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A Ruler Protein in a Complex for Antiviral Defense Determines the Length of Small Interfering CRISPR RNAs

  • Asma Hatoum-Aslan
    Affiliations
    From the Laboratory of Bacteriology, The Rockefeller University, New York, New York 10065
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  • Poulami Samai
    Footnotes
    Affiliations
    From the Laboratory of Bacteriology, The Rockefeller University, New York, New York 10065
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  • Inbal Maniv
    Affiliations
    From the Laboratory of Bacteriology, The Rockefeller University, New York, New York 10065
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  • Wenyan Jiang
    Affiliations
    From the Laboratory of Bacteriology, The Rockefeller University, New York, New York 10065
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  • Luciano A. Marraffini
    Correspondence
    Supported by the Searle Scholars Program, the Rita Allen Scholars Program, an Irma T. Hirschl Award, and National Institutes of Health Director's New Innovator Award 1DP2AI104556-01. To whom correspondence should be addressed. Laboratory of Bacteriology, The Rockefeller University, 1230 York Ave., New York, NY 10065. Tel.: 212-327-7014; Fax: 212-327-8262;.
    Affiliations
    From the Laboratory of Bacteriology, The Rockefeller University, New York, New York 10065
    Search for articles by this author
  • Author Footnotes
    This article contains a supplemental table.
    1 Supported by a Helmsley Postdoctoral Fellowship for Basic and Translational Research on Disorders of the Digestive System at The Rockefeller University.
Open AccessPublished:August 09, 2013DOI:https://doi.org/10.1074/jbc.M113.499244
      Small RNAs undergo maturation events that precisely determine the length and structure required for their function. CRISPRs (clustered regularly interspaced short palindromic repeats) encode small RNAs (crRNAs) that together with CRISPR-associated (cas) genes constitute a sequence-specific prokaryotic immune system for anti-viral and anti-plasmid defense. crRNAs are subject to multiple processing events during their biogenesis, and little is known about the mechanism of the final maturation step. We show that in the Staphylococcus epidermidis type III CRISPR-Cas system, mature crRNAs are measured in a Cas10·Csm ribonucleoprotein complex to yield discrete lengths that differ by 6-nucleotide increments. We looked for mutants that impact this crRNA size pattern and found that an alanine substitution of a conserved aspartate residue of Csm3 eliminates the 6-nucleotide increments in the length of crRNAs. In vitro, recombinant Csm3 binds RNA molecules at multiple sites, producing gel-shift patterns that suggest that each protein binds 6 nucleotides of substrate. In vivo, changes in the levels of Csm3 modulate the crRNA size distribution without disrupting the 6-nucleotide periodicity. Our data support a model in which multiple Csm3 molecules within the Cas10·Csm complex bind the crRNA with a 6-nucleotide periodicity to function as a ruler that measures the extent of crRNA maturation.
      Background: CRISPR immune systems protect prokaryotes from their viruses using small interfering RNAs (crRNAs), which require maturation events during their biogenesis.
      Results: In Staphylococcus epidermidis, crRNAs undergo maturation in a Cas10·Csm ribonucleoprotein complex; Csm3 modulates the extent of maturation.
      Conclusion: Csm3 acts as a ruler for crRNAs.
      Significance: Investigating CRISPR immunity is important to understand prokaryotic ecology and to develop biotechnological applications.

      Introduction

      CRISPR sequences (clustered regularly interspaced short palindromic repeats) are an essential component of a prokaryotic immune system that protects against phage infection and other invading genetic elements (
      • Barrangou R.
      • Fremaux C.
      • Deveau H.
      • Richards M.
      • Boyaval P.
      • Moineau S.
      • Romero D.A.
      • Horvath P.
      CRISPR provides acquired resistance against viruses in prokaryotes.
      ,
      • Brouns S.J.
      • Jore M.M.
      • Lundgren M.
      • Westra E.R.
      • Slijkhuis R.J.
      • Snijders A.P.
      • Dickman M.J.
      • Makarova K.S.
      • Koonin E.V.
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      Small CRISPR RNAs guide antiviral defense in prokaryotes.
      ,
      • Marraffini L.A.
      • Sontheimer E.J.
      CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.
