CRISPR-Cas Systems in Bacteria and Archaea - Carnegie Institution

27 Pages·2012·0.67 MB·English

GE45CH13-Bhaya ARI 1October2011 14:42
CRISPR-Cas Systems
in Bacteria and Archaea:
Versatile Small RNAs
y. for Adaptive Defense
nl
o
e
s
orgnal u and Regulation
ews.erso
w.annualrevi09/12. For p DanedvaRkoidBohlpayhae,1BMarircahnegloleu3Davison,1,2
w8/
m won 0 e1Cmaariln:[email protected],DepartmentofPlantBiology,Stanford,California94305;
oy
ded frLibrar e2Dmeapila:rsthmelelnbtluo@fsBtaionlfoogryd,.eSdtuanfordUniversity,Stanford,California94305;
wnloadical e3DmAaiNl:IrSoCdoOlp,hUe.SbAar,rIanncg.,oMu@addiasnoinsc,oW.ciosmconsin53716;
DoMe
45:273-297. mpus - Lane ATnhneuA.nRneuva.lGReenvieetw.2o0f1G1e.n4e5ti:c2s7is3–o9n7lineat Kcoeevyowluotriodns,immunity,interference,RNAi,bacteriophage
11.Ca genet.annualreviews.org
et. 20Main T10h.1is1a4r6t/icalnen’sudreovi:-genet-110410-132430 Abstract
Annu. Rev. Gennford University - A0C0lol6pr6yi-gr4hig1th9s7tr/e(cid:2)1cse1r2/v10e21d011b-0y2A7n3n$u2a0l.0R0eviews. BtTCsopaRheccicItsoiSefiaprPcrieaRsaelwa-lnyCniatdialhnshacvsraoycasrhsrpbtiaoeoeemueraans,theeewaxnsvhphveiaoicrenhrovtdnohsemeladvqseebutdnywetndatohcelenfesesotrnrfveresecoeslesmanofenrtiadsndt,vuirsiaerncdegocsiulnv.ulegFadrtigiyornesrognty,fevmttthhiirceeeuceshvhleeoaamrsntsttiaeasctnmciakltness.
Sta (virusorplasmid)intoaregionofitsgenomethatisdistinguishedby
by clusteredregularlyinterspacedshortpalindromicrepeats(CRISPRs).
Second,whenthesesequencesaretranscribedandpreciselyprocessed
into small RNAs, they guide a multifunctional protein complex (Cas
proteins) to recognize and cleave incoming foreign genetic material.
Thisadaptiveimmunitysystem,whichusesalibraryofsmallnoncod-
ingRNAsasapotentweaponagainstfast-evolvingviruses,isalsoused
as a regulatory system by the host. Exciting breakthroughs in under-
standingthemechanismsoftheCRISPR-Cassystemanditspotential
for biotechnological applications and understanding evolutionary dy-
namicsarediscussed.
273
GE45CH13-Bhaya ARI 1October2011 14:42
INTRODUCTION predators (2, 41, 66). In this context, the
discoveryofanoveladaptivedefensesystemin
Bacteriaandarchaeahaveevolvedtocopeand
bacteriaandarchaeahasgeneratedgreatinter-
Clusteredregularly thrive as communities in dynamic environ-
estandencouragedresearchintothemolecular
interspacedshort mentsthatarestressfulandfluctuating.These
palindromicrepeats environmental stressors can be both abiotic basisofthemechanismofaction,consequently
(CRISPR): hallmark therehavebeenseveralrecentbreakthroughs.
(e.g.,nonoptimaltemperaturesornutrientlev-
ofCRISPR-Cas The CRISPR (clustered regularly interspaced
els,redoxstress)andbiotic(e.g.,toxins,viruses,
systems
short palindromic repeats)-Cas (CRISPR-
transmissiblegeneticelements)innature.The
CRISPR-associated associated proteins) defense comprises a
abundantpresenceofvirusesinalmostallenvi-
proteins(Cas):
multistep process by which specific small
diversetypesof ronmentsisaconstantthreattothesurvivalof
fragmentsofforeignnucleicacidsarefirstrec-
proteinsencodedby bacteriaandarchaea(2,87,93,107).Further-
nly. casgenesinthevicinity more,virusescanhavehighratesofmutation ognizedasbeingnonselfandincorporatedinto
e o ofCRISPRs and recombination, so a successful defense thehostgenomebetweenshortDNArepeats.
s
u Subsequently, these fragments or spacers, in
ews.orgersonal saybsitlietmy thoadsetaol wbiethmvualrtiialabyleeraenddafnadst-heavvoelvitnhge casonajusunrcvtieoilnlanwcitehanhdosatdCapatsivperoimteminus,nearseysutseemd
w.annualrevi09/12. For p CDREINSOPRMSINANATDORRNSAAI:NCDODMIMFFOENRENCES brTeyhcoewghCniiRczheISdiPnaRcnod-mCdaienssgtsryofsoytreeedmigonhrapnsoupscsrlieibmiclyarasicilliyednsbceaeerdne.
