVsystemshaveCsf1,anuncharacterizedproteinproposedtoformpartofaCascade-likecomplex,thoughthesesystemsareoftenfoundasisolatedcasgeneswithoutanassociatedCRISPRarray(MakarovaandKoonin,2015).TypeVsystemsalsocontainaCas9-likesinglenuclease,eitherCpf1,C2c1,orC2c3,dependingonthesubtype(Shmakovetal.,2015;Zetscheetal.,2015a).TypeVIsystemshaveC2c2,alargeproteinwithtwopredictedHEPN(highereu-karyotesandprokaryotesnucleotide-binding)RNasedomains(Shmakovetal.,2015).TypeI,III,andIVsystemsareconsidered

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Figure1.FunctionandOrganizationofCRISPRSystems

(A)CRISPRimmunityoccursinthreestages.UponintroductionofforeignDNA,theadaptationma-chineryselectsprotospacersandinsertsthemintotheleaderendoftheCRISPRlocus.DuringcrRNAbiogenesis,theCRISPRlocusistranscribedandsequenceelementsintherepeatsdirectprocess-ingofthepre-crRNAintocrRNAseachwithasinglespacer.ThecrRNAthenassembleswithCasproteinstoformtheeffectorcomplex,whichactsintheinterferencestagetorecognizeforeignnucleicaciduponsubsequentinfectionanddegradeit.

(B)CRISPRsystemsareextremelydiversebutcanlargelybeclassi?edintosixmajortypes.Repre-sentativeoperonsforeachtypeareshownhere.Genesonlypresentinsomesubtypesareshownwithdashedoutlines.Genesinvolvedininterfer-encearecoloredred,thoseinvolvedincrRNAbiogenesisarecoloredyellow,andthoseinvolvedinadaptationarecoloredblue.TypeIVsystemsarenotablefortheirfrequentoccurrenceintheabsenceofCRISPRloci.

Class1systemsbasedontheirmulti-subuniteffectorcom-plexes,whilethesingle-subuniteffectorTypeII,V,andVIsys-temsaregroupedintoClass2(Makarovaetal.,2015;Shmakovetal.,2015).

ThestudyofCRISPRbiologyhasrevealedenzymemecha-nismsthatcanbeharnessedforprecisiongenomeengineeringandotherapplications,leadingtoanexplosionofinterestinbothnativeCRISPRpathwaysandtheuseofthesesystemsforapplicationsinanimals,plants,microbes,andhumans.InthisReview,wediscussrecentadvancementsinthe?eldthatrevealunexpecteddivergence,aswellasunifyingthemesunder-lyingthethreestagesofCRISPRimmunity.Ineachcase,wehighlightthewaysinwhichthesesystemsarebeingharnessedforapplicationsacrossmanyareasofbiology.

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Acquisition:CreatingGeneticRecordsofPastInfections

CRISPRimmunitybeginswiththedetec-tionandintegrationofforeignDNAintothehostcell’schromosome.IntheStrep-tococcusthermophilusTypeII-Asystem,whereacquisitionwas?rstdetectedexperimentally,newspacersfrombacte-riophageDNAareinsertedintotheleaderendoftheCRISPRlocus,causingdupli-cationofthe?rstrepeattomaintaintherepeat-spacerarchitecture(Figure1A)(Barrangouetal.,2007).SubsequentstudiesusingtheE.coliTypeI-Esystemveri?edthatCas1andCas2mediatespaceracquisition(Datsenkoetal.,2012;Swartsetal.,2012;Yosefetal.,2012).Theselectionofnewprotospacersequencesisnonrandomand,inmostsystems,dependsonthepresenceofa2–5nucleotideprotospaceradjacentmotif(PAM)foundnexttotheprotospacer

sequence(Deveauetal.,2008;Mojicaetal.,2009).PAM-speci?cselectionofprotospacersiscriticalforimmunity,ascrRNA-guidedinterferenceinmostsystemsdependsonthePAMsequenceforforeignDNAdetectionanddestruction,whichavoidsself-targetingatthePAM-freeCRISPRlocus.Interest-ingly,spacersoriginatingfromthehostgenomearepresentinalmost20%ofCRISPR-containingorganisms,suggestingalter-nativerolesoftheCRISPR-Casmachineryindirectingotherpro-cessessuchasendogenousgeneregulationandgenomeevolu-tion(Westraetal.,2014).SpaceracquisitionhasbeenobservedexperimentallyinvarioussystemsacrossTypesI–III.Here,wefocusonrecentmechanisticstudiesofacquisitioninTypeI-EandTypeII-Asystems,inwhichthemostcomprehensivestudieshavebeendone.

Figure2.ProtospacerSelectionandInte-grationinAdaptation

(A)Theselectionofprotospacersforacquisitionispoorlyunderstood,butstudiessuggestatleastthreedistinctmechanismsfortheselectionofsubstratesforintegration.InTypeIsystems,primedadaptationoccurswhenCascadebindsapartiallymismatchedtarget.Thenuclease/heli-caseCas3isrecruitedtothetargetsiteandthenlikelytranslocatesalongthetargetDNAtoanewsite.Thenewlocationisthenselectedasapro-tospacertobeusedbyCas1-Cas2intheintegra-tionreaction.InE.coli,naiveadaptationinvolvesthenuclease/helicaseRecBCD.ThedegradationproductsappeartoserveassubstratesforCas1-Cas2,buthowthevariable-lengthsingle-strandedproductsofRecBCDactivityareconvertedintodouble-strandedprotospacersofappropriatesizeisunknown.InTypeIIsystems,Cas9recognizesPAMsitesandlikelyrecruitsCas1-Cas2toacquirethe?ankingsequence.

(B)Cas1-Cas2actasanintegrasetoinsertpro-tospacersintotheCRISPRlocusasnewspacers.Thecomplexwithprotospacerboundrecognizestheleader-adjacentrepeatandcatalyzesapairoftransesteri?cationreactions.The30OHofeachprotospacerstrandmakesanucleophilicattackontherepeatbackbone,oneattheleader-sideandoneatthespacerside.Theresultinggappedproductisthenrepaired,causingduplicationofthe?rstrepeat.

TypeIAcquisition

AcquisitioninE.colioccursviatwomechanisms—naiveandprimed(Figure2A).NaiveacquisitioninitiatesuponinfectionbypreviouslyunencounteredDNAandreliesontheCas1-Cas2integrasecomplextorecognizeandacquirenewspacersfromforeignDNA.OverexpressionofCas1andCas2intheabsenceofotherCasproteinsleadstotheacquisitionof33bpspacersattheleader-proximalendoftheCRISPRarray(Datsenkoetal.,2012;Yosefetal.,2012).ThePAMoftheE.coliCRISPR-Cassystemwasidenti?edas50-AWG-30,withtheGbecomingthe?rstnucleotideoftheintegratedspacer(Dat-′ez-Villasen?oretal.,2013;Levyetal.,senkoetal.,2012;D?

?ezetal.,2014;Savitskayaetal.,2013;Shmakov2015;Nun

etal.,2014;Swartsetal.,2012;Yosefetal.,2012;Yosefetal.,2013).InadditiontothePAM,adinucleotidemotif,AA,foundatthe30endoftheprotospacerwasalsoshowntobepresentinadisproportionatelylargenumberofspacers(Yosefetal.,2013).ArecentcrystalstructureoftheCas1-Cas2complexboundtoanunprocessedprotospacerrevealedsequence-spe-ci?ccontactswiththe50-CTT-30sequenceonthePAM-comple-

mentarystrand,suggestingthatCas1recognizesPAMsitesonpotentialproto-spacersbeforetheyareprocessedforintegration(Wangetal.,2015).

