RESEARCH ARTICLE SUMMARY

CRISPR EVOLUTION

C2c2 is a single-componentprogrammable RNA-guidedRNA-targeting CRISPR effectorOmar O Abudayyeh Jonathan S Gootenberg Silvana Konermann Julia JoungIan M Slaymaker David B T Cox Sergey Shmakov Kira S MakarovaEkaterina Semenova Leonid Minakhin Konstantin Severinov Aviv RegevEric S Lander Eugene V Koonin Feng Zhang

INTRODUCTION Almost all archaea andabout half of bacteria possess clustered reg-ularly interspaced short palindromic repeat(CRISPR)ndashCRISPR-associated genes (Cas) adapt-ive immune systems which protect microbesagainst viruses and other foreignDNA All func-tionally characterized CRISPR systems havebeen reported to target DNAwith somemulti-component type III systems also targeting RNAThe putative class 2 type VI system which hasnot been functionally characterized encom-passes the single-effector protein C2c2 whichcontains two Higher Eukaryotes and Prokary-otes Nucleotide-binding (HEPN) domains com-monly associated with ribonucleases (RNases)suggestingRNA-guidedRNA-targeting function

RATIONALE Existing studies have only estab-lished a role for RNA interference in additionto DNA interference in the multicomponenttype III-A and III-B systems We investigatedthe possibility of C2c2-mediated RNA inferenceby heterologously expressing C2c2 locus fromLeptotrichia shahii (LshC2c2) in the modelsystem Escherichia coli The ability of LshC2c2

to protect against MS2 single-stranded RNA(ssRNA) phage infectionwas assessed by usingevery possible spacer sequence against thephage genome We next developed protocolsto reconstitute purified recombinant LshC2c2protein and test its biochemical activity whenincubatedwith itsmature CRISPRRNA (crRNA)and target ssRNA We systematically evaluatedthe parameters necessary for cleavage Last todemonstrate the potential utility of the LshC2c2complex for RNA targeting in living bacterialcells we guided it to knockdown red fluorescentprotein (RFP) mRNA in vivo

RESULTS This work demonstrates the RNA-guided RNase activity of the putative type VICRISPR-effector LshC2c2 Heterologously ex-pressed C2c2 can protect E coli from MS2phage and by screening against the MS2 ge-nome we identified a H (non-G) protospacerflanking site (PFS) following the RNA targetsite which was confirmed by targeting a com-plementary sequence in the b-lactamase tran-script followed by a degenerate nucleotidesequence Using purified LshC2c2 protein

we demonstrate that C2c2 and crRNA aresufficient in vitro to achieve RNA-guided PFS-dependent RNA cleavage This cleavage prefer-entially occurs at uracil residues in ssRNAregions and depends on conserved catalyticresidues in the two HEPN domains Mutation

of these residues yields acatalytically inactive RNA-binding protein The sec-ondary structure of thecrRNAdirect repeat (DR)stem is required forLshC2c2 activity andmu-

tations in the 3prime region of the DR eliminatecleavage activity Targeting is also sensitive tomultiple or consecutive mismatches in thespacerprotospacer duplex C2c2 targeting ofRFP mRNA in vivo results in reduced fluores-cence The knockdown of the RFP mRNA byC2c2 slowed E coli growth and in agreementwith this finding in vitro cleavage of the targetRNA results in ldquocollateralrdquo nonspecific cleav-age of other RNAs present in the reaction mix

CONCLUSION LshC2c2 is a RNA-guidedRNase which requires the activity of its twoHEPN domains suggesting previously un-identified mechanisms of RNA targeting anddegradation by CRISPR systems Promiscu-ous RNase activity of C2c2 after activationby the target slows bacterial growth and sug-gests that C2c2 could protect bacteria fromvirus spread via programmed cell death anddormancy induction A single-effector RNAtargeting system has the potential to serveas a general chassis for molecular tools forvisualizing degrading or binding RNA in aprogrammable multiplexed fashion

RESEARCH

SCIENCE sciencemagorg 5 AUGUST 2016 bull VOL 353 ISSUE 6299 557

The list of author affiliations is available in the full article onlineCorresponding author Email zhangbroadinstituteorg(FZ) kooninncbinlmnihgov (EVK)Cite this article as O O Abudayyeh et al Science 353aaf5573 (2016) DOI 101126scienceaaf5573

C2c2 is an RNA-guided RNase that provides protection against RNA phage CRISPR-C2c2 from L shahii can be reconstituted in E coli tomediate RNA-guided interference of the RNA phageMS2 Biochemical characterization of C2c2 reveals crRNA-guided RNA cleavage facilitated by thetwo HEPN nuclease domains Binding of the target RNA by C2c2-crRNA also activates a nonspecific RNase activity which may lead to promiscuouscleavage of RNAs without complementarity to the crRNA guide sequence

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RESEARCH ARTICLE

CRISPR EVOLUTION

C2c2 is a single-componentprogrammable RNA-guidedRNA-targeting CRISPR effectorOmar O Abudayyeh1234 Jonathan S Gootenberg2345 Silvana Konermann234Julia Joung234 Ian M Slaymaker234 David B T Cox12346 Sergey Shmakov78

Kira S Makarova8 Ekaterina Semenova9 Leonid Minakhin9 Konstantin Severinov7910

Aviv Regev26 Eric S Lander256 Eugene V Koonin8dagger Feng Zhang1234dagger

The clustered regularly interspaced short palindromic repeat (CRISPR)ndashCRISPR-associatedgenes (Cas) adaptive immune system defends microbes against foreign genetic elementsvia DNA or RNA-DNA interference We characterize the class 2 type VI CRISPR-Cas effectorC2c2 and demonstrate its RNA-guided ribonuclease function C2c2 from the bacteriumLeptotrichia shahii provides interference against RNA phage In vitro biochemical analysis showsthat C2c2 is guided by a single CRISPR RNA and can be programmed to cleave single-strandedRNA targets carrying complementary protospacers In bacteria C2c2 can be programmed toknock down specific mRNAs Cleavage is mediated by catalytic residues in the two conservedHigher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains mutations of whichgenerate catalytically inactive RNA-binding proteinsThese results broaden our understanding ofCRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools

Almost all archaea and about half of bacteriapossess clustered regularly interspaced shortpalindromic repeats (CRISPRs) and CRISPR-associated genes (CRISPR-Cas)ndashadaptive im-mune systems (1 2) which protectmicrobes

from viruses and other invading DNA throughthree steps (i) adaptationmdashinsertion of foreignnucleic acid segments (spacers) into the CRISPRarray in between pairs of direct repeats (DRs)(ii) transcription and processing of the CRISPRarray to producemature CRISPRRNAs (crRNAs)and (iii) interference by which Cas enzymes areguided by the crRNAs to target and cleave cog-nate sequences in the respective invader genomes(3ndash5) All CRISPR-Cas systems characterized todate follow these three steps although themech-anistic implementation and proteins involved inthese processes display extensive diversity

The CRISPR-Cas systems are broadly dividedinto two classes on the basis of the architectureof the interference module Class 1 systems relyonmultisubunit protein complexes whereas class2 systems use single-effector proteins (1) Withinthese two classes types and subtypes are delineatedaccording to the presence of distinct signaturegenes protein sequence conservation and orga-nization of the respective genomic loci Class1 systems include type I in which interferenceis achieved through assembly of multiple Cas pro-teins into the Cascade complex and type III sys-tems which rely on either the Csm (type III-AD)or Cmr (Type III-BC) effector complexeswhich aredistantly related to the Cascade complex (1 6ndash11)Class 2 CRISPR systems comprise type II sys-

tems characterized by the single-component ef-fector protein Cas9 (12ndash17) which contains RuvCandHNHnuclease domains and type V systemswhich use single RuvC domainndashcontaining ef-fectors such as Cpf1 (18) C2c1 and C2c3 (19)All functionally characterized systems to datehave been reported to target DNA and only themulticomponent type III-A and III-B systemsadditionally target RNA (7 20ndash25) However theputative class 2 type VI system is characterizedby the presence of the single-effector proteinC2c2 which lacks homology to any known DNAnuclease domain but contains two Higher Eu-karyotes and Prokaryotes Nucleotide-binding(HEPN) domains (19) Given that all functionallycharacterized HEPN domains are ribonucleases(RNases) (26) there is a possibility that C2c2 func-tions solely as an RNA-guided RNA-targetingCRISPR effector