      ). CRISPR loci harbor an archive of short sequences (known as spacers) derived from past invaders that provide the specificity for CRISPR immunity. These sequences encode small CRISPR RNAs (crRNAs)
      The abbreviations used are: crRNA
      CRISPR RNA
      Cas
      CRISPR-associated
      nt
      nucleotide(s)
      NEB
      New England Biolabs.
      that together with CRISPR-associated (Cas) proteins, can locate and destroy foreign nucleic acids by an antisense targeting mechanism (
      • Garneau J.E.
      • Dupuis M.È.
      • Villion M.
      • Romero D.A.
      • Barrangou R.
      • Boyaval P.
      • Fremaux C.
      • Horvath P.
      • Magadán A.H.
      • Moineau S.
      The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.
      ,
      • Hale C.R.
      • Zhao P.
      • Olson S.
      • Duff M.O.
      • Graveley B.R.
      • Wells L.
      • Terns R.M.
      • Terns M.P.
      RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex.
      ,
      • Jinek M.
      • Chylinski K.
      • Fonfara I.
      • Hauer M.
      • Doudna J.A.
      • Charpentier E.
      A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
      ,
      • Westra E.R.
      • van Erp P.B.
      • Künne T.
      • Wong S.P.
      • Staals R.H.
      • Seegers C.L.
      • Bollen S.
      • Jore M.M.
      • Semenova E.
      • Severinov K.
      • de Vos W.M.
      • Dame R.T.
      • de Vries R.
      • Brouns S.J.
      • van der Oost J.
      CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3.
      ). The CRISPR immune system can also build a memory of past infections by incorporating new invader-derived sequences into CRISPR loci (
      • Barrangou R.
      • Fremaux C.
      • Deveau H.
      • Richards M.
      • Boyaval P.
      • Moineau S.
      • Romero D.A.
      • Horvath P.
      CRISPR provides acquired resistance against viruses in prokaryotes.
      ,
      • Cady K.C.
      • Bondy-Denomy J.
      • Heussler G.E.
      • Davidson A.R.
      • O'Toole G.A.
      The CRISPR/Cas adaptive immune system of Pseudomonas aeruginosa mediates resistance to naturally occurring and engineered phages.
      ). Found in >40% of bacteria and nearly all archaea (
      • Godde J.S.
      • Bickerton A.
      The repetitive DNA elements called CRISPRs and their associated genes: evidence of horizontal transfer among prokaryotes.
      ,
      • Grissa I.
      • Vergnaud G.
      • Pourcel C.
      The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats.
      ,
      • Haft D.H.
      • Selengut J.
      • Mongodin E.F.
      • Nelson K.E.
      A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes.
      ,
      • Makarova K.S.
      • Grishin N.V.
      • Shabalina S.A.
      • Wolf Y.I.
      • Koonin E.V.
      A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action.
      ), CRISPR-Cas systems exhibit remarkable mechanistic and functional diversity. They can be classified in three main types (I–III) that are defined based upon cas gene content and differences in the mechanism of immunity (
      • Makarova K.S.
      • Haft D.H.
      • Barrangou R.
      • Brouns S.J.
      • Charpentier E.
      • Horvath P.
      • Moineau S.
      • Mojica F.J.
      • Wolf Y.I.
      • Yakunin A.F.
      • van der Oost J.
      • Koonin E.V.
      Evolution and classification of the CRISPR-Cas systems.
      ).
      crRNA biogenesis is the essential first step in the CRISPR immunity pathway. Spacers range from 24–48 nucleotides in length and are interrupted by similarly sized repeat sequences (see Fig. 1A). This repeat-spacer array is transcribed as a long precursor that is subsequently processed to liberate mature crRNAs. In all CRISPR-Cas systems, the first step in crRNA biogenesis, known as primary processing, entails endoribonucleolytic cleavage within repeats. In type I and III systems, Cas6 is considered the primary processing endonuclease (
      • Brouns S.J.
      • Jore M.M.
      • Lundgren M.
      • Westra E.R.
      • Slijkhuis R.J.
      • Snijders A.P.
      • Dickman M.J.
      • Makarova K.S.
      • Koonin E.V.
      • van der Oost J.
      Small CRISPR RNAs guide antiviral defense in prokaryotes.