Afteraspacerisacquiredfromanewinvader,theresultingcrRNAassembleswithCasproteinstoformCascade,theinterferencecomplexcapableoftargetingPAM-adjacentDNAsequencesmatchingthespacersequenceofthecrRNA(Brounsetal.,2008;Joreetal.,2011;Lint-neretal.,2011).Upontargetbinding,thehelicase/nucleaseCas3isrecruitedtothesiteandprocessivelydegradestheforeignDNA(Hochstrasseretal.,2014;MulepatiandBailey,2011;Sinkunasetal.,2011;Sinkunasetal.,2013;Westraetal.,2012).Strikingly,whenCascadeencountersamutantPAMorprotospacerthatpreventsCas3degradation,hyperac-tivespaceracquisitionfromthetargetedplasmidorgenomeistriggeredinaprocesscalled‘‘priming’’(Figure2A)(Datsenkoetal.,2012;Lietal.,2014;Richteretal.,2014;Savitskayaetal.,2013;Swartsetal.,2012).Primingincreasesthehost’srepertoireoffunctionalspacers,allowingthehosttoadapttoin-vadersthatevadetheCRISPR-Cassystembymutation.Cascadeiscapableofbindingescapemutanttargetsites,andrecentsingle-moleculestudiesshowedthatthepresenceofCas1andCas2allowsfortherecruitmentofCas3tothesesites(Blosseretal.,2015;Reddingetal.,2015;Richteretal.,2014).TherecruitedCas3canthentranslocateineitherdirection,incontrasttotheunidirectionalmovementobservedatperfecttar-gets,withoutdegradingthetargetDNA(Reddingetal.,2015).Cas1andCas2mayaccompanythetranslocatingCas3and

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beactivatedforprotospacerselection,allowingforrobustacqui-sitiononeithersideofthetargetsite.

PrimedacquisitionhasalsobeenshownexperimentallyintheP.atrosepticumTypeI–Fsystem,inwhichCas2andCas3arenaturallyfusedasasinglepolypeptidethatassociateswithCas1,aswellasintheHaloarculahispanicaTypeI-Bsystem,wherenaiveacquisitionwasnotexperimentallyobserved(Lietal.,2014;Richteretal.,2014;Richteretal.,2012).AcquisitioninH.hispanicaalsorequiresCas4,a50/30exonucleasefoundinmostTypeIsubtypesaswellasTypeII-BandTypeVsystems,andwhichmightbeinvolvedingenerating30overhangsonpro-tospacerspriortointegration(Lemaketal.,2013;Lietal.,2014;Makarovaetal.,2015).AlthoughCas1andCas2maybethemin-imalproteinsrequiredforspaceracquisitioninsomesystems,theassociationofCas1,Cas2,andtheinterferencemachineryallowsthehosttocoordinaterobustadaptiveimmunityinTypeIsystems.

Self-versusNon-Self-Recognition

ThemechanismunderlyingthepreferenceforforeignoverselfDNAduringprotospacerselectionremainedpoorlyunderstooduntilarecentstudyonspaceracquisitionduringnaiveacquisi-tion.SpaceracquisitioninE.coliwasshowntobehighlydepen-dentonDNAreplication,andforeign-derivedspacerswerepreferredoverself-derivedspacersbyabout100-to1,000-fold(Levyetal.,2015).Analysisofthesourceofself-derivedspacersdemonstratedthatprotospacerswereacquiredlargelyfromgenomiclocipredictedtofrequentlygeneratestalledrepli-cationforksanddouble-strandedDNAbreaks(Levyetal.,2015).SuchharmfuldsDNAbreaksarerepairedbythehelicase/nucleaseRecBCDcomplex,whichdegradesthebrokenendsuntilreachingaChi-site,afterwhichonlythe50endisdegraded(DillinghamandKowalczykowski,2008).DuetothelowerfrequencyofChisitesinforeignDNA,RecBCDispredictedtopreferentiallydegradeplasmidsandviralDNA,resultinginthegenerationofcandidateprotospacersubstratesforCas1andCas2(Levyetal.,2015)(Figure2A).RecBCDdegradesDNAasymmetrically,yieldingsingle-strandedfragmentsrangingfromtenstohundredsofnucleotideslongfromonestrandandkilobaseslongfromtheother(DillinghamandKowalczykowski,2008).ItisunclearhowCas1-Cas2substrates,whichare33bplongandpartiallydoublestrandedwith30overhangs,are

generatedfromRecBCDproducts(Nun

?ezetal.,2015a;Nun?ezetal.,2015b;Wangetal.,2015).ItispossiblethatssDNAprod-uctsre-annealtoproducepartialduplexes,followedbyprocess-ingto33bpbyanunknownmechanismpriortointegrationintotheCRISPRlocus.RecentcrystalstructuresofCas1-Cas2withboundprotospacerrevealthatthecomplexde?nesthelengthoftheduplexregionoftheprotospacerviaarulermechanismand

maycleavethe30overhangstotheir?nallength(Nun

?ezetal.,2015b;Wangetal.,2015).Theinvolvementofahelicase/nucleaseinbothTypeI-Eprimedandnaiveacquisition(Cas3andRecBCD,respectively),aswellasinCas4-containingsub-types,hintsataconservedmechanismforprotospacergenera-tion.ItisalsoworthnotingthatRecBCDisconservedprimarilyinGram-negativebacteria,whileGram-positivebacteriaandarchaearelyonAddABandHerA-NurA,respectively,to?llasimilarrole(Blackwoodetal.,2013;DillinghamandKowalczy-kowski,2008).WhetherCRISPR-Cassystemsintheseorgan-32Cell164,January14,2016a2016ElsevierInc.

ismshaveevolvedtocooperatewiththeseevolutionarilydistinctmachineriesremainstobetested.

MechanismofProtospacerIntegration

Cas1andCas2playcentralrolesintheacquisitionofnew

spacers,wheretheyfunctionasacomplex(Nun

?ezetal.,2014).CrystalstructuresofCas1andCas2,withorwithoutboundprotospacer,revealedtwocopiesofaCas1dimer

bridgedbyacentralCas2dimer(Nun

?ezetal.,2014;Nun?ezetal.,2015b;Wangetal.,2015).Cas1functionscatalytically,whileCas2appearstoserveaprimarilystructuralrole(Arslan

etal.,2014;Datsenkoetal.,2012;Nun

?ezetal.,2014;Yosefetal.,2012).

The?rstinsightintothemechanismofprotospacerintegrationwasgainedbySouthernblotanalysisofthegenomicCRISPRlocusofE.colicellsoverexpressingCas1andCas2(Arslanetal.,2014).Thisrevealedintegrationintermediatesconsistentwithtwotransestere?cationreactions,whereeachstrandoftheprotospacerisintegratedintooppositesidesoftheleader-proximalrepeat(Figure2B).Thisintegrase-likemodelwasfurtherbolsteredbytheinvitroreconstitutionofprotospacerintegrationintoaplasmid-encodedCRISPRlocususingpuri?ed

Cas1-Cas2complex(Nun

?ezetal.,2015a).Theintegrationreac-tionrequireddouble-strandedDNAprotospacerswith30-OHendsthatareintegratedintoplasmidDNAviaadirectnucleo-philictransesteri?cationreaction,reminiscentofretroviralinte-grasesandDNAtransposases(Engelmanetal.,1991;MizuuchiandAdzuma,1991).

Althoughdeepsequencingofinvitrointegrationproductsre-vealedpreferentialprotospacerintegrationadjacenttothe?rstrepeat,con?rmingthatCas1-Cas2directlyrecognizetheCRISPRlocus,integrationalsooccurredatthebordersofevery

repeatatvaryinglevels(Nun

?ezetal.,2015a).Thiscontrastswithspaceracquisitiononlyoccurringatthe?rstrepeatinE.coliinvivo(Datsenkoetal.,2012;Swartsetal.,2012;Yosefetal.,2012).TodetermineiftheCas1-Cas2complexhassequencespeci?cityfortheleader-repeatsequence,arecentstudytookadvantageoftheCas1-catalyzeddisintegrationreac-tion,areversaloftheintegrationreactionalsoobservedwithretroviralintegrasesandtransposases(Chowetal.,1992;Rollieetal.,2015).Disintegrationactivitywasstimulatedwhenusingthecorrectleader-repeatbordersequences,highlightingintrinsicsequence-speci?crecognitionbyCas1.Furthermore,disintegra-tionwasfasterattheleader-repeatjunctioncomparedtotherepeatdistalend(Rollieetal.,2015).Takentogether,protospacerintegrationlikelybeginsattheleader-repeatjunctionviasequence-speci?crecognitionbyCas1,followedbyasecondnucleophilicattackattherepeatdistalend.Thisensurespreciseduplicationofthe?rstrepeat,asobservedinvivo,afterDNArepairbyhostproteins.Theintegrationmechanismishypothe-sizedtobehighlyspeci?c,asalmostallacquiredspacerswithacorrespondingAAGPAMareorientedwiththe50-Gattheleader-proximalend,leadingtofunctionalcrRNA-dependenttar-getingbyCascadeandCas3(Shmakovetal.,2014).Apreferenceforintegrationintheproperorientationwasobservedinvitro

whenprotospacerswitha50-Gwereused(Nun

?ezetal.,2015a);however,inclusionofpartofthePAMinspacershasonlybeenobservedinE.coli,raisingthequestionofhowCas1-Cas2inothersystemsproperlyorienttheintegrationreaction.