HEPN domains are also found in other Casproteins Csm6 a component of type III-A sys-tems and the homologous protein Csx1 in typeIII-B systems each contain a single HEPN domainand have been biochemically characterized assingle-stranded RNA (ssRNA)ndashspecific endoribo-nucleases (endoRNases) (21 27 28) In additiontype III systems contain complexes of other Casenzymes that bind and cleave ssRNA throughacidic residues associated with RNA-recognitionmotif (RRM) domains These complexes (Cas10-Csm in type III-A andCmr in type III-B) carry outRNA-guided cotranscriptional cleavage of mRNAin concert with DNA target cleavage (22 29 30)In contrast the roles of Csm6 and Csx1 whichcleave their targets with little specificity areless clear although in some cases RNA cleavageby Csm6 apparently serves as a second line ofdefense when DNA-targeting fails (21) Addition-ally Csm6 and Csx1 have to dimerize to form acomposite active site (27 28 31) but C2c2 con-tains two HEPN domains which suggests that itfunctions as a monomeric endoRNaseAs is common with class 2 systems type VI

systems are simply organized In particular thetype VI locus in Leptotrichia shahii contains Cas1Cas2 C2c2 and a CRISPR array which is ex-pressed and processed into mature crRNAs (19)In all CRISPR-Cas systems characterized to dateCas1 and Cas2 are exclusively involved in spaceracquisition (32ndash37) which suggests that C2c2 isthe sole effector protein that uses a crRNA guideto achieve interference likely targeting RNA

Reconstitution of the L shahii C2c2locus in Escherichia coli confersRNA-guided immunity

We explored whether LshC2c2 could confer im-munity to MS2 (25) a lytic ssRNA phage withoutDNA intermediates in its life cycle that infects Ecoli We constructed a low-copy plasmid carryingthe entire LshC2c2 locus (pLshC2c2) so as to al-low for heterologous reconstitution inE coli (figS1A) Because expressed mature crRNAs fromthe LshC2c2 locus have amaximum spacer lengthof 28 nucleotides (nt) (fig S1A) (19) we tiled allpossible 28-nt target sites in the MS2 phagegenome (Fig 1A) This resulted in a library of3473 spacer sequences (along with 588 non-targeting guides designed to have a Levenshteindistance of ge8with respect to theMS2 and E coligenomes) which we inserted between pLshC2c2DRs After transformation in of this constructinto E coli we infected cells with varying dilu-tions of MS2 (10minus1 10minus3 and 10minus5) and analyzedsurviving cells to determine the spacer sequencescarried by cells that survived the infection Cellscarrying spacers that confer robust interferenceagainstMS2 are expected to proliferate faster thanthose that lack such sequences After growth for16 hours we identified a number of spacers thatwere consistently enriched across three inde-pendent infection replicas in both the 10minus1 and10minus3 dilution conditions suggesting that theyenabled interference against MS2 Specifically152 and 144 spacers showed gt08 log2-fold en-richment in all three replicates for the 10minus1 and

RESEARCH

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1Department of Health Sciences and Technology MassachusettsInstitute of Technology Cambridge MA 02139 USA 2BroadInstitute of MIT and Harvard Cambridge MA 02142 USA3McGovern Institute for Brain Research at MIT Cambridge MA02139 USA 4Departments of Brain and Cognitive Science andBiological Engineering Massachusetts Institute of TechnologyCambridge MA 02139 USA 5Department of Systems BiologyHarvard Medical School Boston MA 02115 USA 6Departmentof Biology Massachusetts Institute of Technology CambridgeMA 02139 USA 7Skolkovo Institute of Science and TechnologySkolkovo 143025 Russia 8National Center for BiotechnologyInformation National Library of Medicine National Institutes ofHealth Bethesda MD 20894 USA 9Waksman Institute forMicrobiology Rutgers The State University of New JerseyPiscataway NJ 08854 USA 10Institute of Molecular GeneticsRussian Academy of Sciences Moscow 123182 RussiaThese authors contributed equally to this work daggerCorrespondingauthor Email zhangbroadinstituteorg (FZ) kooninncbinlmnihgov (EVK)

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10minus3 phage dilutions respectively of these twogroups of top enriched spacers 75 are shared(Fig 1B fig S2 A to G and table S1) Addition-ally no nontargeting guides were found to beconsistently enriched among the three 10minus1 10minus3or 10minus5 phage replicates (fig S2 D and G) Wealso analyzed the flanking regions of proto-spacers on the MS2 genome corresponding tothe enriched spacers and found that spacers witha G immediately flanking the 3prime end of theprotospacer were less fit relative to all othernucleotides at this position (A U or C) sug-gesting that the 3prime protospacer-flanking site (PFS)affects the efficacy of C2c2-mediated targeting(Fig 1C and figs S2 E and F and S3) Althoughthe PFS is adjacent to the protospacer target wechose not to use the commonly used protospacer-adjacent motif (PAM) nomenclature because ithas come to connote a sequence used in self ver-sus nonself differentiation (38) which is irre-levant in a RNA-targeting system It is worthnoting that the avoidance of G by C2c2 echoesthe absence of PAMs observed for other RNA-targeting CRISPR systems and effector proteins(20 22 24 25 39 40)That only ~5 of crRNAs are enriched may

reflect other factors influencing interferenceactivity such as accessibility of the target sitethat might be affected by RNA-binding proteinsor secondary structure In agreement with thishypothesis the enriched spacers tend to clusterinto regions of strong interference where theyare closer to each other than onewould expect byrandom chance (fig S3 F and G)To validate the interference activity of the en-

riched spacers we individually cloned four top-enriched spacers into pLshC2c2 CRISPR arraysand observed a 3- to 4-log10 reduction in plaqueformation which is consistent with the level ofenrichment observed in the screen (Fig 1B andfig S4) We cloned 16 guides targeting distinctregions of the MS2 mat gene (four guides perpossible single-nucleotide PFS) All 16 crRNAsmediated MS2 interference although higherlevels of resistance were observed for the C Aand U PFS-targeting guides (Fig 1 D and E andfig S5) indicating that C2c2 can be effectivelyretargeted in a crRNA-dependent fashion to siteswithin the MS2 genomeTo further validate the observed PFS prefer-

ence with an alternate approach we designed aprotospacer site in the pUC19 plasmid at the 5primeend of the b-lactamase mRNA which encodesampicillin resistance in E coli flanked by fiverandomized nucleotides at the 3prime end Significantdepletion and enrichment was observed for theLshC2c2 locus (P lt 00001) as comparedwith the pACYC184 controls (fig S6A) Analysisof the depleted PFS sequences confirmed thepresence of a PFS preference of H (fig S6B)

C2c2 is a single-effector endoRNasemediating ssRNA cleavage with a singlecrRNA guide

We purified the LshC2c2 protein (fig S7) andassayed its ability to cleave an in vitrondashtranscribed173-nt ssRNA target (Fig 2A and fig S8) containing

aaf5573-2 5 AUGUST 2016 bull VOL 353 ISSUE 6299 sciencemagorg SCIENCE

Fig 1 Heterologous expression of the L shahii C2c2 locus mediates robust interference of RNAphage in E coli (A) Schematic for the MS2 bacteriophage interference screen A library consisting ofspacers targeting all possible sequences in the MS2 RNA genome was cloned into the LshC2c2 CRISPRarray Cells transformed with the MS2-targeting spacer library were then treated with phage and platedand surviving cells were harvestedThe frequency of spacers was compared with an untreated control(no phage) and enriched spacers from the phage-treated condition were used for the generation ofPFS preference logos (B) Box plot showing the distribution of normalized crRNA frequencies for thephage-treated conditions and control screen (no phage) biological replicates (n = 3)The box extendsfrom the first to third quartile with whiskers denoting 15 times the interquartile range The mean isindicated by the red horizontal bar The 10minus1 and 10minus3 phage dilution distributions are significantlydifferent than each of the control replicates [P lt 00001 bymeans of analysis of variance (ANOVA)with multiple hypothesis correction] (C) Sequence logo generated from sequences flanking the 3prime endof protospacers corresponding to enriched spacers in the 10minus3 phage dilution condition revealing thepresence of a 3prime H PFS (not G) (D) Plaque assay used to validate the functional importance of the HPFS in MS2 interference All protospacers flanked by non-G PFSs exhibited robust phage interferenceSpacer were designed to target the MS2 mat gene and their sequences are shown above the plaqueimages the spacer used in the nontargeting control is not complementary to any sequence in eitherthe E coli or MS2 genome Phage spots were applied as series of half-log dilutions (E) Quantitation ofMS2 plaque assay validating the H (non-G) PFS preference Four MS2-targeting spacers were de-signed for each PFS Each point on the scatter plot represents the average of three biological replicatesand corresponds to a single spacer Bars indicate the mean of four spacers for each PFS and standarderror (SEM)