      ,
      • Makarova K.S.
      • Haft D.H.
      • Barrangou R.
      • Brouns S.J.
      • Charpentier E.
      • Horvath P.
      • Moineau S.
      • Mojica F.J.
      • Wolf Y.I.
      • Yakunin A.F.
      • van der Oost J.
      • Koonin E.V.
      Evolution and classification of the CRISPR-Cas systems.
      ,
      • Carte J.
      • Wang R.
      • Li H.
      • Terns R.M.
      • Terns M.P.
      Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes.
      ,
      • Haurwitz R.E.
      • Jinek M.
      • Wiedenheft B.
      • Zhou K.
      • Doudna J.A.
      Sequence- and structure-specific RNA processing by a CRISPR endonuclease.
      ). One exception appears to be the type I-C system in Bacillus halodurans, where Cas5d was recently shown to catalyze the cleavage of repeat sequences (
      • Nam K.H.
      • Haitjema C.
      • Liu X.
      • Ding F.
      • Wang H.
      • DeLisa M.P.
      • Ke A.
      Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in subtype I-C/Dvulg CRISPR-Cas system.
      ). In contrast, primary processing in type II systems relies upon an antisense trans-encoded crRNA and the host RNase III to cleave within repeats (
      • Deltcheva E.
      • Chylinski K.
      • Sharma C.M.
      • Gonzales K.
      • Chao Y.
      • Pirzada Z.A.
      • Eckert M.R.
      • Vogel J.
      • Charpentier E.
      CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.
      ). Primary processing generates crRNA intermediates that consist of a single spacer that is flanked on both ends by partial repeats. Whereas no further processing is known to occur in type I CRISPR-Cas systems, in type II and III systems, the crRNA intermediates are subject to a final maturation step that eliminates repeat and spacer sequences at the 5′ or 3′ end of the intermediate, respectively (
      • Deltcheva E.
      • Chylinski K.
      • Sharma C.M.
      • Gonzales K.
      • Chao Y.
      • Pirzada Z.A.
      • Eckert M.R.
      • Vogel J.
      • Charpentier E.
      CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.
      ,
      • Hale C.
      • Kleppe K.
      • Terns R.M.
      • Terns M.P.
      Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus.
      ,
      • Hatoum-Aslan A.
      • Maniv I.
      • Marraffini L.A.
      Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site.
      ).
      Figure thumbnail gr1
      FIGURE 1Mature crRNAs are measured in a Cas10·Csm complex. A, organization of the type III-A CRISPR system in S. epidermidis RP62A. This system contains 9 CRISPR-associated (cas and csm) genes, 4 direct repeats (black boxes), and 3 spacers (colored boxes), the first of which targets the nickase gene in staphylococcal conjugative plasmids. B, a ruler mechanism determines the length of mature crRNAs. Transcription of the repeat-spacer array generates a precursor crRNA that is subject to two cleavage events: primary processing within repeats to yield ∼71-nt intermediates (filled triangles), and maturation through trimming of the 3′ end of the intermediate (empty triangles). A ruler mechanism anchored at the primary processing site determines the extension of maturation to generate 37- and 43-nt-long mature crRNA. C, the type III-A Cas10·Csm complex. His6 tags were placed on the indicated (N or C) terminus of each of the genes involved in crRNA biogenesis. Constructs were expressed in S. epidermidis LM1680, and whole cell lysates were subject to Ni2+ affinity chromatography. Complexes were resolved by SDS-PAGE and visualized using coomassie G-250 staining. Red asterisks indicate each of the tagged species. D, each tagged Cas10·Csm subunit was visualized by Western blot of cell extracts using Ni2+-HRP. E, RNA was extracted from each of the His6- Cas10·Csm complexes, radiolabeled at the 5′ end, and resolved using denaturing PAGE.
      Type III CRISPR-Cas systems have been classified into two subtypes: III-A, containing the csm module of cas genes, and III-B, harboring the cmr module (
      • Makarova K.S.
      • Haft D.H.
      • Barrangou R.
      • Brouns S.J.
      • Charpentier E.
      • Horvath P.
      • Moineau S.
      • Mojica F.J.
      • Wolf Y.I.
      • Yakunin A.F.
      • van der Oost J.