TypeIIAcquisition

WhilemostmechanisticworkonacquisitionhasbeenperformedinTypeIsystems,recentstudiesinTypeIIsystemshavealsoshedlightonkeyaspectsofspaceracquisition.Onegeneraliz-able?ndinginTypeIIsystemsisthedependenceofacquisitiononinfectionbydefectivephage(Hynesetal.,2014).Asigni?cantproblemwithCRISPRimmunityisthetimerequiredforforeignDNAtobeidenti?ed,integratedintotheCRISPRlocus,tran-scribed,processed,andassembledintoaninterferencecom-plexthatmustthenbeginthesearchforappropriatetargets.Sincelyticphagecankillcellswithin20min,providinginsuf?-cienttimeforthismulti-stepprocess,Hynesandcolleaguestestedthehypothesisthatinitialimmunizationtakesplacefrominfectionbyadefectivephage.SupplementationofactivephagewithUV-irradiatedphageorphagesusceptibletoarestriction-modi?cationsystemstimulatedspaceracquisitioncomparedtothatobservedwithactivephagealone(Hynesetal.,2014).Theauthorsspeculatethatacquisitionfromcompromisedphagemightalsorepresentthedominantmodeofacquisitioninwildpopulations,allowingforasmallsubsetofthepopulationtoac-quireresistanceandescapewithoutneedingtooutpacearapidlyreproducingphage.

TypeIIAcquisitionMachinery

TypeIIsystemsaresubdividedintoII-A,II-B,andII-Cbasedonthepresenceorabsenceofanadditionalcasgenealongsidetheminimalcomplementofcas1,cas2,andcas9.TypeII-Asystemscontaincsn2,whileTypeII-Bsystems,whichareleastcommonlyfound,containcas4(Chylinskietal.,2014;Makarovaetal.,2011b).TypeII-Csystemscompriseonlytheminimalthreegenes.Csn2hasbeenshowntobeessentialforacquisitioninseveralTypeII-Asystems(Barrangouetal.,2007;Heleretal.,2015;Weietal.,2015b).Itformsatetramerwithatorroidalarchi-tecturethatbindsandslidesalongfreeDNAends,thoughitsfunctioninCRISPRsystemsisunclear(Arslanetal.,2013;Ellingeretal.,2012;Kooetal.,2012;Leeetal.,2012).Cas4,discussedabove,islikelyinvolvedinacquisitioninTypeII-Bsys-tems.TypeII-Csystems,whichconstitutethemajorityofidenti-?edTypeIIsystems(Chylinskietal.,2014;Makarovaetal.,2015),arepossiblyfunctionalforacquisitionintheabsenceofauxiliaryacquisitionfactors,thoughinthecaseoftheCampylo-bacterjejunisystem,acquisitionwasonlyobservedfollowinginfectionbyphageencodingaCas4homolog(HootonandCon-nerton,2014).

Recently,twosimultaneousstudiesdemonstratedthat,inadditiontoCas1,Cas2,andCsn2,Cas9playsanecessaryroleintheacquisitionofnewspacersinTypeIIsystems(Heleretal.,2015;Weietal.,2015b).Bothgroups,oneworkingwiththeCRISPR1TypeII-AsystemofS.thermophilus,theotherwiththeTypeII-AsystemofStreptococcuspyogenesandtheCRISPR3systemofS.thermophilus,alsoTypeII-A,showedthatwild-typeorcatalyticallyinactiveCas9(dCas9)supportedrobustspaceracquisition,whereasdeletionofCas9abolishedspaceracquisition.ItisproposedthatCas9servestorecognizePAMsitesinpotentialprotospacersandmarkthemforrecogni-tionbyCas1andCas2(Figure2A).Thishypothesiswascon?rmedbymutatingthePAM-interactingresiduesofCas9,re-sultingincompletelossinPAM-speci?cityinthenewlyacquiredspacers(Heleretal.,2015).ThispresentsastrikingcontrasttotheE.coliTypeI-Esystem,whereCas1-Cas2recognizePAMsequencesindependently.

Intriguingly,expressionofdCas9resultsintheacquisitionofprimarilyself-targetingspacers,suggestingthatmanyacquisi-tioneventsleadtoself-targetingandsuicide(Weietal.,2015b).MicrobialpopulationsmayrelyonafewindividualstoacquirephageresistancewhiletherestsuccumbtoinfectionorCRISPR-mediatedsuicide.Somesystems,suchasthatfoundinE.coli,mayevolvetousehostprocessestobiasacquisitionawayfromself-targeting.Alternatively,S.thermophilusmighthavemechanismsofself-non-self-discriminationthatweremaskedinthestrainoverexpressingCRISPRproteins.Phagechallengeexperimentswithwild-typeS.thermophilusrevealedthatsomesequenceswereacquiredasspacersdisproportion-atelyoftenacrossmultipleexperiments,suggestingthattheTypeIIacquisitionmachineryhaspreferencesinadditiontoCas9-dependentPAMselection,thoughnoclearpatternemergedwithrespecttothegenomiclocationorsequenceofprotospacersthatindicatedabasisforthepreferences(Paez-Espinoetal.,2013).

Additionally,itwasdemonstratedthatthefourproteinsoftheS.pyogenesCRISPRsystem(Cas1,Cas2,Csn2,andCas9)formacomplex,suggestingthatCas9directlyrecruitstheacquisitionproteinstopotentialtargets(Heleretal.,2015).WhiledrawingcomparisonsbetweentheinvolvementofCas9inacquisitionandprimedacquisitioninTypeIsystemsistempting,neithergroupsawevidencethatacquisitionwasaffectedbythepres-enceofexistingspacersmatchingorcloselymatchingtheinfect-ingphageorplasmid(Heleretal.,2015;Weietal.,2015b).Inaddition,whilethetrans-activatingcrRNA(tracrRNA)thatformsacomplexwithCas9andthecrRNAisnecessaryforacquisition,itisunclearwhetheracorrespondingcrRNAisalsorequired(He-leretal.,2015;Weietal.,2015b).FuturemechanisticworkwillberequiredtoshedlightonthesimilaritiesbetweenCas9-mediatedspaceracquisitionandtheprimedacquisitioninTypeIsystems.TypeIIProtospacerIntegration

ThesequencerequirementsforprotospacerintegrationinTypeII-AsystemswererecentlydemonstratedinS.thermophilus(Weietal.,2015a).SimilartoE.coli,theleaderandasinglerepeatweresuf?cienttodirectintegration.Furthermore,onlythetennucleotidesoftheleaderproximaltothe?rstrepeatarerequiredtolicensetheintegrationofnewspacers,incontrasttothe60ntminimalrequirementinE.coli(Weietal.,2015a;Yo-sefetal.,2012).Alimitedmutationalstudyoftherepeatshowedthatthe?rsttwonucleotidesarenecessaryforacquisition,whilethe?naltwonucleotidescanbemutatedwithoutconsequence(Weietal.,2015a).Thus,Cas1-Cas2-catalyzedintegrationattheleader-repeatjunctionissequencespeci?c,whiletheattackattherepeat-spacerjunctionisdeterminedbyarulermecha-nism,inagreementwithobservationsfromexperimentsinthe

E.colisystem(D?