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a C PFS (ssRNA target 1 with protospacer 14)Mature LshC2c2 crRNAs contain a 28-nt DR anda 28-nt spacer (fig S1A) (19) We therefore gen-erated an in vitrondashtranscribed crRNAwith a 28-ntspacer complementary to protospacer 14 on ssRNAtarget 1 LshC2c2 efficiently cleaved ssRNA in aMg2+- and crRNA-dependent manner (Fig 2Band fig S9) We then annealed complementaryRNA oligos to regions flanking the crRNA targetsite This partially double-stranded RNA (dsRNA)substrate was not cleaved by LshC2c2 which sug-gests that it is specific for ssRNA (fig S10 A and B)We tested the sequence constraints of RNA

cleavage by LshC2c2 with additional crRNAscomplementary to ssRNA target 1 in which proto-spacer 14 is preceded by each PFS variant Theresults of this experiment confirmed the prefer-ence for C A and U PFSs with little cleavageactivity detected for the G PFS target (Fig 2C)Additionally we designed five crRNAs for eachpossible PFS (20 total) across the ssRNA target1 and evaluated cleavage activity for LshC2c2pairedwith each of these crRNAs As expected weobserved less cleavage activity forGPFSndashtargetingcrRNAs as compared with other crRNAs tested(Fig 2D)We then generated a dsDNA plasmid library

with protospacer 14 flanked by seven randomnucleotides so as to account for any PFS prefer-ence When incubated with LshC2c2 protein anda crRNA complementary to protospacer 14 nocleavage of the dsDNA plasmid library was ob-served (fig S10C) We also did not observe cleav-age when targeting a ssDNA version of ssRNAtarget 1 (fig S10D) To rule out cotranscriptionalDNA cleavage which has been observed in typeIII CRISPR-Cas systems (22) we recapitulatedthe E coli RNA polymerase cotranscriptionalcleavage assay (fig S11A) (22) expressing ssRNAtarget 1 from a DNA substrate This assay in-volving purified LshC2c2 and crRNA targetingssRNA target 1 did not show any DNA cleavage(fig S11B) Together these results indicate thatC2c2 cleaves specific ssRNA sites directed by thetarget complementarity encoded in the crRNAwith a H PFS preference

C2c2 cleavage depends on local targetsequence and secondary structure

Given that C2c2 did not efficiently cleave dsRNAsubstrates and that ssRNA can form complexsecondary structures we reasoned that cleavageby C2c2might be affected by secondary structureof the ssRNA target Indeed after tiling ssRNAtarget 1 with different crRNAs (Fig 2D) we ob-served the same cleavage pattern regardless ofthe crRNA position along the target RNA Thisobservation suggests that the crRNA-dependentcleavage pattern was determined by features ofthe target sequence rather than the distancefrom the binding site We hypothesized that theLshC2c2-crRNA complex binds the target andcleaves exposed regions of ssRNA within the sec-ondary structure elements with potential pref-erence for certain nucleotidesIn agreement with this hypothesis cleavage

of three ssRNA targets with different sequences

SCIENCE sciencemagorg 5 AUGUST 2016 bull VOL 353 ISSUE 6299 aaf5573-3

U

ndash

ssRNA 1

ssRNA 1

protospacer 14

protospacer 141 41 72

5 ndash ndash 3176

5 ndash

LshC2c2

crRNA

EDTA

+ + + ndash + + + ndash

+ + ndash + + + ndash +

ndash + ndash ndash ndash + ndash ndash

5 -end label

5rsquo-end label

3 -end label

3rsquo-end label

ndash 3ndash 3

5 ndash ndash 3

C G A U C G A

crRNA + + + + ndash ndash ndash

5 ndash [X]

crRNA

CG

32

15 17 21 29

18 26

28 26 25 20 33 31 27 17 18 34 30 24 22 19 15 29 16 21 23

A U

C A U5 ndash ndash 3 G

16

25

24

23

32

27

20 28

22

19

30

31

33

34

5 ndash ndash 3

PFS

uncleaved

cleaved

uncleaved

cleaved

IR800 Cy5 IR800

IR800

ndash

103nt

136nt

103nt86nt73nt

90nt

73nt

40nt

103nt90nt

73nt

40nt

uncleaved

cleaved

103nt90nt

73nt

40nt

PFS

PFS

PFS

Fig 2 LshC2c2 and crRNA mediate RNA-guided ssRNA cleavage (A) Schematic of the ssRNA sub-strate being targeted by the crRNAThe protospacer region is highlighted in blue and the PFS is indicatedby the magenta bar (B) A denaturing gel demonstrating crRNA-mediated ssRNA cleavage by LshC2c2after 1 hour of incubation The ssRNA target is either 5prime labeled with IRDye 800 or 3prime labeled with Cy5Cleavage requires the presence of the crRNA and is abolished by addition of EDTA Four cleavage sites areobserved Reported band lengths arematched fromRNA sequencing (C) A denaturing gel demonstratingthe requirement for an H PFS (not G) after 3 hours of incubation Four ssRNA substrates that are identicalexcept for the PFS (indicated by the magenta ldquoXrdquo in the schematic) were used for the in vitro cleavagereactions ssRNA cleavage activity is dependent on the nucleotide immediately 3prime of the target site Re-ported band lengths are matched from RNA sequencing (D) Schematic showing five protospacers foreach PFS on the ssRNA target (top) Denaturing gel showing crRNA-guided ssRNA cleavage activity after1 hour of incubation crRNAs correspond to protospacer numbering Reported band lengths are matchedfrom RNA sequencing

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flanking identical 28-nt protospacers resulted inthree distinct patterns of cleavage (Fig 3A) RNA-sequencing of the cleavage products for the threetargets revealed that cleavage sites mainly local-ized to uracil-rich regions of ssRNA or ssRNA-dsRNA junctions within the in silicondashpredictedcofolds of the target sequence with the crRNA(Fig 3 B and C and fig S12 A to D) To testwhether the LshC2c2-crRNA complex preferscleavage at uracils we analyzed the cleavage ef-ficiencies of homopolymeric RNA targets (a 28-ntprotospacer extendedwith 120 As or Us regularlyinterspaced by single bases of G or C to enableoligo synthesis) and found that LshC2c2 prefer-entially cleaved the uracil target compared withadenine (fig S12 E and F) We then tested cleav-age of a modified version of ssRNA 4 that had itsmain site of cleavage a loop replaced with eachof the four possible homopolymers and foundthat cleavage only occurred at the uracil homo-polymer loop (fig S12G) To further test whether

cleavage was occurring at uracil residues wemutated single-uracil residues in ssRNA 1 thatshowed cleavage in the RNA-sequencing (Fig 3B)to adenines This experiment showed that bymutating each uracil residue we could modulatethe presence of a single cleavage band which isconsistent with LshC2c2 cleaving at uracil resi-dues in ssRNA regions (Fig 3D)