      • Koonin E.V.
      Evolution and classification of the CRISPR-Cas systems.
      ). In both subtypes, mature crRNAs display an invariant 5′ end containing 8 nt of repeat sequence (the crRNA tag) but variable 3′ ends that match the targeted sequence in the phage or plasmid genome (
      • Carte J.
      • Wang R.
      • Li H.
      • Terns R.M.
      • Terns M.P.
      Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes.
      ,
      • Hatoum-Aslan A.
      • Maniv I.
      • Marraffini L.A.
      Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site.
      ). Primary processing cleaves the repeat sequence immediately upstream of the crRNA tag and maturation occurs at the 3′-end of the intermediate crRNA. Importantly, the extent of maturation at the 3′-end determines the cleavage site within the target sequence (
      • Hale C.R.
      • Zhao P.
      • Olson S.
      • Duff M.O.
      • Graveley B.R.
      • Wells L.
      • Terns R.M.
      • Terns M.P.
      RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex.
      ), and its mechanism remains poorly understood.
      The type III-A system of Staphylococcus epidermidis RP62a contains nine cas/csm genes (see Fig. 1A). Previously, we showed that a ruler mechanism anchored at the primary processing site generates mature crRNAs of discrete lengths (37 and 43 nucleotides, see Fig. 1B) and that csm2, csm3, and csm5 are required for crRNA maturation (
      • Hatoum-Aslan A.
      • Maniv I.
      • Marraffini L.A.
      Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site.
      ). Here, we show that Csm2, Csm3, and Csm5, along with Csm4 and the type III signature protein Cas10, are part of a ribonucleoprotein complex analogous to the Cascade (CRISPR-associated complex for antiviral defense) complex described for Escherichia coli and other organisms (
      • Brouns S.J.
      • Jore M.M.
      • Lundgren M.
      • Westra E.R.
      • Slijkhuis R.J.
      • Snijders A.P.
      • Dickman M.J.
      • Makarova K.S.
      • Koonin E.V.
      • van der Oost J.
      Small CRISPR RNAs guide antiviral defense in prokaryotes.
      ,
      • Hale C.R.
      • Zhao P.
      • Olson S.
      • Duff M.O.
      • Graveley B.R.
      • Wells L.
      • Terns R.M.
      • Terns M.P.
      RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex.
      ,
      • Nam K.H.
      • Haitjema C.
      • Liu X.
      • Ding F.
      • Wang H.
      • DeLisa M.P.
      • Ke A.
      Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in subtype I-C/Dvulg CRISPR-Cas system.
      ,
      • Wiedenheft B.
      • van Duijn E.
      • Bultema J.B.
      • Waghmare S.P.
      • Zhou K.
      • Barendregt A.
      • Westphal W.
      • Heck A.J.
      • Boekema E.J.
      • Dickman M.J.
      • Doudna J.A.
      RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions.
      ,
      • Zhang J.
      • Rouillon C.
      • Kerou M.
      • Reeks J.
      • Brugger K.
      • Graham S.
      • Reimann J.
      • Cannone G.
      • Liu H.
      • Albers S.V.
      • Naismith J.H.
      • Spagnolo L.
      • White M.F.
      Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity.
      ). This complex, here named Cas10·Csm, is enriched with mature crRNAs that range from 31 to 67 nucleotides, measured precisely in 6-nucleotide increments. We show that Csm3 is essential for the formation of this complex, and demonstrate that mutating conserved residues or changing overall levels of Csm3 in vivo alters the size distribution of the crRNAs without disrupting their 6-nucleotide periodicity. Furthermore, recombinant Csm3 binds RNA in vitro in a sequence-independent manner, producing gel-shift patterns that suggest that each protein binds 6 nucleotides of substrate. Our observations support a model in which multiple Csm3 molecules bind the crRNA with a 6-nucleotide periodicity to function as a ruler that measures the extent of crRNA maturation within the S. epidermidis Cas10·Csm complex.

      DISCUSSION

      Here, we show that a Cas10·Csm ribonucleoprotein complex, similar to the E. coli Cascade (
      • Brouns S.J.
      • Jore M.M.
      • Lundgren M.
      • Westra E.R.
      • Slijkhuis R.J.
      • Snijders A.P.