′ez-Villasen?oretal.,2013).Together,these?nd-ingssupportthefunctionalconservationoftheCas1-Cas2inte-grasecomplexdespitedivergentmechanismsofprotospacerselectionbetweenTypesIandIICRISPR-Cassystems.CRISPRIntegrasesasGenome-ModifyingTools

AswithmanyotherCasproteins,theCas1-Cas2integrasecomplexshowspromiseforuseinmodifyinggenomes.WhileCas1-Cas2catalyzeareactionsimilartothatofmanyintegrases

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andtransposases,theyexhibitseveralfundamentaldifferencesthatmakethemuniquelysuitedtocertainapplications.Cas1-Cas2complexeslacksequencespeci?cityfortheDNAsubstratetobeintegrated,apropertythatcouldmakethesystemidealforbarcodinggenomes.Genomebarcodingallowsfortrackinglin-eagesoriginatingfromindividualcells,facilitatingstudiesofpop-ulationevolution,cancer,development,andinfection(BlundellandLevy,2014).Cas1-Cas2complexesintegrateshortDNAse-quences,incontrastwithcurrenttechniquesbasedonrecombi-nasesthatintegrateentireplasmids,resultinginpotential?tnesscostsandunwantednegativeselection(BlundellandLevy,2014).Interestingly,invitrointegrationofDNAsubstratesintoplasmidtargetsrevealedintegrationintonon-CRISPRsites(Nu-n

?ezetal.,2015a),suggestingthatCas1-Cas2canbeharnessedtointegrateintoawidearrayoftargetsequences.Agreaterun-derstandingoftheminimalfunctionalrecognitionmotifforvariousCas1-Cas2integraseswillfacilitatethedevelopmentofthistechnology.

crRNPBiogenesis:GeneratingMolecularSentinelsfortheCell

CRISPRimmunesystemsuseRNA-programmedproteinstopa-trolthecellinsearchofDNAmoleculesbearingsequencescom-plementarytothecrRNA.AssemblyofthesemolecularsentinelsbeginswithtranscriptionoftheCRISPRlocustogeneratelong,precursorCRISPRRNAs(pre-crRNAs),followedbyprocessingintoshortcrRNAguides(Brounsetal.,2008;Carteetal.,2008).ThepromoterisembeddedwithintheAT-richleadersequenceupstreamoftherepeat-spacerarray,orsometimeswithintherepeatsequences(Zhangetal.,2013).Here,webrie?yreviewtheprocessingofpre-crRNAscatalyzedbytheCas6en-doribonucleasefamilyinTypeIandIIIsystemsandadistinctprocessingpathwayinTypeIIsystemsthatinvolvesendoge-nousRNaseIII,Cas9,andatracrRNA.ThecrRNAbiogenesispathwayhasbeenextensivelyreviewedelsewhere(Charpentieretal.,2015;HochstrasserandDoudna,2015).ProcessingbyCas6Endoribonucleases

TypeIandTypeIIIsystemsemployCas6endoribonucleasestocleavepre-crRNAssequencespeci?callywithineachrepeat(Brounsetal.,2008;Carteetal.,2008;Haurwitzetal.,2010).AlthoughCas6homologsarevariableinsequence,theyshareaconservedcleavagemechanismthatresultsincrRNAguidescomprisinganentirespacersequence?ankedbyportionsoftherepeatsequenceonthe50and30ends.MaturecrRNAguidesconsistofan8nt50handlederivedfromtherepeatsequenceandvariablelengthsoftherepeatatthe30handle,whichisfurthertrimmedbyas-yet-unidenti?edcellularnuclease(s)inTypeIIIsystems(Haleetal.,2008).AnotableexceptionisinTypeI-Csystems,whichutilizeaCas5variantforcrRNApro-cessing,leavingan11nt50handleand21–26ntatthe30end(Garsideetal.,2012;Nametal.,2012b).InotherTypeIsystems,Cas5subunitsserveanon-catalyticrolecappingthe50endofthecrRNAinCascadecomplexes.

InTypeI-C,I-D,I-E,andI-Fsystems,therepeatsformstablehairpinstructuresthatallowforstructure-andsequence-spe-ci?ccleavagebyCas6atthebaseofthehairpin(Gesneretal.,2011;Haurwitzetal.,2010;Sashitaletal.,2011).Aftercleavage,thehairpinconstitutesthe30handleofthecrRNA.TheCas6pro-34Cell164,January14,2016a2016ElsevierInc.

teinsinHaloferaxvolcanii(Cas6b),E.coliandT.thermophilus(Cas6e),andPseudomonasaeruginosa(Cas6f)remainstablyboundtothe30handleandeventuallybecomepartoftheCascadecomplex(Brendeletal.,2014;Brounsetal.,2008;Ges-neretal.,2011;Haurwitzetal.,2010;Sashitaletal.,2011).

TypeI-A,I-B,III-A,andIII-Brepeatsequencesarenon-palin-dromicandpredictedtobeunstructuredinsolution(Kuninetal.,2007).Thus,therespectiveCas6isthoughttorelyonsequenceforspeci?cityratherthanstructure.Interestingly,acrystalstruc-tureoftheTypeI-ACas6boundtoitscognateRNAstructurere-vealsCas6inducinga3bphairpinintheRNAthatpositionsthescissilephosphateintheenzymeactivesite(ShaoandLi,2013).ItremainsunknownwhetherotherCas6sthatrecognizenon-palindromicrepeatshaveasimilarmechanismofRNAstabiliza-tion.FollowingorconcurrentwiththematurationofthecrRNAs,theCasproteinsinvolvedininterferenceassembleintothe?naleffectorcomplexthatfunctionstorecognizeanddestroytargetsbearingsequencecomplementaritytothecrRNA.InsystemswhereCas6remainsboundtothecrRNA,itmayservetonucleatetheassemblyofthesubunitsthatconstitutetheeffectorcomplexbackbonealongthecrRNA.IntypeIIIsystems,thenumberofbackbonesubunitsde?ningthecomplexlengthisvar-iable,andanyunprotectedcrRNAremainingisdegraded(Haleetal.,2008;Staalsetal.,2014).ProcessinginTypeIISystems

TypeIIsystemsrelyonadifferentmechanismtoprocesspre-crRNAs.InTypesII-AandII-B,pre-crRNAcleavagespeci?cityisaidedbyatracrRNAthathassequencecomplementaritytotheCRISPRrepeatsequence(Deltchevaetal.,2011).ThegeneencodingthetracrRNAistypicallylocatedeitherproximaltoorwithintheCRISPR-caslocus(Chylinskietal.,2014).UponcrRNA:tracrRNAbasepairing,whichisstabilizedbyCas9,endogenousRNaseIIIcleavesthepre-crRNAattherepeat.TherelianceonRNaseIII,whichisnotfoundinarchaea,mayexplainwhyTypeIIsystemsarelimitedtobacteria(Garrettetal.,2015).Anunknownnucleasetrimsthe50endofthecrRNAtoremovethe?ankingrepeatsequenceandportionsofthespacer.InS.pyogenes,the30ntspacersequenceistrimmedtothe20ntthatbase-pairswithcomplementaryforeignsequencesduringinterference(Deltchevaetal.,2011;Jineketal.,2012).

IntheNeisseriameningitidisandC.jejuniTypeII-Csystems,eachrepeatsequenceencodesapromoter,resultinginvaryinglengthsofpre-crRNAsdependingonthetranscriptionstartsite(Dugaretal.,2013;Zhangetal.,2013).AlthoughRNaseIII-medi-atedpre-crRNAprocessingcanstilloccur,RNaseIIIisdispens-ableforinterferenceinthesesystems(Zhangetal.,2013).Thus,Cas9isabletocomplexwiththepre-crRNAandunprocessedtracrRNAforfunctionaltargetinterferencewithoutfurtherpro-cessingofthepre-crRNAs.

Cas6asaBiotechnologyTool

TheCas6homologfromTypeI-Fsystems,Cas6f(alsoknownasCsy4),wasthe?rstCasproteintoberepurposedasatool.Followingdemonstrationofthesequencespeci?cityofCas6fbindingandcleavage,theproteinhasbeenusedforthepuri?ca-tionoftaggedRNAtranscriptsfromcells(Haurwitzetal.,2010;Leeetal.,2013;Salvail-Lacosteetal.,2013;Sternbergetal.,2012).SubsequentstudiesshowedthatCas6fcouldbeusedtoalterthetranslationandstabilityoftaggedmRNAs,allowing

Figure3.InterferencebyClass1Systems

(A)InterferenceinTypeIsystemsiscarriedoutbyCascadeandCas3.CascadeisalargecomplexcomposedofthecrRNA,boundateitherendbyCas5andCas6,multipleCas7subunitsalongthecrRNA,alargesubunit(Cse1,Csy1,Cas8,orCas10),andsometimessmallsubunits(Cse2andCsa5).TheTypeI-Ecomplexisschematizedhere.ThelargesubunitrecognizesthePAMinforeignDNAandinitiatesunwindingofthetargetDNAandannealingtothecrRNA.Cas3isrecruitedtotheresultingR-loopandmakesanick.Itthentrans-locatesalongthedisplacedstrandandproc-essivelydegradesit.