The HEPN domains of C2c2 mediateRNA-guided ssRNA-cleavage

Bioinformatic analysis of C2c2 has suggestedthat the HEPN domains are likely to be respon-sible for the observed catalytic activity (19) Eachof the two HEPN domains of C2c2 contains adyad of conserved arginine and histidine resi-dues (Fig 4A) which is in agreement with thecatalytic mechanism of the HEPN endoRNAse(26ndash28) We mutated each of these putative cat-alytic residues separately to alanine (R597AH602A R1278A H1283A) in the LshC2c2 locus

plasmids and assayed for MS2 interference Noneof the four mutant plasmids were able to protectE coli from phage infection (Fig 4B and fig S13)(Single-letter abbreviations for the amino acidresidues are as follows A Ala C Cys D Asp EGlu F Phe G Gly H His I Ile K Lys L LeuMMet N Asn P Pro Q Gln R Arg S Ser T ThrV Val W Trp and Y Tyr In the mutants otheramino acids were substituted at certain locationsfor example R597A indicates that arginine at po-sition 597 was replaced by alanine)We purified the four single-point mutant pro-

teins and assayed their ability to cleave 5prime endndashlabeled ssRNA target 1 (Fig 4C) In agreementwith our in vivo results all four mutations abol-ished cleavage activity The inability of either ofthe two wild-type HEPN domains to compensatefor inactivation of the other implies cooperationbetween the twodomains These results agreewithobservations that several bacterial and eukaryoticsingle-HEPNproteins function as dimers (27 28 41)

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Fig 3 C2c2 cleavage sites aredetermined by secondary structureand sequence of the target RNA(A) Denaturing gel showing C2c2-crRNAndashmediated cleavage after 3 hoursof incubation of three nonhomopoly-meric ssRNA targets (1 4 5 black blueand green respectively in Fig 3 B andC and fig S12 A to D) that share thesame protospacer but are flanked bydifferent sequences Despite identicalprotospacers different flanking se-quences resulted in different cleavagepatterns Reported band lengths arematched fromRNA sequencing (B) Thecleavage sites of nonhomopolymerssRNA target 1 were mapped with RNA-sequencing of the cleavage productsThe frequency of cleavage at each baseis colored according to the z-score andshown on the predicted crRNA-ssRNAcofold secondary structure Fragmentsused to generate the frequency analysiscontained the complete 5prime endThe 5primeand 3prime end of the ssRNA target areindicated by blue and red outlines onthe ssRNA and secondary structurerespectivelyThe 5prime and 3prime end of thespacer (outlined in yellow) is indicatedby the blue and orange residues high-lighted respectivelyThe crRNA nucleo-tides are highlighted in orange (C) Plotof the frequencies of cleavage sites foreach position of ssRNA target 1 for allreads that begin at the 5prime endTheprotospacer is indicated by the bluehighlighted region (D) Schematic of amodified ssRNA 1 target showing sites(red) of single U-to-A flips (left)Denaturing gel showing C2c2-crRNAndashmediated cleavage of each of these singlenucleotide variants after 3 hours of incu-bation (right) Reported band lengthsare matched from RNA sequencing

position on ssRNA 1 (nt)

fold

cha

nge

in c

leav

age

even

t

ssRNA 1

0 20 40 60 80 100 120 140 160

5

0

15

10

201 47 74 176

ssRNA 1

crRNA

ssRNA

crRNA+ ndash + ndash + ndash

1 4 4 5 5

+ ndash + ndash + ndash

1 1 4 4 5 5

modified ssRNA 1

crRNA

G G G G G C C A G UG

A A UUC

GAGCUCGGUACCC

G G G GAU

CCUCUAGAAAUA U G G A U U A C U U G G U A G A A C A G C A A U C U A

CUCGACCUG

CA

GGCA

U GC

AAG

CU

UG

GCGU

A A UCA

UGG

UC

AUA

GC

UG

UU

UCC

UG

U

G U UUAU C C G C U CA

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CCACA

CA

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CGAGCCGGAAGC

AUAAA

GUAGAUUGCUGUUCUACCAAGUAAUCCAU

1

10

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160170

U to A position

crRNA +

ndash

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40 73 90 103

ndash

ndash

ndash ndash ndash ndash

40 73 90103

100 z-score

G G G G G C C A G UG

A A UUC

GAGCUCGGUACCC

G G G GAU

CCUCUAGAAAUA U G G A U U A C U U G G U A G A A C A G C A A U C U A

CUCGACCUG

CA

GGCA

U GC

AAG

CU

UG

GCGU

A A UCA

UGG

UC

AUA

GC

UG

UU

UCC

UG

U

G U UUAU C C G C U CA

CA A U U C

CACA

CAACAU

AC

GAGCCGGAAGCA

UAAAG

UAGAUUGCUGUUCUACCAAGUAAUCCAU

1

10

20

U40U73

U90

U103

30

40

50 60 70

80

90

100

110

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130 140

160170crRNA

U40

U90

U103

U73

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cleaved

103nt90nt

73nt

40nt

uncleaved

cleaved

103nt 121nt90nt73nt

40nt

55nt

uncleaved

cleaved

136nt103nt86nt73nt

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Catalytically inactive variants of Cas9 retaintarget DNA binding allowing for the creationof programmable DNA-binding proteins (12 13)Electrophoretic mobility shift assays (EMSAs) onboth the wild-type (Fig 4D) and R1278A mutantLshC2c2 (Fig 4E) in complex with crRNA showedthe wild-type LshC2c2 complex binding strongly[dissociation constant (Kd) ~ 46 nM] (fig S14A)and specifically to 5prime endndashlabeled ssRNA target10 but not to the 5prime endndashlabeled nontarget ssRNA(the reverse complement of ssRNA target 10)The R1278A mutant C2c2 complex showed evenstronger (Kd ~ 7 nM) (fig S14B) specific bindingindicating that this HEPN mutation results in acatalytically inactive RNA-programmable RNA-binding protein The LshC2c2 protein or crRNAalone showed reduced levels of target affinity asexpected (fig S14 C to E) Additionally no spe-cific binding of LshC2c2-crRNA complex to ssDNAwas observed (fig S15)These results demonstrate that C2c2 cleaves

RNA via a catalytic mechanism distinct fromother known CRISPR-associated RNases In par-ticular the type III Csm and Cmr multiproteincomplexes rely on acidic residues of RRM do-mains for catalysis whereas C2c2 achieves RNAcleavage through the conserved basic residues ofits two HEPN domains

Sequence and structural requirementsof C2c2 crRNA

Similar to the type V-A (Cpf1) systems (18) theLshC2c2 crRNA contains a single stem loop inthe DR suggesting that the secondary struc-ture of the crRNA could facilitate interactionwith LshC2c2 We thus investigated the lengthrequirements of the spacer sequence for ssRNAcleavage and found that LshC2c2 requires spacersof at least 22 nt length to efficiently cleave ssRNAtarget 1 (fig S16A) The stem-loop structure ofthe crRNA is also critical for ssRNA cleavagebecause DR truncations that disturbed the stemloop abrogated target cleavage (fig S16B) Thusa DR longer than 24 nt is required to maintainthe stem loop necessary for LshC2c2 to mediatessRNA cleavageSingle-base-pair inversions in the stem that

preserved the stem structure did not affect theactivity of the LshC2c2 complex In contrastinverting all four G-C pairs in the stem elimi-nated the cleavage despite maintaining the dup-lex structure (fig S17A) Other perturbations suchas those that introduced kinks and reduced orincreased base-pairing in the stem also elim-inated or drastically suppressed cleavage Thissuggests that the crRNA stem length is impor-tant for complex formation and activity (fig S17A)We also found that loop deletions eliminatedcleavage whereas insertions and substitutionsmostlymaintained some level of cleavage activity(fig S17B) In contrast nearly all substitutions ordeletions in the region 3prime to the DR preventedcleavage by LshC2c2 (fig S18) Together theseresults demonstrate that LshC2c2 recognizesstructural characteristics of its cognate crRNAbut is amenable to loop insertions andmost testedbase substitutions outside of the 3prime DR region