      • Dickman M.J.
      • Makarova K.S.
      • Koonin E.V.
      • van der Oost J.
      Small CRISPR RNAs guide antiviral defense in prokaryotes.
      ) and Pyrococcus furiosus and Sulfolobus solfataricus (
      • Hale C.R.
      • Zhao P.
      • Olson S.
      • Duff M.O.
      • Graveley B.R.
      • Wells L.
      • Terns R.M.
      • Terns M.P.
      RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex.
      ,
      • Zhang J.
      • Rouillon C.
      • Kerou M.
      • Reeks J.
      • Brugger K.
      • Graham S.
      • Reimann J.
      • Cannone G.
      • Liu H.
      • Albers S.V.
      • Naismith J.H.
      • Spagnolo L.
      • White M.F.
      Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity.
      ) Cmr complexes, mediates type III-A CRISPR immunity in staphylococci. This complex is composed of Cas10, Csm2, Csm3, Csm4, and Csm5 and mature crRNAs that, through base pair interactions with a cognate DNA sequence, guide the complex to its target on the genome of plasmids and bacteriophages. Unexpectedly, multiple mature crRNA species differing by 6-nt increments at the 3′ end are present in this complex. Previously, we demonstrated that the final length of mature crRNAs is determined by a ruler mechanism that measures from the 5′ end primary processing cleavage site (
      • Hatoum-Aslan A.
      • Maniv I.
      • Marraffini L.A.
      Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site.
      ). Here, we demonstrate that Csm3 is the ruler that determines the mature crRNA length. Csm3 is essential for the formation of the complex and seems to be present in multiple copies. In vitro, multiple copies of Csm3 bind the RNA in a sequence-independent manner, one protein every 6 nt of substrate. Alanine substitution of a conserved aspartate residue, Asp-100, impacts Csm3 folding and/or ability to bind the crRNA and prevents the accumulation of longer mature crRNA species. However, overexpression of Csm3 leads to the accumulation of longer species. Altogether, these results allow us to propose that Csm3 binds to the crRNAs in the complex at multiple sites, once every 6 nt, with each additional copy extending the crRNA length by 6 nt.
      The nucleases involved in the biogenesis of crRNAs remain to be determined. In other type III and type I CRISPR-Cas systems, primary processing is carried out by Cas6 (
      • Brouns S.J.
      • Jore M.M.
      • Lundgren M.
      • Westra E.R.
      • Slijkhuis R.J.
      • Snijders A.P.
      • Dickman M.J.
      • Makarova K.S.
      • Koonin E.V.
      • van der Oost J.
      Small CRISPR RNAs guide antiviral defense in prokaryotes.
      ,
      • Haurwitz R.E.
      • Jinek M.
      • Wiedenheft B.
      • Zhou K.
      • Doudna J.A.
      Sequence- and structure-specific RNA processing by a CRISPR endonuclease.
      ,
      • Carte J.
      • Pfister N.T.
      • Compton M.M.
      • Terns R.M.
      • Terns M.P.
      Binding and cleavage of CRISPR RNA by Cas6.
      ), which cleaves within repeats at the base of a hairpin and defines the 5′ end for all crRNAs. Therefore, S. epidermidis Cas6 is a strong candidate to cleave the crRNA precursor into 71-nt intermediates. The identity of the nuclease responsible for crRNA maturation in type III CRISPR-Cas systems is less clear. The fact that the same type III-A complex, when expressed in S. epidermidis and E. coli, produce mature species of different lengths and that this complex is unable to direct the maturation of a crRNA substrate in vitro, suggests that a host-encoded endo- or exoribonuclease might be responsible for the degradation of the 3′ end of crRNA intermediates. Several ribonucleases are annotated in the S. epidermidis RP62a genome (
      • Gill S.R.
      • Fouts D.E.
      • Archer G.L.
      • Mongodin E.F.
      • Deboy R.T.
      • Ravel J.
      • Paulsen I.T.
      • Kolonay J.F.
      • Brinkac L.
      • Beanan M.
      • Dodson R.J.
      • Daugherty S.C.
      • Madupu R.
      • Angiuoli S.V.
      • Durkin A.S.
      • Haft D.H.
      • Vamathevan J.
      • Khouri H.