(B)TypeIIIsystemscontaineitherCsmorCmrcomplexes,whichshareasimilararchitecture.TheCsmcomplexfromTypeIII-Asystemsisshownhere.ThecrRNAisboundateitherendbyCsm5/Cmr1andCsm4/Cmr3,whichhavehomologytoCas6andCas5,respectively.Csm3/Cmr4formthebackboneofthecomplex,Cas10servesasthelargesubunit,andCsm2/Cmr5arethesmallsub-unit.ThesecomplexescantargetbothRNAandactivelytranscribedDNA.Cas10catalyzescleav-ageoftargetDNA,whilethebackbonesubunitcatalyzescleavageofthetargetRNAateverysixthbase,whichisunpairedwiththecrRNA.RatherthanrecognizingaPAMsequence,thesecom-plexesonlycleaveifthe50and30handlesofthecrRNAdonotannealtothetarget.

forpost-transcriptionalregulationofproteinexpression(Borch-ardtetal.,2015;Duetal.,2015;Nissimetal.,2014).Cas6fhasalsobeenusedalongsideCas9toprocessmultipleguideRNAsfromasingletranscript,greatlyfacilitatingmultiplexeded-iting(Tsaietal.,2014).

Interference:Precise,ProgrammableDNABindingandCleavage

ImplementationofCRISPRsystemstoprovideimmunityin-volvesRNA-guidedrecognitionandprecisioncuttingofDNAmolecules,apropertythatmakesthemusefulforgenomeengi-neeringandcontrolofgeneexpression.TheextremediversityofthecrRNPtargetingcomplexesislargelyresponsibleforthevari-abilityobservedindifferentCRISPRtypes.WhereasTypesIandIIIusemulti-proteincomplexes,TypesIIandVrelyonasingleproteinforinterference.Extensivestudieshaveelucidatedthemechanismsandstructuresofseveralcomplexesfromeachofthethreemajortypes,revealingthecommonalityoftargetbind-ingthroughcrRNAbase-pairingandhighdivergenceinthema-chineriesandmodesoftargetcleavage.Formorein-depthrecentreviewsfocusedexclusivelyonCRISPRinterference,refertoTsuiandLi(2015)andPlagensetal.(2015).TypeIInterference

InTypeIsystems,therolesoftargetDNArecognitionanddegra-dationaresegregatedintotwodistinctcomponents.ThecrRNA-guidedCascadecomplexbindsandunwindstheDNAtargetsequence(Brounsetal.,2008)andthenrecruitsCas3todegradethetargetinaprocessivemannerthroughthecombinedactionofitsHDnucleaseandhelicasedomains(Figure3A)(Makarovaetal.,2011b;MulepatiandBailey,2013;Sinkunasetal.,2013;Westraetal.,2012).EachTypeIsubtype(I-AthroughI-F)hasadistinctcomplementofCascadecomponentsand,insomecases,signi?cantvariationofthecas3gene(Makarovaetal.,2011b).

TheE.coliCascadecomplexhasservedasthemodelsystemforunderstandingthemechanismofTypeIinterference.Inaddi-tiontothecentral61ntcrRNAbearingthe32ntspacer,thecom-plexcomprises?veproteinsindifferentstoichiometries:(Cse1)1,(Cse2)2,(Cas5)1,(Cas7)6,and(Cas6)1.TheCas7subunitsformthe‘‘backbone’’thatpolymerizesalongthecrRNAanddeter-minesthecrescent-shaped,semi-helicalarchitectureseeninallstructurallycharacterizedCascadecomplexes(Hochstrasseretal.,2014;Jacksonetal.,2014;Joreetal.,2011;Mulepatietal.,2014;Wiedenheftetal.,2011a;Zhaoetal.,2014).Cas6(Cas6einTypeI-Esystems)remainsboundtothe30hairpinfollowingCRISPRmaturation,whileCas5bindsthe50handle(Brounsetal.,2008;Joreetal.,2011).A‘‘smallsubunit’’(Cse2inTypeI-E)isoftenfoundintwocopiesformingthe‘‘belly’’ofthestruc-tureandhelpsstabilizethecrRNAandtargetDNA(Jacksonetal.,2014;Mulepatietal.,2014;Zhaoetal.,2014).A‘‘largesub-unit’’(Cse1inTypeI-E,Cas8inmostothersubtypes)bindsatthe50endofthecrRNAandrecognizesthePAMsequencesandre-cruitsCas3toanauthenticatedtarget(Figure3A)(Hochstrasseretal.,2014;Sashitaletal.,2012).WhileCas6doesnotalwaysremainwiththecomplexandthesmallsubunitisoftenfoundasafusionwiththelargesubunit,theoverallarchitectureofCascadecomplexesisgenerallyconserved(Makarovaetal.,2011b;Plagensetal.,2012;Sokolowskietal.,2014).

Cascadepre-arrangesthespacersegmentofthecrRNAinsix?ve-basesegmentsofpseudoA-formconformation,withthesixthbase?ippedoutandboundbyaCas7subunit(Jacksonetal.,2014;Mulepatietal.,2014;Zhaoetal.,2014).Toinitiate

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Figure4.InterferencebyClass2Systems

(A)InTypeIIsystems,Cas9formstheeffectorcomplexwithacrRNAandatracrRNA.Cas9iscomposedofthenucleaselobeandthea-helicallobe.ThenucleaselobecontainsboththeHNHandRuvC-likenucleasedomainsaswellasthePAM-interactingdomain.The30hairpinsofthetracrRNAbindthenucleaselobe,whilethestemloopandspacerlinethechannelbetweenthetwolobes.Bindingtoamatching,PAM-adjacenttargetcausestheHNHdomaintomoveintopositiontocleavetheannealedstrand,whilethedis-placedstrandisfedintotheRuvCactivesiteforcleavage.

(B)Cpf1istheeffectorproteininTypeV-Asystems,thebestcharacterizedTypeVsubtype.ItbindsthecrRNAalone.ThestructureofCpf1isunknown,butitcontainsanactiveRuvC-likenucleasedomainfortargetcleavage.Cpf1recognizesaPAMandmakestwostaggeredcutsinamatchingsequence.IthasbeenproposedthatCpf1actsasadimer,witheachmonomerprovidingaRuvCactivesite,thoughtheremaybeanotherunidenti?ednucleasedomain.

interference,Cascade?rstrecognizestrinucleotidePAMsitesinthetargetstrandofforeignDNAthroughspeci?cinteractionswithCse1(Sashitaletal.,2012).UponPAMbinding,theDNAtargetisunwoundstartingatthePAM-proximalendoftheproto-spacertoformanRloopstructure(Hochstrasseretal.,2014;Rollinsetal.,2015;Rutkauskasetal.,2015;Sashitaletal.,2012;Szczelkunetal.,2014;vanErpetal.,2015).Eachstretchof?veexposedbasesinthecrRNAisfreetobindthetargetDNA,leadingtoastablebuthighlydistortedanddiscontinuouscrRNA:targetstrandduplex(Mulepatietal.,2014;Szczelkunetal.,2014).CascadeundergoesaconformationalchangeupontargetbindingthatenablesrecruitmentofCas3tothe

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Cse1subunit(Hochstrasseretal.,2014;Mulepatietal.,2014).Cas3bindsandnicksthedisplacedstrandusingitscatalyticcenteroftheHDnucleasedomain(Gongetal.,2014;Huoetal.,2014;MulepatiandBailey,2013;Sinkunasetal.,2013;Westraetal.,2012).TheATP-dependenthelicaseactivityofCas3isthenactivated,causingmetal-andATP-dependent30/50translocationandprocessivedegradationofthenon-targetstrand(Gongetal.,2014;Huoetal.,2014;Westraetal.,2012).Cas3initiallydegradesonly200–300ntofthenontargetstrand,thoughitcontinuestranslocatingformanykilobases(Reddingetal.,2015).ExposedssDNAonthetargetstrandmaythenbecomeasubstrateforotherssDNAnucleasesoranadditionalCas3moleculetocompletethedegradationofforeignDNA(MulepatiandBailey,2013;Reddingetal.,2015;Sinkunasetal.,2013).InadditiontothePAM,targetinterferencealsoreliesonaseedregionatthe30endofthespacersegmentofthecrRNA(Semenovaetal.,2011;Wiedenheftetal.,2011b).SinglepointmutationsoftheseedregionoftheE.coliCascadecom-plex,atthe1to5and7to8positionofthespacer,isenoughtodecreasetargetDNAbindingandsubsequentinterference(Semenovaetal.,2011).