SCIENCE sciencemagorg 5 AUGUST 2016 bull VOL 353 ISSUE 6299 aaf5573-5

R597A

H602A

R1278

A

H1283

A

wtLsh

C2c2

pACYCY18

4100

101

102

103

fold

res

ista

nce

LshC2c2 Cas1 Cas2Leptotrichia shahii

C2c2 locus(1389 aa)

mixed alphabeta mixed alphabeta

HEPN HEPN

R597 H602 R1278 H1283

catalytic residues

C2c2(R1278A)-crRNA + ssRNA 10 ssRNA C2c2(R1278A)-crRNA + ssRNA 10(rc) ssRNAC2c2-crRNA(01pM-10microM)

C2c2-crRNA(01pM-10microM)

protospacer 35MS2 mat

C2c2-crRNA(02pM-2microM) C2c2-crRNA

(02pM-2microM)

C2c2(WT)-crRNA + ssRNA 10 ssRNA C2c2(WT)-crRNA + ssRNA 10(rc) ssRNA

bound

unbound

bound

unbound

bound

unbound

bound

unbound

PFS

C2c2(WT)

C2c2(R1278A)

5 ndash ndash 3

LshC2c2 ndash R597AWT H602A

R1278A

H1283A

uncleaved

cleaved

IR800ssRNA 1 5rsquo-end label

1 2 3 4 5 6

CRISPR array

KD = 46nM

KD = 7nM

spacerdirect repeat (DR)

103nt90nt

73nt

40nt

Fig 4 The two HEPN domains of C2c2 are necessary for crRNA-guided ssRNA cleavage but notfor binding (A) Schematic of the LshC2c2 locus and the domain organization of the LshC2c2 proteinshowing conserved residues in HEPN domains (dark blue) (B) Quantification of MS2 plaque assaywith HEPN catalytic residue mutants For each mutant the same crRNA targeting protospacer 35 wasused (n = 3 biological replicates P lt 00001 compared with pACYC184 by Studentrsquos t test Barsrepresent mean plusmn SEM) (C) Denaturing gel showing conserved residues of the HEPN motif indicatedas catalytic residues in (A) are necessary for crRNA-guided ssRNA target 1 cleavage after 3 hours ofincubation Reported band lengths are matched from RNA sequencing (D) Electrophoretic mobility shiftassay (EMSA) evaluating affinity of the wild-type LshC2c2-crRNA complex against a targeted (left) and anontargeted (right) ssRNA substrate The nontargeted ssRNA substrate is the reverse-complement ofthe targeted ssRNA 10 EDTA is supplemented to reaction condition in order to reduce any cleavageactivity (E) Electrophoretic mobility shift assay with LshC2c2(R1278A)-crRNA complex against on-targetssRNA 10 and nontargeting ssRNA [same substrate sequences as in (D)]

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These results have implications for the futureapplication of C2c2-based tools that require guideengineering for recruitment of effectors or mod-ulation of activity (42ndash44)

C2c2 cleavage is sensitive to doublemismatches in the crRNA-target duplex

We tested the sensitivity of the LshC2c2 systemto single mismatches between the crRNA guideand target RNA by mutating single bases acrossthe spacer to the respective complementary bases(for example A to U) We then quantified plaqueformation with these mismatched spacers in theMS2 infection assay and found that C2c2was fullytolerant to single mismatches across the spacerbecause suchmismatched spacers interfered withphage propagation with similar efficiency as fullymatched spacers (figs S19A and S20) However

when we introduced consecutive double substi-tutions in the spacer we found a ~3-log10ndashfoldreduction in the protection for mismatches inthe center but not at the 5prime or 3prime end of the crRNA(figs S19B and S20) This observation suggests thepresence of a mismatch-sensitive ldquoseed regionrdquo inthe center of the crRNA-target duplexWe generated a set of in vitrondashtranscribed

crRNAs with mismatches similarly positionedacross the spacer region When incubated withLshC2c2 protein all single mismatched crRNAsupported cleavage (fig S19C) which is in agree-ment with our in vivo findingsWhen testedwitha set of consecutive and nonconsecutive double-mutant crRNAs LshC2c2 was unable to cleavethe target RNA if the mismatches were posi-tioned in the center but not at the 5prime or 3prime end ofthe crRNA (figs S19D and S21A) further sup-

porting the existence of a central seed regionAdditionally no cleavage activity was observedwith crRNAs containing consecutive triple mis-matches in the seed region (fig S21B)

C2c2 can be reprogrammed to mediatespecific mRNA knockdown in vivo

Given the ability of C2c2 to cleave target ssRNAin a crRNA sequencendashspecific manner we testedwhether LshC2c2 could be reprogrammed to de-grade selected nonphage ssRNA targets and par-ticularly mRNAs in vivo We cotransformed Ecoli with a plasmid encoding LshC2c2 and acrRNA targeting the mRNA of red fluorescentprotein (RFP) as well as a compatible plasmidexpressing RFP (Fig 5A) For optical density(OD)ndashmatched samples we observed an ~20 to92 decrease in RFP-positive cells for crRNAs

aaf5573-6 5 AUGUST 2016 bull VOL 353 ISSUE 6299 sciencemagorg SCIENCE

Fig 5 RFP mRNA knockdown byretargeting LshC2c2 (A) Schematicshowing crRNA-guided knockdownof RFP in E coli heterologouslyexpressing the LshC2c2 locus ThreeRFP-targeting spacers were selectedfor each non-G PFS and eachprotospacer on the RFP mRNA isnumbered (B) RFP mRNA-targetingspacers effected RFP knockdownwhereas DNA-targeting spacers(targeting the noncoding strand ofthe RFP gene on the expressionplasmid indicated as ldquorcrdquo spacers) didnot affect RFP expression (n = 3biological replicates P lt 00001compared with nontargeting guide bymeans of ANOVA with multiplehypothesis correction Bars representmean plusmn SEM) (C) Quantification ofRFP knockdown in E coli Threespacers each targeting C U orA PFS-flanking protospacers [ninespacers numbered 5 to 13 as indi-cated in (A)] in the RFP mRNA wereintroduced and RFP expression wasmeasured with flow cytometry Eachpoint on the scatter plot representsthe average of three biological repli-cates and corresponds to a singlespacer Bars indicate the mean ofthree spacers for each PFS anderrors bars are shown as the SEM(D) Timeline of E coli growth assay(E) Effect of RFP mRNA targeting onthe growth rate of E coli transformedwith an inducible RFP expressionplasmid as well as the LshC2c2 locuswith nontargeting RNA targeting(spacer complementary to the RFPmRNA or RFP gene coding strand)and pACYC control plasmid at differ-ent anhydrotetracycline (aTc)concentrations

0

20

40

60

80

100

perc

entR

FP+

cel

ls

PFS NA NA TU GC AAPFSRFP mRNA

C2c2locus

plasmid

RFP

RFPplasmid

C2c2 degraded mRNA

C2c2

crRNA analysis by flow

cytometry

protospacers79 138

RFP mRNA

RFP mRNA

0

20

40

60

80

100

perc

entR

FP+

cel

ls

RFP mRNA11

1213

C AU

target ndash + + + ++ + +

protospacer ndash NT 7(rc) 8(rc) 9(rc)7 8 9

RNA-targeting

non-targeting crRNA pACYC184

DNA-targeting

construct

OD

600

minutes post-induction minutes post-induction minutes post-induction

RFP RNA targeting (7)

aTC concentration(ngmL)0

5 x 1025 x 10155 x 1015 x 10055 x 1005 x 10-055 x 10-1

01

02

03

04

05

06

00

01

02

03

04

05

06

00

01

02

03

04

05

06

00100 200 400 500 600 700300 100 200 400 500 600 700300 100 200 400 500 600 700300

seed culture

16 hrs 1 hr 12 hrs

dilute to OD 01induce

expressionmeasure OD600

in shaking reader

C U APFS

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targeting protospacers flanked by C A or U PFSs(Fig 5 B and C) As a control we tested crRNAscontaining reverse complements (targeting thedsDNA plasmid) of the top performing RFPmRNA-targeting spacers As expected we ob-served no decrease in RFP fluorescence by thesecrRNAs (Fig 5B) We also confirmed that mu-tation of the catalytic arginine residues in eitherHEPN domain to alanine precluded RFP knock-down (fig S22) Thus C2c2 is capable of gen-eral retargeting to arbitrary ssRNA substratesgoverned exclusively by predictable nucleic-acidinteractionsWhen we examined the growth of cells carry-

ing the RFP-targeting spacer with the greatestlevel of RFP knockdown we noted that the growthrate of these bacteria was substantially reduced(Fig 5A spacer 7) We investigated whether theeffect on growthwasmediated by theRFPmRNAndashtargeting activity of LshC2c2 by introducingan inducible-RFP plasmid and an RFP-targetingLshC2c2 locus into E coli Upon induction ofRFP transcription cells with RFP knockdownshowed substantial growth suppression not ob-served in nontargeting controls (Fig 5 D andE) This growth restriction was dependent onthe level of the RFP mRNA as controlled by theconcentration of the inducer anhydrotetracyclineIn contrast in the absence of RFP transcriptionwe did not observe any growth restriction nor didwe observe any transcription-dependent DNA-targeting in our biochemical experiment (figS11) These results indicate that RNA-targetingis likely the primary driver of this growth re-striction phenotype We therefore surmised thatin addition to the cleavage of the target RNA