      • Utterback T.
      • Lee C.
      • Dimitrov G.
      • Jiang L.
      • Qin H.
      • Weidman J.
      • Tran K.
      • Kang K.
      • Hance I.R.
      • Nelson K.E.
      • Fraser C.M.
      Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain.
      ), RNase H-II and -III, RNase III, RNase II/VacB and RNase R, RNase BN, RNase P, Cbf1, and YjgF. Any of these could participate in crRNA maturation. Future biochemical and genetic experiments will determine the role of host RNases in crRNA maturation.
      The three different types of CRISPR-Cas systems can be distinguished according to their crRNA biogenesis pathways. Type II systems display a distinct mechanism for the generation of crRNAs that requires the pairing of the precursor crRNA with an antisense trans-encoded crRNA to ensure RNase III cleavage of the precursor (
      • Deltcheva E.
      • Chylinski K.
      • Sharma C.M.
      • Gonzales K.
      • Chao Y.
      • Pirzada Z.A.
      • Eckert M.R.
      • Vogel J.
      • Charpentier E.
      CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.
      ). Type I and III systems, however, have comparable crRNA biogenesis pathways; the main difference being the lack of further processing of the intermediate crRNAs in the former. This may be attributed to the different properties of the type I Cas6 homologs. In these systems, the endonuclease is part of the Cascade complex (
      • Brouns S.J.
      • Jore M.M.
      • Lundgren M.
      • Westra E.R.
      • Slijkhuis R.J.
      • Snijders A.P.
      • Dickman M.J.
      • Makarova K.S.
      • Koonin E.V.
      • van der Oost J.
      Small CRISPR RNAs guide antiviral defense in prokaryotes.
      ,
      • Westra E.R.
      • van Erp P.B.
      • Künne T.
      • Wong S.P.
      • Staals R.H.
      • Seegers C.L.
      • Bollen S.
      • Jore M.M.
      • Semenova E.
      • Severinov K.
      • de Vos W.M.
      • Dame R.T.
      • de Vries R.
      • Brouns S.J.
      • van der Oost J.
      CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3.
      ,
      • Wiedenheft B.
      • Lander G.C.
      • Zhou K.
      • Jore M.M.
      • Brouns S.J.
      • van der Oost J.
      • Doudna J.A.
      • Nogales E.
      Structures of the RNA-guided surveillance complex from a bacterial immune system.
      ) and remains tightly bound to the 3′ end of its product after cleavage (
      • Haurwitz R.E.
      • Jinek M.
      • Wiedenheft B.
      • Zhou K.
      • Doudna J.A.
      Sequence- and structure-specific RNA processing by a CRISPR endonuclease.
      ). The strong and continuous protection of the crRNA by the type I Cas6 homologs may prevent further nucleolysis of the 3′ end. In contrast, Cas6 is not part of the S. epidermidis type III-A ribonucleoprotein complex or of the well characterized type III-B P. furiousus and S. solfataricus Cmr complex, which also contain mature crRNAs differing by 6-nt increments at the 3′ end (
      • Hale C.R.
      • Zhao P.
      • Olson S.
      • Duff M.O.
      • Graveley B.R.
      • Wells L.
      • Terns R.M.
      • Terns M.P.
      RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex.
      ,
      • Hale C.
      • Kleppe K.
      • Terns R.M.
      • Terns M.P.
      Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus.
      ,
      • Zhang J.
      • Rouillon C.
      • Kerou M.
      • Reeks J.
      • Brugger K.
      • Graham S.
      • Reimann J.
      • Cannone G.
      • Liu H.
      • Albers S.V.
      • Naismith J.H.
      • Spagnolo L.
      • White M.F.
      Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity.
      ,
      • Hale C.R.
      • Majumdar S.
      • Elmore J.
      • Pfister N.
      • Compton M.
      • Olson S.
      • Resch A.M.
      • Glover 3rd, C.V.
      • Graveley B.R.
      • Terns R.M.
      • Terns M.P.
      Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs.
      ). Due to its homology to Csm3, we suspect that Cmr6 acts as a ruler of the mature crRNA length in P. furiousus.