om wwy on 08/ ThehypothesisthatCRISPR/Cassystemsmightbeanadaptive iDnNveAstig(vaitreudseins iatnsddepfleanssmiviedsr)o,lebuatgaitinssvtefrosraetiiglen,
ded frLibrar iamtamnuannealsoygsytetmotwhaesebuaksaerdyootniciRnNsiAliciontaenrfaelyresensceth(aRtNalAsoi)hminectehd- mulaotdourlyarroalrechinitheoctsutrceelmlsa.yTahleloawppitatroenptlapyaraarlleegl-s
wnloadical anism(74).Althoughtherearecommondenominatorsbetween to the eukaryotic RNA interference (RNAi)
DoMe crRNA and RNAi-based mechanisms of action, there are also system (see sidebar, CRISPRs and RNAi:
et. 2011.45:273-297. Main Campus - Lane sCmaopifrgRreoinndbtIieuiSofiaicP-ntclenaResdunicictanblnaveydcdooiisflpdfmvRerseor.NadetlTlneAiincnhnieoe-pstnrch.reocieTmomadbhrapiieernoliegglfmxyueRnntocacNeloretsngiciAosesunistlsoaatwsfrleohsqscfmiiuocmtehmahnillelimcaneirfnoi-actnstcoiepeatnreslfjticeuthbiirnfiaeeitcsnctwtgbibcoeoleReenttanhwNvweatAaiehgtrsnheee, CtgpmnheraooeontmudvepreirmdaolalettoceeseonndnaetvDviiuianorelntolnueiunqortsmueimeosesenti.nnointbaTpstepohbtoowrierorsettiCauenennncRcdhiItohnySDnooPttsilroRtfofoge-aolCrylnbeedadsnhescavsrevesvisreyte)utsiatsaanelnnsgimnddos
Geny - as well as mechanistic and structural commonalities between because acquisition and immunity occur on
Annu. Rev. nford Universit tCOfohRnreeIeCSuPRnkoRaIStr-ayPabosRlsteio-cicndRiatieNtfferedfAree-rcineonncmdecupeclieesisxdtdfshoiselrDenaNfncaictAnitv,girtachalotlahmtdoeputflhegeenhxs(epmRr(IRiCSmNCAaSA)r,yCacAntadaDnrtgEhbe)eet. saFfohgirnogearritelglsyntsi,imoDtnehNsesAccaaalanebnsidlbiatnesyudbdteseoevdqiududceeyennndctaelmyioniufcsaspeloalymisttetagocecqnfiauesgiethrisect.
Sta targeted by some CRISPR/Cas systems. Some key proteins do off invading genetic material has elements of
by not have direct functional/ structural equivalents, notably the an acquired and heritable immunity system,
eukaryoticArgonaute(ARO),andsomeoftheCRISPRuniversal reflectingaLamarckianmodeofevolution(64).
and“signature”proteins.Overall,althoughCRISPRisarguably We begin with a personal perspective of
mostsimilartoPIWI-interactingRNAs(piRNAs),whichprotect the discovery of the CRISPR-Cas system and
genomeintegrityfromparasitessuchastransposons(103),fur- thensynthesizeresultsinthisfast-movingfield,
therstudiesthatexaminethemolecularbasisforCRISPR-based including an attempt to categorize the bewil-
interference will likely expand the list of mechanistic idiosyn- dering variety of Cas proteins and their func-
crasiesandnotabledifferences.Nevertheless,evidenceofsmall tions. We focus on bacteria, although studies
RNA-baseddefenseandregulatorysystemsinallthreedomains witharchaea,mostofwhichcontainaCRISPR-
oflifeshouldfosterresearchthattranscendtheseboundaries. Cas system, have provided important insights
into its mode of action, diversity and unique
· ·
274 Bhaya Davison Barrangou
GE45CH13-Bhaya ARI 1October2011 14:42
characteristics(forreviewswithafocusonAr- 1990s, bioinformatics-based tools allowed for
chaea, see 32, 69, 99, 108). We present new easyidentificationofthesehighlyconservedar-
evidenceoftheregulatoryroleofCRISPR-Cas raysofpalindromicrepeatsasbacterialandar-
Spacer: small,variable
systems in bacteria, which functions via small chaealgenomesequencing/annotationprojects
sequences(flankedby
noncoding RNAs. The evolutionary implica- greatlyexpanded(16,47,48,59,60,62,77,81, repeats)inthehost
tionsofthisrapidlyevolving,heritableimmune 84,98).Inhindsight,themeagerviralandplas- genomethatare
system in prokaryotes and the opportunity it mid sequence information available hindered acquiredfromforeign
nucleicacidsandplay
affordstostudythecoevolutionofvirusesand the interpretation of the genetic content and
aroleindefense,so
hosts are discussed. We include a section on potentialfunction(s)oftheCRISPRsandeven
thenameisinaccurate
progressinengineeringtheCRISPR-Cassys- today remains a limitation. Initial predictions butwidelyadopted
tem for biotechnological and epidemiological based on bioinformatic analyses were made
Bacteriophage:
nly. applications. Several recent reviews have de- suggestinginvolvementinchromosomeparti- virusesthatinfect
e o scribedtheexperimentsthatledtothediscov- tioning(81)andDNArepair(73),andthebold bacteria,alsoknownas
us ery of this new defense system (5, 24, 49, 58, conjecture was put forward by Makarova and phage
ews.orgersonal 1o0f4a,ct1io1n3)oofrthheavCeRfIoScPuRse-dCoasnstyhsteemmeicnhbaancistem- cboellaeadgeufeesnstehastytshteemCRakIiSnPtRo-Ceuaksasryysotetimc mRNigAhti
w.annualrevi09/12. For p rhainaave(ev5o6elx,ua7tmi6o)innoaerrdyintpheaerrscCpheRaceItSaivP(e3R(2-6,C39,a91s,1s12y0)s8toe)rm;poltfahrcoeemrds (t7h4reT).ehgeryoeuaprs20in0d5empeanrdkeedntalyturrenpionrgtepdoitnhtawthtehne
m wwon 08/ iwtoinrldth(e58c)o.ntextoftheburgeoningsmallRNA hogypyetrovavriiraubsleess(poarcebrascstheroiwopedhasgeeqsu)eonrcephlaosmmoidl-s
oy
ded frLibrar aanteddhpyrpootethinessizceoduldthaptlaCyRaISroPlResinanidmmasusnocitiy-
DownloaMedical MBIEO´NINAFGOERAMTARTOICISS:, a9g0a).inRsetsteraarncshmgisrsoibulpesgweonrektiincgelwemithengtesn(e1t3ic,a8l0ly,
45:273-297. mpus - Lane BBBAIrOiCefCTHHEiERstMIoOrIyPSoTHfRAthYGe,EASND taemrnxaepdcne/ttroaiibrnmlgveeitrnhbatlaeacglateebenvriloiiidamtleynesoscyfeswtt.heeAmererCsesoRpaoonInSrdtPaiabRnvl-ae2Cit0laoa0bs7plserydosohvtceiodumse-t
et. 2011.Main Ca CAtRimISelPinRe-oCfuansrSelyastetdemobservationsmadeover tino2p0ro0v8idsheovwirianlgretshiestaabnicleit(y1o1f)CanRdIaSPpuRbslitcoaptiroen-
Geny - the past twenty years provides a compelling ventplasmidtransfer(75)providedtheimpetus
Annu. Rev. nford Universit ehabnxiyoadpmcohgtpehelmeensoieocmsafle,tghsecetnoreusnlrctoaetwtuxertdablpu,srtaoendsleadilctyitgsifoeyfnnrionsemtgi(c7p3sda,etahqt8au0fer)tnohctmaoet tiapnolesgroivntaqvorueiaisictbmiklgelpyaoatrenretdaathnlrietazpemndideetlwcyhheeaavpdnoovilsatvmeninnctgeoisafg.leaoRncfteeittsoihencae,rlsocelechhaiedyar--ss
Sta substantiated these initial ideas (11, 30). An a valuable tool for genotyping closely related
by unintendedconsequenceofthistrajectoryhas pathogenicorenvironmentallyrelevantstrains
beentheuseofseveralconfusingacronymsand andthisisanareaofactiveresearch.