C2c2 CRISPR systems might prevent virus re-production also via nonspecific cleavage of cel-lular mRNAs causing programmed cell death(PCD) or dormancy (45 46)

C2c2 cleaves collateral RNA in additionto crRNA-targeted ssRNA

Cas9 and Cpf1 cleave DNA within the crRNA-target heteroduplex at defined positions re-verting to an inactive state after cleavage Incontrast C2c2 cleaves the target RNA outsideof the crRNA binding site at varying distancesdepending on the flanking sequence presum-ably within exposed ssRNA loop regions (Fig3 B and C and fig S12 A to D) This observedflexibility with respect to the cleavage distanceled us to test whether cleavage of other non-target ssRNAs also occurs upon C2c2 target-binding and activation Under this model theC2c2-crRNA complex once activated by bindingto its target RNA cleaves the target RNA as wellas other RNAs nonspecifically We carried outin vitro cleavage reactions that included in ad-dition to LshC2c2 protein crRNA and its targetRNA one of four unrelated RNA molecules with-out any complementarity to the crRNA guide(Fig 6A) These experiments showed that where-as the LshC2c2-crRNA complex did not mediatecleavage of any of the four collateral RNAs in theabsence of the target RNA all four were effi-ciently degraded in the presence of the targetRNA (Fig 6B and fig S23A) Furthermore R597Aand R1278A HEPNmutants were unable to cleavecollateral RNA (fig S23B)To further investigate the collateral cleavage

and growth restriction in vivo we hypothesized

that if a PFS preference screen for LshC2c2 wasperformed in a transcribed region on the trans-formed plasmid thenwe should be able to detectthe PFS preference due to growth restrictioninduced by RNA-targeting We designed a pro-tospacer site flanked by five randomized nucleo-tides at the 3prime end in either a nontranscribedregion or in a region transcribed from the lacpromoter (fig S24A) The analysis of the depletedand enriched PFS sequences identified a H PFSonly in the assay with the transcribed sequencebut no discernable motif in the nontranscribedsequence (fig S24 B and C)These results suggest aHEPN-dependentmech-

anism bywhich C2c2 in a complex with crRNA isactivated upon binding to target RNA and sub-sequently cleaves nonspecifically other availablessRNA targets Such promiscuous RNA cleavagecould cause cellular toxicity resulting in the ob-served growth rate inhibition These findingsimply that in addition to their likely role in directsuppression of RNA viruses type VI CRISPR-Cassystems could function as mediators of a distinctvariety of PCD or dormancy induction that isspecifically triggered by cognate invader genomes(Fig 7) Under this scenario dormancy wouldslow the infection and supply additional time foradaptive immunity Such a mechanism agreeswith the previously proposed coupling of adapt-ive immunity and PCD during the CRISPR-Casdefensive response (47)

Conclusions

The class 2 type VI effector protein C2c2 is aRNA-guided RNase that can be efficiently pro-grammed to degrade any ssRNA by specifyinga 28-nt sequence on the crRNA (fig S10) C2c2cleaves RNA through conserved basic residueswithin its two HEPN domains in contrast tothe catalytic mechanisms of other known RNasesfound in CRISPR-Cas systems (25 48) Alaninesubstitution of any of the four predicted HEPNdomain catalytic residues converted C2c2 intoan inactive programmable RNA-binding protein(dC2c2 analogous to dCas9) Many differentspacer sequences work well in our assays al-though further screening will likely define prop-erties and rules governing optimal functionThese results suggest a broad range of bio-

technology applications and research questions(49ndash51) For example the ability of dC2c2 to bindto specified sequences could be used to (i) bringeffector modules to specific transcripts in orderto modulate their function or translation whichcould be used for large-scale screening construc-tion of synthetic regulatory circuits and otherpurposes (ii) fluorescently tag specific RNAs inorder to visualize their trafficking andor lo-calization (iii) alter RNA localization throughdomains with affinity for specific subcellularcompartments and (iv) capture specific tran-scripts (through direct pull-down of dC2c2) inorder to enrich for proximal molecular part-ners including RNAs and proteinsActive C2c2 also has many potential appli-

cations such as targeting a specific transcriptfor destruction as performed here with RFP

SCIENCE sciencemagorg 5 AUGUST 2016 bull VOL 353 ISSUE 6299 aaf5573-7

A B

collateralRNA

6 7 8 9 6 7 8 9 6 7 8 9

2targetedssRNA

2 2 2 3 3 3 3 ndash ndash ndash ndash

+LshC2c2crRNA

+ + + + + + + + + + +

targetedssRNA

collateral RNA

+ C2c2

cleavage of targetedssRNA and collateral RNA

protospacer

Cy5uncleaved

cleaved

Fig 6 crRNA-guided ssRNA cleavage activates nonspecific RNase activity of LshC2c2 (A) Sche-matic of the biochemical assay used to detect crRNA-bindingndashactivated nonspecific RNase activity onnon-crRNAndashtargeted collateral RNA molecules The reaction consists of C2c2 protein unlabeledcrRNA unlabeled target ssRNA and a second ssRNAwith 3prime fluorescent labeling and is incubated for3 hours C2c2-crRNA mediates cleavage of the unlabeled target ssRNA as well as the 3prime endndashlabeledcollateral RNA which has no complementarity to the crRNA (B) Denaturing gel showing nonspecificRNase activity against nontargeted ssRNA substrates in the presence of target RNA after 3 hours ofincubation The nontargeted ssRNA substrate is not cleaved in the absence of the crRNA-targetedssRNA substrate

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In addition C2c2 once primed by the cognatetarget can cleave other (noncomplementary)RNA molecules in vitro and inhibit cell growthin vivo Biologically this promiscuous RNaseactivity might reflect a PCDdormancyndashbasedprotection mechanism of the type VI CRISPR-Cas systems (Fig 7) Technologically it mightbe used to trigger PCD or dormancy in specificcells such as cancer cells expressing a particulartranscript neurons of a given class or cellsinfected by a specific pathogen

Further experimental study is required toelucidate the mechanisms by which the C2c2system acquires spacers and the classes of path-ogens against which it protects bacteria Thepresence of the conserved CRISPR adaptationmodule consisting of typical Cas1 and Cas2 pro-teins in the LshC2c2 locus suggests that it iscapable of spacer acquisition Although C2c2systems lack reverse transcriptases which medi-ate acquisition of RNA spacers in some type IIIsystems (52) it is possible that additional host or

viral factors could support RNA spacer acquisi-tion Additionally or alternatively type VI sys-tems could acquire DNA spacers similar to otherCRISPR-Cas variants but then target transcriptsof the respective DNA genomes eliciting PCDand abortive infection (Fig 7)The CRISPR-C2c2 system represent a distinct

evolutionary path among class 2 CRISPR-Cassystems It is likely that other broadly analogousclass 2 RNA-targeting immune systems exist andfurther characterization of the diverse membersof class 2 systems will provide a deeper under-standing of bacterial immunity and provide arich starting point for the development of pro-grammable molecular tools for in vivo RNAmanipulation

Materials and methods

Expanded materials and methods includingcomputational analysis can be found in sup-plementary materials and methods

