PROGRESS CRISPR–Cas systems: beyond adaptive immunity Edze R. Westra, Angus Buckling and Peter C. Fineran

Abstract | The discovery of CRISPR–Cas (clustered, regularly interspaced short palindromic repeats–CRISPR-associated proteins) adaptive immune systems in prokaryotes has been one of the most exciting advances in microbiology in the past decade. Their role in host protection against mobile genetic elements is now well established, but there is mounting evidence that these systems modulate other processes, such as the genetic regulation of group behaviour and virulence, DNA repair and genome evolution. In this Progress article, we discuss recent studies that have provided insights into these unconventional CRISPR–Cas functions and consider their potential evolutionary implications. Understanding the role of CRISPR–Cas in these processes will improve our understanding of the evolution and maintenance of CRISPR–Cas systems in prokaryotic genomes. Bacteria and archaea encode various defence systems to protect against predation by phage and other parasites1,2. Although many defence systems, such as restrictionmodification and abortive-infection systems, provide innate immunity, approximately one-half of bacteria and most archaea also have adaptive immunity, which is encoded by the CRISPR–Cas (clustered, regularly interspaced short palindromic repeats–CRISPRassociated proteins) system. This system provides protection against invading mobile genetic elements (MGEs), such as plasmids and phages, by integrating invader-derived sequences into the CRISPR locus3. Precursor CRISPR RNA (pre-crRNA) transcripts are processed into short CRISPR RNAs (crRNAs), which, together with the Cas proteins, detect and degrade MGEs that carry a complementary sequence (known as a protospacer; BOX 1), via a mechanism that is similar to RNA interference (RNAi; a process that involves the inhibition of gene expression by RNA molecules) in eukaryotes3. Although all CRISPR–Cas variants that have been discovered so far share some basic mechanistic features, there are substantial differences between the systems, which has resulted in their classification into three main CRISPR– Cas types and eleven subtypes4 (BOX 1).

The role of CRISPR–Cas systems in defence against MGEs is now well established3. The first direct evidence that these systems function in defence came from a study in which Streptococcus thermophilus acquired resistance to phage infection by the incorporation of phage-derived sequences (known as spacers) into its CRISPR loci5. Spacer acquisition occurs in several archaeal and bacterial species in natural environments6–9 and CRISPR resistance has been well studied under laboratory conditions using many different host species and a wide range of invasive DNA elements5,10–12. However, some CRISPR–Cas systems, such as the Escherichia coli type I‑E system10, are not active under laboratory conditions11,12, as the cas genes are silenced by the repressor protein H-NS13. In such strains, a functional immune response against invading DNA is only observed in the absence of hns or in engineered strains that overexpress the CRISPR–Cas system11,12,14. Bioinformatic analyses have revealed that this CRISPR–Cas system is evolving very slowly (the CRISPR arrays have remained unchanged for 103–105 years15,16) compared with the rapid evolution of CRISPR–Cas systems in other species6. Crucially, this slow evolution is inconsistent with the assumed strong and

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continually changing selective pressures that are imposed by parasitic viruses and plasmids15,17. Thus, the maintenance of this type I‑E CRISPR–Cas system in diverse E. col­i genomes suggests that it might have a function other than immunity15,16. Consistent with this, several additional functions of CRISPR–Cas have indeed been identified in other organisms (TABLE 1). From a historical perspective, the discovery that CRISPR–Cas systems have functions other than defence is reflective of how the RNAi field developed. Although RNAi was initially considered to be involved in immunity, it was only later discovered that it affects many cellular processes, such as gene regulation and heterochromatin formation18. In this Progress article, we discuss the additional functions of CRISPR–Cas systems and consider their potential implications from an evolutionary perspective — whether these additional functions are the direct result of natural selection or whether they are simply by‑products of their role in adaptive immunity. We conclude that some additional functions, such as the regulation of biofilm formation in Pseudomonas aeruginosa and genome evolution, are by‑products of the canonical CRISPR–Cas function in defence, whereas others, such as the regulation of development in Myxococcus xanthus and virulence in Francisella novicida and Listeria monocytogenes, are selected functions. Endogenous gene regulation Besides their role in adaptive immunity, the regulation of gene expression is the most widely reported function of CRISPR–Cas systems. In this section, some recent examples of CRISPR–Cas-mediated regulation of endogenous genes that control group behaviour and virulence are discussed. In most, if not all, of these examples, gene regulation seems to involve the independent activity of either CRISPR transcripts or Cas proteins.