synonyms[seeMakarova(72)andDeveauetal.
(24) for clarification]. Fifteen years elapsed THECASTOFCHARACTERS
between the initial report of the presence of
NovelFeaturesoftheCRISPR-Cas
DNArepeatarraysintheintergenicregionad-
DefenseSystem
jacenttothealkalinephosphatase(iap)genein
EscherichiacoliK12(52)andthecoiningofthe The CRISPR-Cas defense system has the
CRISPRacronymin2002,followingtheobser- novel ability to incorporate short sequences
vationthatsucharraysofrepeatswerecommon of nonself genetic material known as spacers
in bacteria and archaea (53, 54, 81). In the at specific locations within CRISPRs in the
www.annualreviews.org • CRISPR-CasSystems 275
GE45CH13-Bhaya ARI 1October2011 14:42
host genome. Spacers are transcribed and locus;thus,positionalinformationrepresentsa
processed into small noncoding RNAs, which timelineofspaceracquisitionevents.Toindi-
inconjunctionwithspecificCasproteincom- catethesequenceontheviralgenomethatcor-
CRISPRlocus/array:
plexes can bind to incoming foreign genetic respondstoaspacer,thetermprotospacerwas
regionongenomeor
plasmidcontaining materialifthereisacloseorabsolutesequence coined(23).Inseveral,butnotall,cases,avery
CRISPRs match between the small RNA and incoming short stretch of conserved nucleotides in the
Leader: 5(cid:2)endof nucleic acid. This sequence-specific recogni- immediatevicinityoftheprotospacer,referred
crRNAprecedingthe tion process culminates in destruction of the to as the protospacer adjacent motif (PAM),
firstCRISPRrepeat, invadingnucleicacidandrequiresseveralCas or CRISPR motif, appears to be a recogni-
whichcancontainlong
proteins. The surveillance and attack process tionmotifrequiredforacquisitionoftheDNA
AT-richtractsand
exploitspreviousexposuretoavirusorplasmid fragment(24,79)(Figure 2).Thisfirstphase
includeapromoter
y. to target incoming foreign DNA (or RNA). minimally requires two nucleases, Cas1 and
nl Protospacer:
e o sequenceinforeign This provides the host heritable immunity to Cas2,bothofwhichareuniversallypresentin
s
ews.orgersonal u DaincNtqouAitrhweedhhiaocshsatigsspeancoemre rbsyeescetenenmttle.yrmHdeeotdwecaetnveedar,dfoasrpoetmiigvene oDorbNvaicAoquuasnirddedihsteiimnnccmetiuohnnaess gssyyessnttoeemmm.easndthcaatnhbaveecoanfsuidnecrtieodnhalalClmRaIrSkPsRof/Cthaes
om www.annualreviy on 08/09/12. For p Pa(ctohPosfresnAoaositMpmceorrisam)ovp:teteeaodsddcshspieoaemartqrc-etouevteriinftchcineaititnyis bsfasapiceccqatatwculetievrhreesean)rttnatethenhbwederaCtCtsheRpRiasIicmIStSePimrPmRsRue-(n--CorCereaassrscoesolsysvnpysetovsdetenmersamseceatlniiycnvda,icntltlyuohrdseaeeelaclodlotawihlsldye-s eCfNrxoRepmxIrIntSe,sPttshhsRieopeneCsc,ReiRcfiNoaIcSnAPdepRnrs(pitdmalroogearce-iruc,byrsroRenbftNueyrrcaARrlnee)sNadcsriAetsiospptat,corslaleCynoamsrRvceeIrrSipabtPrsheeeRde-.