Bacterial phage interference

The C2c2 CRISPR locus was amplified fromDNAfrom Leptotrichia shahii DSM 19757 (ATCCManassas VA) and cloned for heterologous ex-pression in E coli For screens a library of allpossible spacers targeting the MS2 genomewere cloned into the spacer array for individ-ual spacers single specific spacers were clonedinto the array Interference screens were per-formed in liquid culture and plated survivingcolonies were harvested for DNA and spacerrepresentationwas determined by next-generationsequencing Individual spacers were tested byspotting on top agar

bndashlactamase and transcribednon-transcribed PFS preference screens

Sequences with randomized nucleotides adja-cent to protospacer 1 were cloned into pUC19 incorresponding regions Libraries were screenedby co-transformation with LshC2c2 locus plas-mid or pACYC184 plasmid control harvesting ofthe surviving colonies and next-generation se-quencing of the resulting regions

RFP-targeting assay

Cells containing anRFP expressing plasmidweretransformedwith an LshC2c2 locus plasmidwithcorresponding spacers grown overnight andanalyzed for RFP fluorescence by flow cytom-etry The growth effects of LshC2c2 activity werequantified by titrating inducible RFP levels withdilutions of anhydrotetracycline inducer andthen measuring OD600

in vitro nuclease and electrophoreticmobility shift assays

LshC2c2 protein and HEPN mutants were puri-fied for use in in vitro reactions RNA were syn-thesized via in vitro transcription For nucleaseassays protein was co-incubated with crRNA andeither 3prime or 5prime-labeled targets and analyzed viadenaturing gel electrophoresis and imaging orby next-generation sequencing For electropho-retic mobility shift assays protein and nucleic

aaf5573-8 5 AUGUST 2016 bull VOL 353 ISSUE 6299 sciencemagorg SCIENCE

C2c2 Cas1 Cas2generalizedtype VI locus 1 2 3 4

CRISPR array

PFS

C2c2

crRNA processing

mature crRNA

spacer

DR

phage phage RNA

host mRNA

binding of target ssRNA activatesoff-target cleavage of host RNAs

collateral effectabortive infectioncleavage of ssRNA

cleavage of host RNAs inducescell deathdormancy

Fig 7 C2c2 as a putative RNA-targeting prokaryotic immune system The C2c2-crRNA complexrecognizes target RNA via base pairing with the cognate protospacer and cleaves the target RNA Inaddition binding of the target RNA by C2c2-crRNA activates a nonspecific RNase activity which maylead to promiscuous cleavage of RNAs without complementarity to the crRNA guide sequence Throughthis nonspecific RNase activity C2c2 may also cause abortive infection via programmed cell death ordormancy induction

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acid were co-incubated and then resolved by gelelectrophoresis and imaging

REFERENCES AND NOTES

1 K S Makarova et al An updated evolutionary classification ofCRISPR-Cas systems Nat Rev Microbiol 13 722ndash736 (2015)doi 101038nrmicro3569 pmid 26411297

2 K S Makarova et al Evolution and classification of theCRISPR-Cas systems Nat Rev Microbiol 9 467ndash477 (2011)doi 101038nrmicro2577 pmid 21552286

3 A V Wright J K Nuntildeez J A Doudna Biology andapplications of CRISPR systems Harnessing naturersquos toolboxfor genome engineering Cell 164 29ndash44 (2016) doi 101016jcell201512035 pmid 26771484

4 L A Marraffini CRISPR-Cas immunity in prokaryotes Nature526 55ndash61 (2015) doi 101038nature15386 pmid 26432244

5 J van der Oost M M Jore E R Westra M LundgrenS J Brouns CRISPR-based adaptive and heritable immunity inprokaryotes Trends Biochem Sci 34 401ndash407 (2009)doi 101016jtibs200905002 pmid 19646880

6 S J Brouns et al Small CRISPR RNAs guide antiviral defensein prokaryotes Science 321 960ndash964 (2008) doi 101126science1159689 pmid 18703739

7 C R Hale et al RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex Cell 139 945ndash956 (2009) doi 101016jcell200907040 pmid 19945378

8 R N Jackson et al Crystal structure of the CRISPR RNA-guided surveillance complex from Escherichia coli Science345 1473ndash1479 (2014) doi 101126science1256328pmid 25103409

9 L A Marraffini E J Sontheimer CRISPR interference limitshorizontal gene transfer in staphylococci by targeting DNAScience 322 1843ndash1845 (2008) doi 101126science1165771pmid 19095942

10 S Mulepati A Heacuteroux S Bailey Crystal structure of a CRISPRRNA-guided surveillance complex bound to a ssDNA targetScience 345 1479ndash1484 (2014) doi 101126science1256996pmid 25123481

11 T Sinkunas et al In vitro reconstitution of Cascade-mediatedCRISPR immunity in Streptococcus thermophilusEMBO J 32 385ndash394 (2013) doi 101038emboj2012352pmid 23334296

12 G Gasiunas R Barrangou P Horvath V Siksnys Cas9-crRNAribonucleoprotein complex mediates specific DNA cleavagefor adaptive immunity in bacteria Proc Natl Acad Sci USA109 E2579ndashE2586 (2012) doi 101073pnas1208507109pmid 22949671

13 M Jinek et al A programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunity Science 337816ndash821 (2012) doi 101126science1225829 pmid 22745249

14 E Deltcheva et al CRISPR RNA maturation by trans-encodedsmall RNA and host factor RNase III Nature 471 602ndash607(2011) doi 101038nature09886 pmid 21455174

15 R Sapranauskas et al The Streptococcus thermophilusCRISPRCas system provides immunity in Escherichia coliNucleic Acids Res 39 9275ndash9282 (2011) doi 101093nargkr606 pmid 21813460

16 J E Garneau et al The CRISPRCas bacterial immune systemcleaves bacteriophage and plasmid DNA Nature 468 67ndash71(2010) doi 101038nature09523 pmid 21048762

17 R Barrangou et al CRISPR provides acquired resistanceagainst viruses in prokaryotes Science 315 1709ndash1712 (2007)doi 101126science1138140 pmid 17379808

18 B Zetsche et al Cpf1 is a single RNA-guided endonucleaseof a class 2 CRISPR-Cas system Cell 163 759ndash771 (2015)doi 101016jcell201509038 pmid 26422227

19 S Shmakov et al Discovery and functional characterization ofdiverse class 2 CRISPR-Cas systems Mol Cell 60 385ndash397(2015) doi 101016jmolcel201510008 pmid 26593719

20 C R Hale et al Essential features and rational design ofCRISPR RNAs that function with the Cas RAMP modulecomplex to cleave RNAs Mol Cell 45 292ndash302 (2012)doi 101016jmolcel201110023 pmid 22227116

21 W Jiang P Samai L A Marraffini Degradation of phagetranscripts by CRISPR-associated RNases enables type IIICRISPR-Cas immunity Cell 164 710ndash721 (2016) doi 101016jcell201512053 pmid 26853474

22 P Samai et al Co-transcriptional DNA and RNA cleavageduring type III CRISPR-Cas immunity Cell 161 1164ndash1174(2015) doi 101016jcell201504027 pmid 25959775

23 R H Staals et al Structure and activity of the RNA-targetingtype III-B CRISPR-Cas complex of Thermus thermophilus MolCell 52 135ndash145 (2013) doi 101016jmolcel201309013pmid 24119403

24 R H Staals et al RNA targeting by the type III-A CRISPR-CasCsm complex of Thermus thermophilus Mol Cell 56 518ndash530(2014) doi 101016jmolcel201410005 pmid 25457165

25 G Tamulaitis et al Programmable RNA shredding by the typeIII-A CRISPR-Cas system of Streptococcus thermophilus MolCell 56 506ndash517 (2014) doi 101016jmolcel201409027pmid 25458845

26 V Anantharaman K S Makarova A M BurroughsE V Koonin L Aravind Comprehensive analysis of the HEPNsuperfamily Identification of novel roles in intra-genomic conflictsdefense pathogenesis and RNA processing Biol Direct 8 15(2013) doi 1011861745-6150-8-15 pmid 23768067