Regulation of group behaviour. M. xanthus is a predatory Deltaproteobacterium that is ubiquitous in soil and is a model organism for the study of fruiting body development and sporulation19 (FIG. 1a). Fruiting body formation is a multistep process that is triggered by starvation and is initiated by VOLUME 12 | MAY 2014 | 317

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PROGRESS Box 1 | The basics of CRISPR–Cas interference The CRISPR–Cas (clustered, regularly interspaced short palindromic nucleic acids in invading MGEs. The target nucleic acid (known as a repeats–CRISPR-associated proteins) system consists of two main protospacer; purple) is usually double-stranded DNA (dsDNA) (in type I, components: the Cas proteins, which function as the catalytic core of type II and type III‑A systems)93–95, but the type III‑B system targets the system, and the CRISPR locus, which functions as genetic memory. complementary single-stranded RNA (ssRNA)43,45. Target cleavage is A CRISPR array consists of repetitive sequences (repeats) that are carried out either by the Cas–crRNA ribonucleoprotein complex itself separated by variable sequences (spacers) derived from invading mobile (in type II and type III‑B systems) or by recruiting a Cas nuclease (in type I genetic elements (MGEs). The repeats and spacers are both typically and type III‑A systems)96. In type I systems, the surveillance complex (which 25–40 nucleotides in length. The cas genes are often located adjacent is composed of Cascade and the crRNA) binds to dsDNA97 and then recruits to the CRISPR locus but can also be encoded elsewhere on the genome. the Cas3 nuclease to degrade the target95. In type II systems, Cas9 (which CRISPR–Cas systems are diverse in terms of the content and is loaded with crRNA and tracrRNA) binds to and cleaves target dsDNA39. organization of cas genes and have been classified into three main types In type III‑A systems, a Csm–crRNA complex98 binds to, and presumably and at least 11 subtypes4. There are major mechanistic differences degrades, invader dsDNA93, possibly by recruiting Csm6 (REF. 99). between the variants of the system; nevertheless, the general mode of Type III‑B Cmr–crRNA complexes cleave complementary RNA43. For action of all three types of CRISPR–Cas systems involves three distinct further details, we refer to comprehensive reviews on the mechanism stages: adaptation, expression and interference (see the figure). During and structure of Cas–ribonucleoprotein complexes96,100. adaptation, expansion of the CRISPR array Spacers Repeats occurs by the addition of an MGE-derived CRISPR locus spacer sequence, which involves the Leader duplication of a repeat sequence54. Spacer acquisition occurs in a polarized manner: cas genes Cas1 new spacers are typically integrated at the leader-proximal end of the array, which Cas2 Adaptation involves the duplication of the first repeat of the array54. This process requires Cas1 and New spacer Cas2 (REF. 54), which are encoded by all CRISPR–Cas systems4, but it might also involve additional Cas proteins in some systems. During the expression stage, CRISPR loci are transcribed from an upstream promoter in the AT‑rich leader sequence and Expression the resulting pre-CRISPR RNA (pre-crRNA) is Pre-crRNA processed into short crRNAs by cleavage in the repeat sequences. In type I and type III crRNA processing systems, pre-crRNA cleavage is carried out by 14,91 Cas endoribonucleases . In type II systems, this process involves the expression of a tracrRNA crRNA transactivating crRNA (tracrRNA), which base-pairs with the repeats in the pre-crRNA transcript. The resulting duplexes are cleaved Cas6 (type I-A,B,D,E,F) in the repeat sequences by RNase III in a Cas6 Cas9 RNase III Cas5 (type I-C) Cas9‑dependent reaction29 (see the figure). Thus, in all CRISPR–Cas systems, cleavage Ribonucleoprotein Secondary Secondary of the pre-crRNA occurs in the repeat complex formation processing processing sequences, hence, mature crRNA consists of a spacer (purple) flanked by partial repeats (black). In some systems, further processing of Cascade Cas9 Csm or Cmr the crRNA takes place43,92. In type II systems, the tracrRNA remains bound to the crRNA and the mature crRNA–tracrRNA duplexes are Interference complexed with Cas9. In type I and III systems, mature crRNA is bound by a Cas protein complex. Type I ribonucleoprotein complexes Cascade Cas9 Csm Cmr are known as Cascade, whereas type III‑A and type III‑B complexes are known as Csm and dsDNA Cmr complexes, respectively. During the ssRNA interference stage (see the figure) crRNAs Protospacer function as guides for the Cas proteins, as cleavage Csm6? Cas3 they recognize and bind to complementary Type I Type II Type III-A Type III-B Nature Reviews | Microbiology