ded frLibrar raecqquuiirseitdiofnor itthattoirsesaplsoondevdoylnvianmgicaatllyhitgoharavtiersa.l Fpruerdthateorr- p(crreR-cNrRANs).ABsaseidntoonthsmeiarlflunCctRioInS,PtRheseRsNmAalsl
45:273-297. Downloampus - Lane Medical p(fpCCuprrRRlroele-Id-IlS-SCuecPPncRreRgRRdItSNhaRfrPrtrAoNrRama)yA:nRscNriApt mdmeduaisoouktAdragienerh,ygftusoouepntfriaeschcestc.eivaeorobln-lllsduea,eltcirrooCievnmmRe,dpIiwnSoiiPnmhsecRimcne-hntuCstndarioosetqyefssuyiasisnrtieLeondmtahmfeoohrcariactrasecucdkrttiwibavinnoy- Rs(acC1isrlNAR4e,nSiNAn1cCstiA9enAh)rsg.aDfweIvn(rEeipetahtsnh(ilicCRsneoeNRatbhI(Ame2Sires4Pudn))lRtar(i4-enopa0frdres,osrfioitr7menec4idianm)alttcouseootrdnaamigsgtceyppuo,lrimeddo(x3eekp,0sal[cRe)rc,rxyaNiolbtlftAheeoiddecrs
et. 2011.Main Ca (npcroronRdcNuocdAeind):gbsyRmNcaleAllavage fiteyatu(Freigiusrtehe1)C.RTIhSePRfirlsotcuesa/sailryraryecloocganteizdabolne aasntEiv.ircoalli]decfaennsree)coingnpiazretiacnuldarboasreg-apnaiisrmssp,escuicfih-
Geny - ofpre-crRNA(also the genome (either chromosome or plasmid), callywithregionsofincomingforeignDNA(or
Annu. Rev. nford Universit kgItinhnnucetoiodewpmerrnfoRiencarNgesesnApfsocs)bireRey:iNgwnAhicohr wqfineuhaittirchuehredefvcriiosocnimantaidvtiyniivrsueorsftshoeaergCphrlRyoaspuImSeprPivdoRafDriclaaNobscAluge.se,Tnspwehashecilescoerhcscaoetanencdd-- RpcthrlNeRemoNAte)hAnteh–trafaorhtirahteynaidvg(1e,n4ipfn)et.uhrTfceelhreceiitsca(ioranercimtaidilamitscemoossmacttlppcelehaervexfaseg(bc3ete0)to)cw.foeOtmehnne-
Sta DNAorRNAis codeproteins(genericallycalledCasproteins) thespacerandtargetDNAoriftherearemuta-
by targetedfor requiredforthemultistepdefenseagainstinva- tionsinthePAM,thencleavageisnotinitiated.
destruction;also sivegeneticelements. In this case, DNA is not targeted for attack,
knownasadaptive
The CRISPR-Cas defense process can be replicationofthevirusproceeds,andthehost
immunity
separated into either two or three stages isnotimmunetovirusattack(Figure2).This
CASCADE
(Figure1).Thefirststage,referredtoasadap- leadstohostlysis,andthereleasedviruscanat-
(CRISPR-associated
tation (30, 76), immunization (49), or spacer tackothersusceptiblehostcells.Tooperateasa
complexforantiviral
defense): acquisition (58, 113), involves the recognition defensesystem,allthreephases(i.e.,spacerac-
multisubunitprotein andsubsequentintegrationofspacersbetween quisition,expression,andinterference)mustbe
complexrequiredfor two adjacent repeat units within the CRISPR functional,butitisimportanttonotethateach
interference
locus.Spacersappeartobeintegratedprimar- of these processes can work independently,
ilyatoneend(theleaderend)oftheCRISPR bothmechanisticallyandtemporally.
· ·
276 Bhaya Davison Barrangou
GE45CH13-Bhaya ARI 1October2011 14:42
Virus DNA Plasmid
DNA
Plasmid DNA cleaved
1 Acquisition
y.
nl Leader10 9 8 7 6 5 4 3 2 1
e o cas locus CRISPR array
s
u
w.annualreviews.org09/12. For personal 2 Expression 3 Interference
w8/
m won 0
oy
ded frLibrar Pre-crRNA
wnloadical Cas proteins Virus DNA cleaved
DoMe
45:273-297. mpus - Lane crRNA
11.Ca Key
et. 20Main Protospacer
Geny - PAM
Annu. Rev. ord Universit RSTpreapanceseacrtription start
nf
a Figure1
St
y FeaturesoftheCRISPR-Casadaptiveimmunesystem.Stage1:CRISPRspaceracquisition.Specificfragmentsorprotospacers(withan
b
adjacentprotospacer-associatedmotif;shownasredbar)ofdouble-strandedDNAfromavirusorplasmidareacquiredattheleaderend
ofaCRISPRarrayonhostDNA.ACRISPRarrayconsistsofuniquespacers(coloredboxes;spacersarenumberedsequentiallywiththemost
recentlyacquiredspacerhavingthehighestnumber)interspacedbetweenrepeats(blackdiamonds).Acquisitionoccursbyaprocessthat
minimallyrequiresCas1andCas2,encodedinthecaslocus,usuallylocatedinthevicinityoftheCRISPRarray.Stage2:CRISPR
expression.Pre-CRISPRRNA(Pre-crRNA)istranscribedfromtheleaderregionbyRNApolymeraseandfurthercleavedintosmaller
crRNAsthatcontainasinglespacerandapartialrepeat(hairpinstructureswithcoloredspacers)byCasproteins.Stage3:CRISPR
interference.crRNAcontainingaspacerthathasastrongmatchtoincomingforeignnucleicacid(plasmidorvirus)initiatesacleavage
event(shownbyscissors);Casproteinsarerequiredforthisprocess.DNAcleavageinterfereswithvirusreplicationorplasmidactivityand
impartsimmunitytothehost.InterferencecanbemechanisticallyandtemporallyseparatedfromCRISPRacquisitionandexpression
(depictedbywavywhitebaracrossthecell).ThisfigureisbasedontheCRISPR-CassysteminStreptococcusthermophilus,whichrepresentsa
well-studiedandrelativelysimpleCRISPR-Cassystem.