Differencesinthecas3geneamongTypeIsubtypessuggestsomevariabilityininterferencemechanism.InsomeTypeI-Especies,Cas3isfusedtoCse1byalinkerthatallowsittostablyassociatewiththeCascadecomplex(Westraetal.,2012).InTypeI-Asystems,theCas3helicaseandnucleasedomainsexistasseparatepolypeptidesthatbothassociatewiththeCascadecomplex(Plagensetal.,2014).InTypeI-Fsystems,Cas3isfusedtoCas2,lendingfurthergeneticsupportfortheinteractionbetweentheinterferenceandacquisitionmachineryduringprimedacquisition(Makarovaetal.,2015;RichterandFineran,2013;Richteretal.,2012).Howthesefusionsanddomainsepa-rationsaffecttheprocessivedegradationobservedinTypeI-Esystemsrequiresfurtherstudy.TypeIIInterference

Incontrasttothemulti-subuniteffectorcomplexesseeninTypeIandTypeIIIsystems(butsimilartoCpf1ofTypeVsystems),theTypeIIsignatureproteinCas9functionsasanindividualprotein,alongwithacrRNAandtracrRNA,tointerrogateDNAtargetsanddestroymatchingsequencesbycleavingbothstrandsofthetarget(Figure4A)(Gasiunasetal.,2012;Jineketal.,2012).ExtensivestudiesonCas9haveyieldedarangeofstructuresofS.pyogenesCas9indifferentsubstrate-boundstates,aswellasstructuresofseveralorthologs(Andersetal.,2014;Jiangetal.,2015;Jineketal.,2014;Nishimasuetal.,2015;Nishimasuetal.,2014).Manyofthesestructures,aswellasthemechanismofCas9targetsearchandrecognition,arereviewedelsewhere(vanderOostetal.,2014);here,wefocusonthemostrecentadvances.

StructuresofCas9haverevealedtwodistinctlobes,thenucleaselobeandthea-helicalorREClobe(Andersetal.,2014;Jineketal.,2014;Nishimasuetal.,2015;Nishimasuetal.,2014).ThenucleaselobeiscomposedoftheHNHnucleasedomain,whichcleavesthetargetstrand,aRuvC-likenucleasedomain,whichcleavesthenon-targetstrandandisseparatedintothreedistinctregionsintheprimarysequencebytheinterveninga-helicallobeandtheHNHdomain,andaC-terminalPAM-interactingdomain(Andersetal.,2014;Jinek

etal.,2014;Nishimasuetal.,2015;Nishimasuetal.,2014).Thea-helicallobecontainsanarginine-rich‘‘bridgehelix,’’whichconnectsthetwolobesandinteractswiththeguideRNA,andisthemostvariableregionofCas9,withinsertionsordeletionsaccountingformuchofthewidevariationinsizeseeninCas9orthologs(Chylinskietal.,2014;Jineketal.,2014;Nishimasuetal.,2014).

Cas9initiatesitstargetsearchbyprobingduplexedDNAforanappropriatePAMbeforeinitiatingtargetunwinding(Sternbergetal.,2014).Thetargetunwindsfromtheseedregion,the?rst10–12nucleotidesfollowingthePAM,towardthePAM-distalend(Szczelkunetal.,2014).Aperfectornear-perfectmatchleadstocleavageofbothDNAstrands,withmismatchesbeingmoretoleratedoutsideoftheseedregion(Congetal.,2013;Jiangetal.,2013;Jineketal.,2012;Sternbergetal.,2014).Themechanismbywhichmismatchesdistantfromthecleavagesitepreventcleavageappearstorelyonthestructural?exibilityoftheHNHdomain,whichhasyettobecrystallizedinproximitytothescissilephosphate(Andersetal.,2014;Nishimasuetal.,2015;Nishimasuetal.,2014).FRETassaysshowthattheHNHdomainswingsintoacatalyticallycompetentpositiononlyuponbindingtoacognatedouble-strandedDNAsubstrate,underscoringthemultiplestepsofconformationalcontrolofCas9-catalyzedDNAcleavage(Sternbergetal.,2015).TheRuvCdomainisinturnallostericallyregulatedbytheHNHdomain.Cleavageofthenon-targetstrandrequiresmovementoftheHNHdomainintoanactiveposition,evenwhenthemis-matchedsubstratesallowfullunwindingofthenon-targetstrand(Sternbergetal.,2015).

RecentcrystalstructuresofS.pyogenesCas9-sgRNAsurveil-lancecomplexandofthesmallerStaphylococcusaureusCas9inatarget-boundstateprovidednewinsightsintoCas9function(Jiangetal.,2015;Nishimasuetal.,2015).ThesgRNA-boundstructurerevealedhowbindingofsgRNAshiftsCas9fromtheauto-inhibitedstateobservedintheapoformtoaconformationcompetentfortargetsearch(Jiangetal.,2015;Jineketal.,2014).Aspreviouslyobservedinlow-resolutionelectronmicro-scopystructures,anucleicacidbindingcleftisformedbetweenthetwolobesuponsgRNAbinding(Jineketal.,2014).Further-more,twoPAM-interactingarginineresiduesarepre-positionedtoallowforscanningofpotentialtargetDNA,a?ndingthatmayexplainthenecessityoftracrRNAindirectingPAM-dependentspaceracquisition.Surprisingly,whilethe30hairpinsofthetracrRNAhavebeenshowntoprovidenearlyallofthebindingenergyandspeci?cityforCas9,therepeat-anti-repeatregionofthesgRNAaswellastheseedsequencewererequiredtoinducetheconformationalrearrangement(Brineretal.,2014;Jiangetal.,2015;Wrightetal.,2015).TheseedsequenceofthesgRNAwasalsofoundtobepre-orderedinanA-formhelix,analogoustothepre-orderedseedregionofguideRNAobservedineukaryoticArgonautestructuresandtheTypeIandTypeIIIeffectorcomplexes,wheretheentirecrRNAispre-arrangedinatarget-binding-competentstate(Jacksonetal.,2014;KuhnandJoshua-Tor,2013;Mulepatietal.,2014;Osawaetal.,2015;Tayloretal.,2015;Zhaoetal.,2014).Theobservedpre-orderingoftheguideRNAprovidesanenergeticcompensationfortheunwindingofthetargetduplextofacilitatebinding.Cas9fromtheTypeII-CCRISPRsystemofS.aureuswascrystallizedincomplexwithsgRNAandasingle-strandedDNAtargetsequence,providinginsightintothestructuralvariationbetweenmoredistantlyrelatedCas9(Nishimasuetal.,2015).S.aureusCas9issigni?cantlysmallerthantheCas9ofS.pyogenes(1,053versus1,368aminoacids)andrecognizesasigni?cantlydifferentguideRNAandPAMsite.TheS.aureusCas9structurerevealedasmallera-helicallobe,withdomainsinthemiddleandPAM-proximalsidenotablyabsent,whilethenucleaselobeislargelyconserved(Nishimasuetal.,2015).Theauthorsproposedanewdomaindesignation,thewedgedomain,whichdivergessigni?cantlybetweenthetwoproteinsandappearsintegraltodeterminingguideRNAorthogonality.AnothersmallCas9,thatfromActinomycesnaeslundi,waspre-viouslycrystallizedintheapoform,buttheabsenceofboundsubstrateandsigni?cantdisorderedregionslimiteddetailedexplorationofthedifferencesbetweentheorthologs(Jineketal.,2014).OtherrecentworkwithTypeII-CCas9proteinsfromN.meningitidisandCorynebacteriumdiphtheriae,amongotherTypeII-Corthologs,revealedthattheseenzymeshaveareducedabilitytounwinddsDNAcomparedtoS.pyogenesCas9andexhibitef?cientPAM-independentandinsomecasestracrRNA-independentcleavageofssDNA(Maetal.,2015;Zhangetal.,2015).Thisactivitymayallowformoreef?cientinterferencewithssDNAplasmidorphageorrepresentamoreancestralactivitythatpredatestheexpansionofthea-helicallobetofacilitatemorerobustDNAunwinding.TypeIIIInterference