27 O Niewoehner M Jinek Structural basis for theendoribonuclease activity of the type III-A CRISPR-associatedprotein Csm6 RNA 22 318ndash329 (2016) doi 101261rna054098115 pmid 26763118

28 N F Sheppard C V Glover 3rd R M Terns M P Terns TheCRISPR-associated Csx1 protein of Pyrococcus furiosus is anadenosine-specific endoribonuclease RNA 22 216ndash224(2016) doi 101261rna039842113 pmid 26647461

29 G W Goldberg W Jiang D Bikard L A MarraffiniConditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting Nature 514 633ndash637(2014) doi 101038nature13637 pmid 25174707

30 L Deng R A Garrett S A Shah X Peng Q She A novelinterference mechanism by a type IIIB CRISPR-Cmr module inSulfolobus Mol Microbiol 87 1088ndash1099 (2013) doi 101111mmi12152 pmid 23320564

31 Y K Kim Y G Kim B H Oh Crystal structure and nucleicacid-binding activity of the CRISPR-associated protein Csx1of Pyrococcus furiosus Proteins 81 261ndash270 (2013)doi 101002prot24183 pmid 22987782

32 J K Nuntildeez A S Lee A Engelman J A Doudna Integrase-mediated spacer acquisition during CRISPR-Cas adaptiveimmunity Nature 519 193ndash198 (2015) doi 101038nature14237 pmid 25707795

33 R Heler et al Cas9 specifies functional viral targets duringCRISPR-Cas adaptation Nature 519 199ndash202 (2015)doi 101038nature14245 pmid 25707807

34 J K Nuntildeez et al Cas1-Cas2 complex formation mediatesspacer acquisition during CRISPR-Cas adaptive immunity NatStruct Mol Biol 21 528ndash534 (2014) doi 101038nsmb2820pmid 24793649

35 C Diacuteez-Villasentildeor N M Guzmaacuten C AlmendrosJ Garciacutea-Martiacutenez F J Mojica CRISPR-spacer integrationreporter plasmids reveal distinct genuine acquisitionspecificities among CRISPR-Cas I-E variants of Escherichia coliRNA Biol 10 792ndash802 (2013) doi 104161rna24023pmid 23445770

36 I Yosef M G Goren U Qimron Proteins and DNA elementsessential for the CRISPR adaptation process in Escherichia coliNucleic Acids Res 40 5569ndash5576 (2012) doi 101093nargks216 pmid 22402487

37 K A Datsenko et al Molecular memory of prior infectionsactivates the CRISPRCas adaptive bacterial immunity systemNat Commun 3 945 (2012) doi 101038ncomms1937pmid 22781758

38 L A Marraffini E J Sontheimer Self versus non-self discriminationduring CRISPR RNA-directed immunity Nature 463 568ndash571(2010) doi 101038nature08703 pmid 20072129

39 C R Hale A Cocozaki H Li R M Terns M P Terns TargetRNA capture and cleavage by the Cmr type III-B CRISPR-Caseffector complex Genes Dev 28 2432ndash2443 (2014)doi 101101gad250712114 pmid 25367038

40 J Zhang et al Structure and mechanism of the CMR complexfor CRISPR-mediated antiviral immunity Mol Cell 45 303ndash313(2012) doi 101016jmolcel201112013 pmid 22227115

41 G Kozlov et al Structural basis of defects in the sacsin HEPNdomain responsible for autosomal recessive spastic ataxia ofCharlevoix-Saguenay (ARSACS) J Biol Chem 286 20407ndash20412(2011) doi 101074jbcM111232884 pmid 21507954

42 S Kiani et al Cas9 gRNA engineering for genome editingactivation and repression Nat Methods 12 1051ndash1054 (2015)doi 101038nmeth3580 pmid 26344044

43 S Konermann et al Genome-scale transcriptional activationby an engineered CRISPR-Cas9 complex Nature 517 583ndash588(2015) doi 101038nature14136 pmid 25494202

44 J E Dahlman et al Orthogonal gene knockout and activationwith a catalytically active Cas9 nuclease Nat Biotechnol 331159ndash1161 (2015) doi 101038nbt3390 pmid 26436575

45 K S Makarova Y I Wolf E V Koonin Comprehensivecomparative-genomic analysis of type 2 toxin-antitoxinsystems and related mobile stress response systems inprokaryotes Biol Direct 4 19 (2009) doi 1011861745-6150-4-19 pmid 19493340

46 F Hayes L Van Melderen Toxins-antitoxins Diversityevolution and function Crit Rev Biochem Mol Biol 46386ndash408 (2011) doi 103109104092382011600437pmid 21819231

47 K S Makarova V Anantharaman L Aravind E V KooninLive virus-free or die Coupling of antivirus immunityand programmed suicide or dormancy in prokaryotesBiol Direct 7 40 (2012) doi 1011861745-6150-7-40pmid 23151069

48 C Benda et al Structural model of a CRISPR RNA-silencingcomplex reveals the RNA-target cleavage activity in Cmr4 MolCell 56 43ndash54 (2014) doi 101016jmolcel201409002pmid 25280103

49 Z Abil H Zhao Engineering reprogrammable RNA-bindingproteins for study and manipulation of the transcriptome MolBiosyst 11 2658ndash2665 (2015) doi 101039C5MB00289Cpmid 26166256

50 J P Mackay J Font D J Segal The prospects fordesigner single-stranded RNA-binding proteins Nat StructMol Biol 18 256ndash261 (2011) doi 101038nsmb2005pmid 21358629

ACKNOWLEDGMENTS

We thank P Boutz J Doench P Sharp and B Zetsche forhelpful discussions and insights R Belliveau for overallresearch support J Francis and D OrsquoConnell for generousMiSeq instrument access D Daniels and C Garvie for providingbacterial incubation space for protein purification R Macraefor critical reading of the manuscript and the entire Zhanglaboratory for support and advice We thank N Ranu forgenerously providing plasmid pRFP and D Daniels for providing6-His-MBP-TEV OOA is supported by a Paul and Daisy SorosFellowship a Friends of the McGovern Institute Fellowshipand the Poitras Center for Affective Disorders JSG is supportedby a US Department of Energy (DOE) Computational ScienceGraduate Fellowship SS is supported by the graduate programof Skoltech Data-Intensive Biomedicine and Biotechnology Centerfor Research Education and Innovation IMS is supported by theSimons Center for the Social Brain DBTC is supported by awardT32GM007753 from the National Institute of General MedicalSciences KSM EVK and in part SS are supported by theintramural program of the US Department of Health andHuman Services (to the National Library of Medicine) KS issupported by NIH grant GM10407 Russian Science Foundationgrant 14-14-00988 and Skoltech FZ is a New York Stem CellFoundation Robertson Investigator FZ is supported by the NIHthrough the National Institute of Mental Health (5DP1-MH100706and 1R01-MH110049) NSF the New York Stem Cell SimonsPaul G Allen Family and Vallee Foundations and J PoitrasP Poitras R Metcalfe and D Cheng AR is a member of thescientific advisory board for Syros Pharmaceuticals and ThermoFisher OOA JSG JJ EVK SK ESL KSM LMES KS SS and FZ are inventors on provisionalpatent application 62181675 applied for by the Broad InstituteMIT Harvard NIH Skoltech and Rutgers that covers the C2c2proteins described in this paper Deep sequencing data areavailable at Sequence Read Archive under BioProject accessionno PRJNA318890 The authors plan to make the reagents widelyavailable to the academic community through Addgene subjectto a materials transfer agreement and to provide software toolsvia the Zhang laboratory website (wwwgenome-engineeringorg)and GitHub (httpsgithubcomfengzhanglab)

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2 March 2016 accepted 25 May 2016Published online 2 June 2016101126scienceaaf5573

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C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector

Eugene V Koonin and Feng ZhangShmakov Kira S Makarova Ekaterina Semenova Leonid Minakhin Konstantin Severinov Aviv Regev Eric S Lander Omar O Abudayyeh Jonathan S Gootenberg Silvana Konermann Julia Joung Ian M Slaymaker David B T Cox Sergey

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