www.annualreviews.org • CRISPR-CasSystems 277
GE45CH13-Bhaya ARI 1October2011 14:42
a Viral DNA Protospacer PAM
5ˈ–ACACTTTGCTACTGCATGCCAAGCAAGTTGATATATTTCTCTTTCTTTATAAGAAACGTCGATTACGATCGGTA-3ˈ
3ˈ–TGTGAAACGATGCCGTACGGTTCGTTCAACTATATAAAGAGAAAGAAATATTCTTTGCAGCTAATGCTAGCCAT-5ˈ
Cas1, Cas2
b Host DNA
Repeat Spacer Repeat
5ˈ–GTTTTTGTACTCTCAAGATTTAAGTAACTGTACAACAAGCAAGTTGATATATTTCTCTTTCTTTATGTTTTTGTACTCTCAAGATTTAAGTAACTGTACAAC-3ˈ
3ˈ–CAAAAACATGAGAGTTCTAAATTCATTGACATGTTGTTCGTTCAACTATATAAAGAGAAAGAAATACAAAAACATGAGAGTTCTAAATTCATTGACATGTTG-5ˈ
c Pre crRNA Endo ribonucleases, e.g. cas6e RNA polymerase
5ˈ-GUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACAAGCAAGUUGAUAUAUUUCUCUUUCUUUAUGUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAAC-3ˈ
crRNA
nly. 5'OH
o
e 3'P
s
u
orgnal
w.annualreviews.09/12. For perso HdoV s i1tr as0lp D0acN%eAr mAAaAAGGtcCCAAhAAGGTTTTGGAATTAATTAATTTTTTCCTTCCTTTTTTCCTTTTTTAATTAACGAASACAADE + Cas3 eHo V s≥itr a1slp DmacNeiAsrmAAAAaGGtCCcAAhAAGG (TTiTTnGG AAoTTrAA TTnAAeTTTTaTTrCC PTTCCATTMTTTT)CATTTTTGAATTTAATAAA
m wwon 08/ ForeHigonst D imNAm culneeaved ForeHigonst D nNoAt i nmomt culneeaved
oy
wnloaded frdical Librar FAunigcdlueorrsseetua2pndoifntghoefCthReISCPRRI-SCPaRssTyystpeemI.aSnodmIeIsoyfstthemebs;aositchsetrevpasriinatCioRnsISaPreRs-hCoawsndeinfenFsieguarreed4e.p(aic)tDedohuebrlee-bstarsaenddoendcvuirrarleDntNA(gray)
DoMe withaprotospacer(green)andadjacentprotospacerassociatedmotif(PAM,red).(b)HostDNAwiththenewlyacquiredspacer(blue)
et. 2011.45:273-297. Main Campus - Lane (tsflpbttoheerarceotnacowtnrk3onesee(cid:2)sdcaedeprtanneiabrpdcycymteri(srCRohat.rntaNRAuuirsrIcAptnSetaiuuPnarcrmtnrReiRdsnbiorNtebfefeolrpaAcrnoecreskoaRfi.)tgtCT.Nsns(a(hhdAbnsoil)-sua:iwesccsAnkntlsece))hp.oip;coeAdorwiaercsfcadenqaincudcpbtgicres(eoooeilrtmoiatiteorwahprilnn,emolirwswmso,hiissneitenth.mdgtimhm.aab,ear5yCaktpl(cid:2)cdlAwrhhyioSfahrbftCneeeoedrqrAsteelwpueDnaicert(clEeebeeCnslaraaavCccnoskardarg)RpsePfrC1NooAoalatAlfMseno3pidawn,)rnsaeCepd,rd-radeCefsbeovr2Rypree.eIeqnt(Sinhuctg)PdeincrRRilesnenpNdgaRuavfAccoNaolengerprAeiottc(,hh(blaacyielscunrmiRpedpd)earN,irraoniatnAncitscdeeie)tusribatsflarae.ytparr(enoeeesC)nntsaecMRdcnrecoIitilbSrsideaiemPaoblsvReoahapstnagcrtnuieeyhroc-ppeelcteseivrn.oaeR(sTcnpseNctuthsrurwAuercpc.ailptetnNhuuliertoonatecttteectaihrvutseer)
Geny - thatcrRNAbasepairswiththecomplementarystrandoftargetforeignDNAduringtheinterferencestep(notshownhere).
Rev. versit
Annu. nford Uni CdseaRvteaIbrSaaPlsetRo;doinblsc:ltuoCdResISPR TAlhtheouCgRhItShePsReloLcoicwuesrechristenedinseveral atetriaumspecHifialciaCngRiuISmPRochlorcaucesumin tDhSeMmy1xo43b6ac5-;
Sta identifyandanalyze ways, the term CRISPR locus (or CRISPR NC_013440) (35). Among different species,
by CRISPRs.Maintained array)hasnowbeenwidelyadoptedandrefers thelengthoftherepeatcanvaryfrom21bpto
byUniversiteParis
48 bp, whereas spacers are typically between
Sud(http://crispr.u- toagenomicregioncontainingCRISPRs(24).
26bpand72bp(33,35).Thesequenceofthe
psud.fr/) Dedicateddatabases(CRISPRdbandCRISPI)
repeat units in different CRISPR loci is not
CRISPI: database (35, 94) that identify CRISPRs and Cas
conserved, although there are partially con-
thatidentifiesCas proteins on sequenced genomes indicate that
proteinsand theyarepresentonmostarchaeal(∼90%)and servedsequencessuchasaGTTTg/cmotifat
CRISPRs;includes manybacterial(∼50%)genomesoronresident the5(cid:2)endandaGAAACmotifatthe3(cid:2)end(24,
manyfeatures 33,54,65).Becauseofthepartiallypalindromic
plasmids.Thenumberofspacersinaparticular
complementaryto
natureoftherepeats,itwashypothesizedthat
CRISPRdb(http:// CRISPRlocuscanvarywidely,fromasfewas
transcripts from these regions may form
crispi.genouest.org/) onetoseveralhundred(asmanyas587spacers
stable, highly conserved RNA secondary
· ·
278 Bhaya Davison Barrangou
GE45CH13-Bhaya ARI 1October2011 14:42
structures (65, 74), and recent structure/func- colleagues (38) defined eight subtypes of Cas
tion data supports this (see section entitled proteinsbasedonthephylogenyofthehighly
Interference). conservedCas1proteinandtheoperonicorga-
Repeat-associated
Although the majority of genomes contain nization of cas genes, which were named after
mysteriousprotein
a single CRISPR-Cas locus, there are several eight representative organisms that contained (RAMP): proteins
examples of bacterial or archaeal species that a single CRISPR-Cas locus [e.g., E. coli Cas withRNAseactivity
harbormultipleCRISPRlocionthechromo- proteins were designated cse1 (CRISPR sys- involvedincrRNA
processing
some.Somebacterialgenomescontainasmany tem of E. coli gene1); other subtypes included
as 13–15 CRISPR loci, and archaeal genomes Aeropyrum (csa), Desulfovibrio (csd), Haloarcula TypeI,II,andIII
CRISPR-Cassystem:
alsoharbornumerousCRISPRloci,basedon (csh), Mycobacterium (csm), Neisseria (csn),
newclassificationof
information available in CRISPRdb (35) and Thermotoga(cst),andYersinia(csy)].