TypeIIIsystemsareclassi?edintoTypeIII-AandTypeIII-Bbasedontheireffectorcomplexes(TypeIII-CandIII-Dhavealsobeenidenti?ed,butnotyetcharacterized)(Makarovaetal.,2015).TheformerisconstitutedbytheCsmcomplex,andthelatterbytheCmrcomplex(Makarovaetal.,2011b).PhylogeneticstudiessuggestedthatsomecsmandcmrgenesaredistanthomologsofcasgenesthatcomposetheCascadecomplexofTypeIsystems,andsubsequentstructuralstudieshaverevealedastrikingstructuralconservationbetweenCascadeandtheCsmandCmrcomplexes(Hochstrasseretal.,2014;Jacksonetal.,2014;Makarovaetal.,2013;Mulepatietal.,2014;Osawaetal.,2015;Staalsetal.,2014;Tayloretal.,2015;Zhaoetal.,2014).Foradetaileddiscussionofthestruc-turalsimilaritiesbetweenthesecomplexes,refertoJacksonandWiedenheft(2015).Brie?y,Csm3(inIII-Asystems)orCmr4(inIII-B)polymerizesalongthecrRNAasahelicalbackbone,analogouslytoCas7,whileCsm2orCmr5taketheroleofCse2asthesmallsubunit(Figure3B)(JacksonandWiedenheft,2015).SimilartoCascade,thecrRNAispre-arrangedforbindingwithkinkseverysixnucleotides.Thetargetnucleicacid(RNAinallsolvedTypeIIIstructures)bindsinadistortedmanner,form-ing?ve-nucleotidehelicalstretcheswiththesixthbase?ippedouttoallowfortheextremedeviationfromhelicalnucleicacidobservedinallstructures(Osawaetal.,2015;Tayloretal.,2015).Cmr3andCsm4bindthe50crRNAhandle,whileCas10(alsoreferredtoasCsm1andCmr2)servesasthelargesubunit(Makarovaetal.,2011a;Osawaetal.,2015;Staalsetal.,2014;Tayloretal.,2015).Csm5,Cmr6,andCmr1alsosharehomologywithCas7andcapthehelicalbackboneatthe30endofthecrRNA.InTypeIII-Bsystems,twomajorcrRNAspeciesare

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generallyobserved,differingbysixnucleotides(Juraneketal.,2012;Staalsetal.,2014).Cryo-electronmicroscopycapturedtwoCmrcomplexesofdifferentsizes,withonecomplexhavingonefewerCmr4andCmr5subunit,suggestingthatthedifferentcrRNAlengthsaretheresultofdifferentcomplexsizes,orviceversa(Tayloretal.,2015).

Despitethestructuralsimilarities,theTypeIIIinterferencecomplexesfunctionquitedistinctlyfromCascade.Thesubstratespeci?cityofCsmandCmrcomplexeshasonlyrecentlybeenclari?ed.EarlyinvivogeneticexperimentssuggestedCsmtar-getedDNA,whileinvitrostudiesofCmrshowedbindingandcleavageactivityagainstRNAonly(Haleetal.,2009;Marraf?niandSontheimer,2008),leadingtoamodelwhereinthetwosubtypeshadevolveddistinctandcomplementarysubstratepreferences.ThissimplemodelwassooncomplicatedbytheobservationthatCsmcomplexesinvitroalsobindandcleaveRNAwhileexhibitingnoactivityagainstDNA(Staalsetal.,2014;Tamulaitisetal.,2014).Meanwhile,theinvivoDNA-targetingactivityofIII-Asystemswasshowntodependontranscriptionatthetargetsite,incontrasttothetranscription-in-dependenttargetingseeninTypeIandTypeIIsystems,andasimilaractivitywasobservedforaIII-Bsysteminvivo(Dengetal.,2013;Goldbergetal.,2014).TheseobservationswerereconciledbythediscoverythattheCsmcomplexfromStaphy-lococcusepidermidisexhibitsbothRNAcleavageandDNAcleavagewhendirectedagainstthenon-templatestrandofactivelytranscribedDNA(Samaietal.,2015).

DNAandRNAinterferencearecarriedoutbydistinctsubunitsoftheTypeIIIcomplexes.RNAinterferenceismediatedbythebackbonesubunitCsm3(orCmr4inIII-Bsystems),whichcleavesthetargeteverysixnucleotidesintheactivesiteofaseparatesubunitbyactivatingtheribose20OHfornucleophilicattackinamannertypicalofmetal-independentRNases(Osawaetal.,2015;Staalsetal.,2014;Tamulaitisetal.,2014;Tayloretal.,2015).Cas10cleavesDNAexposedbyatranscriptionbubbleusingasinglecatalyticsiteinitspalmpolymerasedomain(Samaietal.,2015).ThedetailsofDNAtargetingbyCmrhavenotbeenindependentlycon?rmed,buttheconserva-tionofCas10andevidencefortranscription-dependentplasmidclearingsupportsasimilarmechanism(Dengetal.,2013;Makar-ovaetal.,2011b).

ThedistinctbehaviorofTypeIIIsystemsprovidesthehostmicrobewiththeabilitytotoleratetemperatephages(Goldbergetal.,2014).WhileTypeIandTypeIIsystemstargetanddegradeanyprotospacer-containingDNA,TypeIIIsystemsignoreforeignDNAuntiltranscriptionbeginsthatposesathreattothecell.Thishastheadvantageofallowingcellstoacquiread-vantageousgenescontainedinprophages,suchasantibioticresistancegenes,andcausingcellsuicideintheeventthatalysogenicphagebecomeslyticandbeginstranscribinggeneswithmatchingspacers(Goldbergetal.,2014).However,thestrand-speci?cnatureofboththeRNAtargetingandtranscrip-tion-dependentDNAtargetingimposesanadditionalrestrictionontheintegrationstepofacquisition,asonlyonedirectionofintegrationwillyieldproductiveinterference.Themeansbywhichthisapparentlimitationisovercomeareunclear.TypeIIIsystemsarealsofrequentlyfoundcoexistingwithTypeIsys-tems,inwhichcasetheirdistincttargetspeci?citymightallow

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forinterferencewithtargetsthatsomehowavoidrecognitionbyCascade(Makarovaetal.,2011b).

TypeIIIsystemsarealsouniqueintheirlackofaPAM.Ratherthanrecognizingadistinctmotiftoavoidauto-immunityattheCRISPRlocus,theCsmandCmrcomplexesinsteadcheckforcomplementaritybetweentherepeat-derivedregionofthecrRNAwiththetargetanddonotcleaveifafullmatchisdetected(Marraf?niandSontheimer,2010;Samaietal.,2015;Staalsetal.,2014;Tamulaitisetal.,2014).Thespeci?cityofTypeIIIeffectorcomplexesforsingle-strandedtargetsmightprovidearationalefortheirdistinctmodeoftargetauthentication.ForTypeIandTypeIIeffectorcomplexes,whichtargetdsDNA,PAMrecognitionallowsforaninitialbindingeventtofacilitatesubsequentunwindingofthetargettoprobeforcomplemen-taritytothecrRNA(Hochstrasseretal.,2014;Rollinsetal.,2015;Sternbergetal.,2014;Szczelkunetal.,2014;Westraetal.,2012).TypeIIIcomplexescanimmediatelyprobeapoten-tialsingle-strandedtargetforcomplementaritytotheirboundcrRNAwithoutaneedtolicenseinitialunwinding,andtheexposednatureofasingle-strandedtargetfacilitatesthecheckforcomplementaritytotherepeat-derivedregionoftheguide.TypeVInterference

TypeVsystemshaveonlyrecentlybeenclassi?ed,butinitialworkdemonstratedthatthesesystemsarefunctionalforinter-ference(Makarovaetal.,2015;Zetscheetal.,2015a).Thesys-temsappearmostsimilartoTypeIIsystems,possessingonlytheacquisitionmachineryandasingleadditionalprotein(Makar-ovaetal.,2015;Schunderetal.,2013;Vestergaardetal.,2014).ThreesubtypesofClassVsystemshavebeenidenti?edwithwidelyvaryinginterferenceproteins(Shmakovetal.,2015).TypeV-A,V-B,andV-CarecharacterizedbythepresenceofCpf1,C2c1,andC2c3,respectively(Shmakovetal.,2015).Allthreeproteinsareevolvedfromthesamefamilyoftransposon-associatedTpnBproteinsasCas9andhaveaC-terminalRuvCdomainandarginine-richbridgehelix(Shmakovetal.,2015).However,theproteinsshowlittlesimilaritytoeachother,andthephylogeneticgroupingoftheassociatedcas1geneswithvariousbranchesofTypeIandTypeIIIcas1genessuggeststhateachofthesesubtypesoriginatedfromdistinctrecombinationeventsbetweenCRISPRsystemsandtpnBgenes(Shmakovetal.,2015).