      It remains to be known whether the precise measurement of crRNA length by Csm3 is important for CRISPR immunity. One possibility is that the extra trimming of crRNAs is caused by rearrangements in the crRNA:Cas·Csm complex that expose these RNAs to host nucleases and has no consequences for CRISPR immunity. Alternatively, crRNA maturation could be necessary for CRISPR-Cas function. In this scenario, it is possible that repeat sequences at the 3′ end of an intermediate crRNA, which do not anneal with the target and form stem-loop structures, would interfere with target recognition and/or cleavage. In fact, in the type III-B system of P. furiosus, the 3′ end of the mature crRNA determines the cleavage site on the target sequence exactly 14 nt upstream of this end (
      • Hale C.R.
      • Zhao P.
      • Olson S.
      • Duff M.O.
      • Graveley B.R.
      • Wells L.
      • Terns R.M.
      • Terns M.P.
      RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex.
      ); such precise cleavage may not be possible with a guide crRNA containing extra sequences at the 3′ end. Equally unknown is the importance, if any, of the presence of multiple crRNA species. If indeed the target cleavage site of type III CRISPR-Cas systems is determined by the mature 3′ end of the crRNA, then multiple mature crRNA sizes could direct multiple target cleavage events. The advantage of this would be that targets cleaved by complexes containing longer crRNAs could be subjected to a second cleavage event directed by shorter crRNAs, thus providing less chance for repair and stronger immunity. Our results argue that this may not be the case since we demonstrated that the Csm3D100A complex, which produces only the shortest crRNA (31 nt), confers full immunity against plasmid conjugation. In this system, a perfect match exists between the spc1 crRNA and its targeted sequence, the protospacer. The possibility exists that longer crRNAs may be required when there is an imperfect match between the crRNA and the protospacer. In the latter scenario, longer crRNAs would increase the likelihood of establishing sufficient length of complementarity to facilitate interference. The 31 nt-long crRNA contains 8 nt of repeat sequences at the 5′ end plus 23 nt of spacer sequence at the 3′ end. Because the full-length spacer is 36 nt, this result also shows that the 13 nt at the 3′ end of the spacer sequence are not required to specify the target of CRISPR immunity. This suggests that if there is a “seed sequence” in type III systems, a region of homology between the crRNA and the target absolutely required for CRISPR immunity (
      • Wiedenheft B.
      • van Duijn E.
      • Bultema J.B.
      • Waghmare S.P.
      • Zhou K.
      • Barendregt A.
      • Westphal W.
      • Heck A.J.
      • Boekema E.J.
      • Dickman M.J.
      • Doudna J.A.
      RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions.
      ,
      • Semenova E.
      • Jore M.M.
      • Datsenko K.A.
      • Semenova A.
      • Westra E.R.
      • Wanner B.
      • van der Oost J.
      • Brouns S.J.
      • Severinov K.
      Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence.
      ), it must be located at the 5′ end of the crRNA target. Deciphering the mechanisms of crRNA biogenesis and targeting will be important both to understand how this immune system prevents infection in prokaryotes as well as to exploit it for biotechnological applications (
      • Bikard D.
      • Jiang W.
      • Samai P.
      • Hochschild A.
      • Zhang F.
      • Marraffini L.A.
      ,
      • Cong L.
      • Ran F.A.
      • Cox D.
      • Lin S.
      • Barretto R.
      • Habib N.
      • Hsu P.D.
      • Wu X.
      • Jiang W.
      • Marraffini L.A.
      • Zhang F.
      Multiplex genome engineering using CRISPR/Cas systems.
      ,
      • Jiang W.
      • Bikard D.
      • Cox D.
      • Zhang F.
      • Marraffini L.A.
      RNA-guided editing of bacterial genomes using CRISPR-Cas systems.
      ,
      • Mali P.
      • Yang L.
      • Esvelt K.M.
      • Aach J.
      • Guell M.
      • DiCarlo J.E.
      • Norville J.E.
      • Church G.M.
      RNA-guided human genome engineering via Cas9.
      ,
      • Qi L.S.
      • Larson M.H.
      • Gilbert L.A.
      • Doudna J.A.
      • Weissman J.S.
      • Arkin A.P.
      • Lim W.A.
      Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.
      ).

      Acknowledgments

      We thank members of our laboratory for critical discussion of the paper.

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