CRISPR-Cassystems
nly. CRISPI(94).NotallCRISPRlocihaveadjoin- These initial categories, although useful, basedonmultiple
e o ingcasgenes;itispossiblethatonlythesubset cannot easily handle the relationships be- criteria
s
u ofCRISPRlocithathaveadjacentcasgenesare tween homologous but distantly related Cas
ews.orgersonal fsuenntctiinoancatilvlyelaocctii,voe,rtwhhaetroenaesstehteofoCthaesrpsrroetperine-s pinrotceaisnso,ptehreonesx,teonrsivoergvaanriisambislittyhatthactoenxtiasitns
w.annualrevi09/12. For p sipnuleftfirCcaenRssI(fS2oP5rR,t5hl0eo)c.aiIcnotinvciaatsygeseonwforhemelraeet,ettdhheeCrseReaqISruePemnRcuellotoci-fi mcinlaucsllsutiidfipiclneagtCioeRnvIosSyluPsttReiomnloabcrayi.seIrdneloaantionmneuwslhtiiappnlsedocurfintciefioreinad-,
m wwon 08/ soormveeroyfsitmheilaCrR,aInSdPRthreespeematasymuatiylizbeetihdeenstaimcael sseervveerdalpgrrootuepinss(7a2n)dwcoasrkoinpgeroonn CorRgIaSnPizRat-iConas,
oy
ded frLibrar speetatosfhCavaesbpereontecliansssi(fi4e4d,i5n0to, 6at5l)e.aCstR1I2SgPrRourpes-, sthyseteCmRsIShPavRe-Cpraospsoyssetdema ccoannsebnesudsivvidieewd itnhtaot
wnloadical andthereappearstobesomecorrespondence twopartiallyindependentsubsystems.Thefirst
DoMe between certain repeats and groups (or sub- consists of an information processing module
45:273-297. mpus - Lane t(6yp5e,s7)2)o.f Cas proteins associated with them atssepuniabdncsseyr,resCtqeaaumcsiq1reiuassinsrtidethqiCeounaiurs.ne2Tdi,vwhfeorhersiacsplehlrycoaocrpneerdseisn,sienvonogrtlovecefxodperrcieniumptniarveorewy-,
et. 2011.Main Ca CCRasISPPrRotloeciinsoften have groups of conserved CanRdISdPeRgratrdaantisocnripotfs (icnrvRadNinAg) afnodrerigencognnuictlieoinc
Geny - protein-encoding genes, named cas genes, in acid, and is quite diverse. For instance, in
Annu. Rev. nford Universit taindhnueeacnillryetisafievsasiec,bsiC,lnepaitodsylopymrm(o7ate3iern)ia.nsssecBwsha,aesarernaeddcptReroernNidsiAtccic-toebmodinfptdhouietcnlaoigtcniapotsraneoiasn-l, cCtmheAuertlSatciCifrnuRAnNDCctREAioI,SniwsPalhRinpervrseoouatlbsev-ientidynpmieonasty,htehtprhelaetypymrptoheucsilsetsiarssouisnlbiegnu.gnIolinetf
Sta teins, which led to the initial speculation that addition, there are several repeat-associated
by theymaybepartofanovelDNArepairsystem mysterious proteins (RAMPs) that constitute
(73).Theorder,orientation,andgroupingsof a large superfamily of Cas proteins. RAMPs
casgenesappeartobeextremelyvariable,and contain at least one RNA recognition motif
this picture grows ever more complex as the (RRM; it is also called the ferredoxin-fold
number of annotated genomes increases. At- domain), and some have been shown to be
temptstoclassifyCasproteinshavebeenmade, involvedinpre-crRNAprocessing(14,19,26,
but this has proven difficult because of the 42).Basedonthisclassificationthatintegrates
diversityoftheproteinsinvolved(38,72,74). phylogeny, sequence, locus organization, and
Initially, Jansen’s group identified four gene content, three types have been distinguished,
families,cas1–4(53),whichwerethenextended Type I, Type II, and Type III CRISPR-Cas
to include cas5 and cas6 (13, 38). Haft and systems(Table1,Figure3,Figure4).
www.annualreviews.org • CRISPR-CasSystems 279
GE45CH13-Bhaya ARI 1October2011 14:42
Table1 MajorCasproteins(Cas1–Cas10).ForotherCasproteins,pleaserefertoMakarova(72).Blue,red,andpurple
designateuniversal,signature,andtype-specificCasproteins(asinFigure3).