WhilesomeTypeV-Bsystemshaveanidenti?abletracrRNAnecessaryforactivity,TypeV-AandV-CsystemslackbothatracrRNAandCas6orCas5-likeendonuclease,makingitun-clearhowthecrRNAisprocessed(Makarovaetal.,2015;Shma-kovetal.,2015).ThecrRNAofTypeV-Asystemshasaconservedstem-loopandcanbeprocessedtoafunctionalformwhentranscribedinE.coliinthepresenceofCpf1(Zetscheetal.,2015a).WhetherCpf1isalsorequiredforprocessingandthepotentialinvolvementhostfactorsremainsunknown.TheCpf1fromFrancisellanovicidacansuccessfullyinterferewithtransformedplasmidsandrecognizesa50-TTN-30PAMatthe50endoftheprotospacersequence,similartothePAMlocationofTypeIsystemsandincontrasttothe30PAMobservedinTypeIIsystems.Theenzymemakesadouble-strandbreak,re-sultingin?ve-nucleotide50overhangsdistaltothePAMsite(Figure4B).MutationofcatalyticresiduesintheRuvCactivesitepreventscleavageofeitherstrand(Zetscheetal.,2015a).

TheauthorsproposethatCpf1mightactasadimer,witheachmonomerprovidingaRuvCactivesitebutonlyonerecognizingthetarget.Ifthisisthecase,whetheroneorbothmonomershasaboundcrRNAisunclear.Alternatively,anas-of-yetundiscov-eredactivesitemightbepresentintheprotein,inwhichcaseitsactivitymustbetightlycoupledtothatofRuvCtoexplainthephenotypeobservedfortheRuvCmutant.AC2c1,whichalsohasonlyoneidenti?ablenucleasedomain,hasalsobeenshowntobeactiveforcleavageinvivoandinvitro,whereitrec-ognizesa50-TTN-30PAMandrequiresatracrRNA(Shmakovetal.,2015).Manymechanismsinthesenewlydiscoveredsys-tems,bothTypeVandtheessentiallyuncharacterizedTypeVI,remainunknownandopenforfuturestudy.

InterferenceComplexesasGenomeEditingTools

MosttooldevelopmentofCasproteinshasfocusedonexploit-ingtheprogrammablesequence-speci?cDNArecognitionofinterferencecomplexes.Cas9fromS.pyogenesinparticularhasprovenenormouslyusefulforgenomeengineering.TheabilitytorenderCas9atwo-componentsystembyfusingthecrRNAandtracrRNAintoasingleguideRNA(sgRNA)hasal-lowedforitseasyuseforgenomeediting,transcriptionalcon-trol,RNAtargeting,andimaging(forrecentreviews,seeJiangandMarraf?ni,2015;SternbergandDoudna,2015).Cas9hasbeenusedinvariouscelltypesandorganismsrangingfrommiceandmonkeystoprimaryhumanTcellsandstemcells,aswellasplants,bacteria,andfungi(JiangandMarraf?ni,2015;SternbergandDoudna,2015).Recentworkhasfocusedondevelopingvariouschemical-andlight-inducibleCas9con-structstoallowforgreaterspatiotemporalcontrolandonem-ployingCas9orthologswithdifferentPAMsequencesandsmallersizes,allowingforeasierpackaginginadeno-associ-atedvirusvectors(Davisetal.,2015;Nihongakietal.,2015;PolsteinandGersbach,2015;Ranetal.,2015;Zetscheetal.,2015b).

Otherinterferencecomplexeshavealreadybeenusedorhavethepotentialtobeusefulforgenomemanipulationaswell.Althoughthemulti-subunitcompositionofCascademakesitlesstractableforgenomeengineeringcomparedtoCas9,itslargesizeandstablebindinghasbeenusedfortran-scriptionalsilencinginE.coli(Rathetal.,2015).NopublishedworkhasshowntheapplicationofCsmorCmrcomplexes,buteithercouldlikelybeusedforvariousRNAmodulationap-plicationsincells.TwoCpf1homologs,outof16thatweretested,havebeenshowntofacilitategenomeeditinginhumancells(Zetscheetal.,2015a).ThealternatePAMspeci?cityofCpf1mayproveusefulfortargetingsiteswithoutanappro-priatePAMforCas9,andthestaggeredcutsmightprovetofavordistinctpathwaysofDNArepair.However,athoroughinvestigationoftheef?ciencyandoff-targeteditingofCpf1willbeneededtodetermineifthisproteinwillseesigni?cantusealongsideCas9.

WhileCas9hasalreadyseenextensiveuseintheresearchsetting,challengesremainforitsapplicationintheclinic.Whilemakingprogrammedcutshasbecomelargelytrivial,biasingDNArepairtowardhomology-directedrepairratherthannon-ho-mologousendjoiningremainsachallenge(Chuetal.,2015;Mar-uyamaetal.,2015).DeliveryofCas9,eitherasanRNPoronaplasmidorviralvector,toparticulartissuesinwholeorganisms

isanotherchallengethatmustbeaddressedtoenableclinicalapplications(D’Astolfoetal.,2015;Gorietal.,2015;HowesandScho?eld,2015;Linetal.,2014;Zurisetal.,2015).Asthe?eldcontinuestoadvancerapidly,clinicaltrialsmayoccurwithinafewyears,withtherapiespossiblyfollowingwithinadecade.EngineeringofcropplantswithCas9isalreadyunderway;regu-latoryrulingshavesofarconsideredknockoutplantsnottobegeneticallymodi?edorganisms,buttheregulatoryfateofothermodi?cationsiscurrentlybeingconsidered(Servick,2015).ConcludingRemarks

Despitetherapidprogressofthe?eldsincethe?rstdemonstra-tionofCRISPRimmunityin2007,manymechanisticquestionsremainunanswered.Fundamentalaspectsofacquisition,suchashowsubstratesforCas1-Cas2-mediatedintegrationaregener-atedandthemechanismandextentofself-versusnon-self-discriminationindifferentCRISPRsubtypes,arestillamystery.WhilecrRNAbiogenesisandinterferencearereasonablywellun-derstoodforcertainmodelsubtypes(TypeI-E,TypeII-A),thesheerdiversityofCRISPRsystemsmeansthatmanysubtypeswithpotentiallydistinctmechanismsremainunexplored.TypeVandVIsystemshaveonlybeguntobeanalyzed,andTypeIVsys-tems,bearingsomefamiliarcasgenesbutnoidenti?ableCRISPRlocus,haveyettobecharacterizedexperimentallyandalmostcertainlyrelyonmechanismsdistinctfromthoseoftraditionalCRISPRsystems(MakarovaandKoonin,2015).

OtheraspectsofCRISPR-CassystemsliebeyondthescopeofthisReview.Wehavenotdiscussedthenon-immunefunc-tionsofCRISPR-Cassystems,someofwhichappeartohaveevolvedtoserveregulatoryratherthandefenseroles(forre-views,seeWestraetal.,2014,andRatneretal.,2015).PhageevasionofCRISPRimmunityisanotheractiveareaofresearch,withidenti?edmechanismsincludingDNAmodi?cation,special-izedanti-CRISPRproteins,andmutationalescape(Bondy-Den-omyetal.,2013;Bondy-Denomyetal.,2015;Brysonetal.,2015;Deveauetal.,2008;Paez-Espinoetal.,2015;Pawluketal.,2014).Thecontext-dependentregulationofCRISPR-Cassys-temsinresponsetophageinfectionandstresssignalshasalsobeenexploredbutrequiresfurtherstudy(Bondy-DenomyandDavidson,2014;Garrettetal.,2015;Kenchappaetal.,2013;Pattersonetal.,2015;Puletal.,2010).Therapiddevelop-mentoftechnologyderivedfromCRISPR-Cassystems,mostnotablyCas9butalsoCas6f/Csy4,Cascade,andCpf1,hasfu-eledintenseinterestinthe?eld.Thearmsracebetweenbacteriaandbacteriophagehasgeneratedpowerfulmolecularbiologytools,fromrestrictionenzymesthatenabledrecombinantDNAtechnologytoCas9,whichstartedthe‘‘CRISPRrevolution’’inmoderngenomeengineering.CRISPRsystemshavenproventobebothfascinatingandenormouslyuseful.Furtherstudyofbacterialimmunesystems,bothCRISPRsystemsandthoseyetundiscovered,promisestoyieldfurtherunforeseendiscov-eriesandexcitingnewtechnologies.

ACKNOWLEDGMENTS

ThisworkwasfundedbyUSNationalScienceFoundationgrantnumber1244557toJ.A.D.A.V.W.andJ.K.N.areNSFGraduateResearchFellows.MeganHochstrasserprovidedvaluableinputonthemanuscript.

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