Protein Distribution COG Process Function
Cas1 Universal COG1518 Spaceracquisition DNAse,notsequencespecfic,canbindRNA;
presentinallTypes;structureavailablefor
severalCas1proteins
Cas2 Universal COG1343, Spaceracquisition SmallRNAsespecifictoU-richregions;
COG3512 presentinallTypes;structureavailablefrom
ThermusthermophilusandSulfolobus
solfataricusandothers
Cas3 TypeIsignature COG1203,COG2254 Targetinterference DNAhelicase;mostproteinshaveafusionto
y. HDnuclease
nl
o Cas4 TypeI,II COG1468 Spaceracquisition RecB-likenucleasewithexonucleaseactivity
e
us homologoustoRecB
orgnal Cas5 TypeI COG1688,RAMP crRNAexpression RAMPprotein,endoribonucleaseinvolvedin
ews.erso crRNAbiogenesis;partofCASCADE
w.annualrevi09/12. For p Cas6 TypeI,III CCOOGG15558531,,RAMP crRNAexpression RcsAtrrRMuNcPtuAprreboiaotvegaieninl,aeebsnliesd;ofprroaibmrtonoPfu.cCfuleAraiSosesCuiAsnDvoElv;edin
w8/ Cas7 TypeI COG1857, crRNAexpression RAMPprotein,endoribonucleaseinvolvedin
m won 0 COG3649,RAMP crRNAbiogenesis;partofCASCADE
ded froLibrary Cas8 TypeI Notdetermined crRNAexpression LdaorgmeapinroatnedinRwuivthC-MlikcerAn/uHclNeaHse-;npuacrlteaosfe
wnloadical Cas9 TypeIIsignature COG3513 Targetinterference LCarAgSeCmAuDltiEdomainproteinwithMcrA-HNH
DoMe nucleasedomainandRuvC-likenuclease
11.45:273-297. Campus - Lane Cas10 TypeIIIsignature COG1353 craRndNiAnteexrpferreesnsicoen HdtraDiobrmgbneoautnicnc;l;lesenoaasemvecaeegdsehosmaormayionfol,orpgiainelmtsewrdfieotrhmenCaciAne,SaZCnndADE
et. 20Main elements
Geny -
Rev. versit TypeICRISPR-CasSystem believedtotargetDNA,andcleavagerequires
nu. Uni In addition to the presence of the conserved Cas3[whichhasahistidine,asparticacid(HD)
Anord Cas1 and Cas2 proteins, Type I is defined by nuclease domain] or Cas4, a RecB-family
anf theubiquitouspresenceofasignatureprotein, nuclease (102). The Type I CRISPR-Cas
St systeminE.coliisoneofthebestcharacterized
y the Cas3 helicase/nuclease. Cas3 is a large
b (Figures4and5)andrecentexperimentsusing
multidomain protein with distinct DNA nu-
E.coli are described in later sections (14) (57).
cleaseandhelicaseactivities(102).Inaddition,
MultiplestudiesinPseudomonasaeruginosahave
there are multiple Cas proteins that form
also shed light on the Type I CRISPR-Cas
CASCADE-like complexes that are involved
mechanism of action (42, 118, 119). For
in the interference step (Figures 4 and 5).
DNAinterference,CASCADEassociateswith
Many of these proteins are in distinct RAMP
processedcrRNAtoformaribonucleoprotein
superfamilies(Cas5,Cas6,Cas7).Ofthethree
complex that drives the formation of R-loops
systems, Type I, thus far, is the most diverse
in invasive double-stranded DNA (dsDNA)
with six different subtypes (Type I-A through
(Figure5)throughseedsequence–drivenbase
TypeII-F)(72).TheTypeICRISPRsystemis
· ·
280 Bhaya Davison Barrangou
GE45CH13-Bhaya ARI 1October2011 14:42
Type I-E (Escherichia coli)
Protein type
cas3 cse1 cse2 cas7 cas5 cas6e ccaass11 cas2 Universal
Type-dependent
Signature
Type II-B (Streptococcus thermophilus)
cas 9 cas1 cas2 cas4
Type III-B (Pyrococcus furiosus)
y.
nl cmr1 cas10 cmr3 cmr4 cmr5 cas6 cmr6 ccaass11 cas2
o
e
s
u
orgnal
ews.erso
w.annualrevi09/12. For p FCreiapgsrueprsreeont3teeindsbiyntTheypoepIe,roIIn,asntrducItIuIrCeRfrIoSmPRE-sCchaesriscyhsitaecmolsi,.SAtrrcehpetotcyopccaulsTthyepremIo,pIhIi,luasn,danIIdIPsyysrtoecomccsuasrfeuriosus,
w8/ respectively.Theuniversallypresentcas1andcas2genesrequiredforacquisitionareshowninblue.
m won 0 Signaturegenesforeachtype(TypeI,cas3;TypeII,cas9;andTypeIII,cas10)areshowninred.
oy Type-dependentgenes(i.e.,cas4,5,6,7)areinpurple,cas8,whichisnotshownhere,isfoundinTypeI-A,
Downloaded frMedical Librar It(eTy-xBppye,rp-eaedsnseIdiApoIen--nICFadn).edInan/ntodgTrebyinnopetteseh,rIfsTIeeIr-yveBpenr,eaccIlemI;orasf1ingw,dn3haI,iItc4Iuh,rh5eaa,rvge6eernaterwepesoetaaysrtupe-abeis-tnsydvopeoceplisvea.etneTdddeyinnmpteiy-ngsdteteeenrprefieesonru(edTsnepycnpretoe.ptHIerIoioIntwesA)ie.nhvTseaasryr,paetehdtIeyifrphfeeiacrasaerlsneliytxesisxenuctvebooptlfytvipeoednssin
45:273-297. mpus - Lane ptoaitrhiensge.cIattewgaosrirees;cesenetlTyasbhloew1nfoirnCEa.scpolrio(t9e7in)faunndctioncsoamndpoMseakathroevaoeptearlo.n(72()thfoisrfiunrcthluedredsetacailss9., cas1,
et. 2011.Main Ca Pds.DaeNruAgintoasarg(e1t19r)etchoagtnCitAioSnCAbDyEsfeaqcuileitnactee-s ctaysp2e,sa,nTdyepiethIeIrAca(so4roCrAcsSnS2)4.Tthhaetreinacrleudtwesocssunb2-)
Geny - specifichybridizationbetweencrRNAandthe and Type IIB (or CASS4a that includes cas4).
Annu. Rev. Stanford Universit tsTaerqygupeeetnDcIeIN)CaAtRtohIveSe5rP(cid:2)aRen7-d–C8oafbstphSesyesspqtauecemenrc(eF(itghuerese5e)d. TtpmliorschiohodecvecidbDudesteNshttdha-Aetesfrta(eum1tndr1osaip,eenhd3asi-0glTue)ans.yi,ncpIwtosetdhwIbeIiaacdsschystsamehtlresaaiomsollprbCiehsecRateehgnInaeSttsPalhoynRofdeSwRsptntrNlaaebtpsAo---
by This system is typified by the Cas9 signature (tracrRNA)isinvolvedintheprocessingofpre-
protein, a large multifunctional protein with crRNAintocrRNAinTypeIIsystemsthrough
theabilitytogeneratecrRNA,aswellastarget theformationofaduplexwiththeCRISPRre-
phageandplasmidDNAfordegradation(30). peat sequence (22). Mature crRNA, together
Cas9appearstocontaintwonucleasedomains, withCas9,interfereswithmatchinginvasiveds-
oneattheNterminus(RuvC-likenuclease)and DNAbyhomology-drivencleavagewithinthe
an HNH (McrA-like) nuclease domain in the protospacersequence,inthedirectvicinityof
middlesection(whichmightbeinvolvedintar- thePAM(30).Mismatchesatthe3(cid:2)endofthe tracrRNA: Trans-
getcleavagebasedonitsendonucleaseactivity) protospacer and/or in the PAM allow phages encodedsmallRNA
requiredforcrRNA
(Table1).TypeIIisthesimplestofthethree andplasmidstocircumventCRISPR-encoded
maturation
CRISPR-Castypes,withonlyfourgenes that immunity(24,30).
www.annualreviews.org • CRISPR-CasSystems 281
GE45CH13-Bhaya ARI 1October2011 14:42
Transcription
Pre-crRNA
Type I Type II Type III
CASCADE
Cas9 RNase III Cas6
crRNA
Cas6e/Cas6f
tracrRNA
y.
nl Csm/Cmr
o Cas3
e
s
u
orgnal
ews.erso
ww.annualrevi8/09/12. For p PAM Target DNA Target DNA Target DNA or RNA
m won 0
oy Figure4
Rev. Genet. 2011.45:273-297. Downloaded frversity - Main Campus - Lane Medical Librar M(pptftfowbacIpooyhnrnrrrahapRrrreeoosdseeniie-ccdNcuCc-igcf-elhgeabcopereasAlnlrRtraascstRyo8sv3irasNnipr-faiNsunasaunsegpTAbduncwAftepIsiogl,byIfeeei)eolrfwpuItxeqieambh-fcen)thurrByetiieatieIattnna,hcncR,onrhtsciehtgIathNidabIoeaiirenm,pnestianeAarr3nprcaDtptonoer(cid:2)lptdehiceduarcNneaoeeanportnhlIsvaAsTsrygIdrsaettfoImne.rneiydtcooguCiIhrpdeezfnnribcaelroeeRetecytadTonudIr(IsrasC,fvrRSeyeatteiItdPoqpanaNiIhtsias,euRhdfate6tAffahie-IeoeirsctneIsCrephi5lr,diselscRePne(cid:2)aoCnlfctsIeeAwNu3tshIianamlfiurI(cid:2)MeseyvAdtbc9edsehe,Pi.yn,tcnCdaebyfIsAsdholrhpntyaboa.MaeloetsllynIntmTwrlohncIgi.ihswnmys-oseySTomE.wpmesiumpsInedtiytbnroeptoepoIhrtbmRrlfdITeyeedyIbNdca.psxu,Iyrc.ytTe)Icpp(RahtI,CctiCsIrehneoNiaoeIoomeanIIsmTctnIA,Aspr.fierI6tyapaITsonhcficpn.cslnfaaaeeirieseAnnlnn-mmarsmdeIsnguspt(,taendsbubuaeotrncccilrtupblotrfurottygiRrtabiieocdrpepyesr)areNnuetepytRbacsidabeDuoInrAltlNuyIRallsnIdNIytnrm-wtAs-N,gFopiaBAitaCierfa,tArnl,tgChrealttaswtehoDcnqsR.ACtl9eoudhTecNNSDAfiearpiCrChsetvrSAAreNheaearsiAsCmatmeega(AieDCstqAnenrarucsiftedaaDEronouerispycfrRri6rbErebssefttR,tuaNceridarynabtalrbNrepelAfendugrtosoaenyeAsstpesnvrcpgctIot)awgreetIehingiohIdipeweI-stniIihtBsib,(Itos-hlactpeAeae)npn.
Annu. ord Uni tmdaeromgdeoitfinResdtNrfaArtoeadmn;doMpaaePknaAtrrMoivaandgeolteesaslnr.eo(7pt2rae)p.speFneialtlreadtcottivrbiieatynregthqleuastirrheeadpsrfneosorettnhyteentaubccetlieevnaitsyiedoaecfntTtiivfiyitepyde.tIhIaItshysatsebmese.nTehxipsefirigmureentiaslly
nf
a
St
y
b TypeIIICRISPR-CasSystem trimming.Theuniversalcas1andcas2genesare
mostly in operon-like structures with the rest
Thissystemhasanumberofrecognizablefea-
ofthecasgenesbutarenotalwaysinthesame
tures, including the signature RAMP protein,
operonastheRAMPproteinsintheTypeIII
Cas10,whichislikelyinvolvedintheprocess-
systems.Sofar,twotypeIIIsystemshavebeen
ingofcrRNAandpossiblyalsointargetDNA
distinguished(TypeIIIAandIIIB).InPyrococ-
cleavage(6),andissomewhatfunctionallyanal-
cus furiosus, a Type IIIA system, the target of
ogoustotheTypeICASCADE.TheTypeIII
CRISPR interference is mRNA (40), whereas
system also contains the signature Cas6, in-
in Staphylococcus epidermidis, a Type IIIB sys-
volved in crRNA processing and additional
tem, the target is DNA (75). This highlights
RAMPproteinslikelytobeinvolvedincrRNA
· ·
282 Bhaya Davison Barrangou