the coordinated rippling movement of cells, which results in the formation of foci of cell aggregates. The cells then form mounds and differentiate into myxospores, which

leads to the development of the fruiting body. This process is tightly regulated by various intercellular quorum-sensing signals and intracellular signalling cascades19. The

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two main signals involved are A‑signal, which consists of amino acids and peptides that are produced during starvation, and C‑signal, which is a 17 kDa protein that www.nature.com/reviews/micro

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PROGRESS Table 1 | Unconventional functions of CRISPR–Cas systems Function

CRISPR–Cas Mechanism type

Cas genes involved

CRISPR involved

Species

Experimental evidence

References

Gene regulation III‑B

Cleavage of complementary Yes mRNA

Yes

Pyrococcus furiosus

No

43, 46

Gene regulation I‑F of group behaviour I‑C

Based on partial complementarity

Yes

Yes

Pseudomonas aeruginosa Yes

40, 41

Unknown

Yes

Unknown

Myxococcus xanthus

Yes

20–22

Virulence gene regulation

II‑C

Cas9‑dependent cell surface modification

Yes

No

Campylobacter jejuni

Yes

27

II‑B

Cas9‑mediated downregulation of BLP production

Yes

No

Francisella novicida

Yes

28

II‑B

Unknown

Yes

No

Legionella pneumophila

Yes

31

Orphan Regulation of feoAB operon No CRISPR locus by partial complementarity

Yes

Listeria monocytogenes

Yes

33, 34

Genome remodelling

I‑F

Removal of genomic regions Yes by self-targeting

Yes

Pectobacterium atrosepticum

Yes

36

DNA repair

I‑E

DNA repair by Cas1

Yes

No*

Escherichia coli

Yes

59

Competition between MGEs

I‑F

Sequence-specific targeting Yes of competing MGE

Yes

Vibrio cholerae phage ICP1

Yes

68

Cell dormancy

Not specified Cas1 and Cas2 function analogously to a TA system to trigger dormancy (and eventual cell death) following phage infection

No

Not specified

No

70

Yes

BLP, bacterial lipoprotein; CRISPR–Cas, clustered, regularly interspaced short palindromic repeats–CRISPR-associated proteins; MGE, mobile genetic elements; TA, toxin–antitoxin.*Deletion mutants that lack the CRISPR locus show increased susceptibility to DNA damage. However, the DNA-repair function of Cas1 seems to be independent of CRISPR, as Cas1 localizes to DNA-damage sites.

is encoded by csgA and signals cell–cell contact. A‑signal induces the expression of the key developmental response regulator, encoded by fruA, whereas C‑signalling activates FruA. The type I‑C CRISPR–Cas system of M. xanthu­s strain DK1622 consists of seven cas genes (also known as dev genes in M. xanthu­s) and a downstream CRISPR locus that contains 22 spacers (FIG. 1b). Initial evidence that the cas genes are involved in development came from transposon mutagenesis experiments, which showed that the disruption of cas7 (also known as devR) and cas5 (also known as devS) resulted in strongly reduced sporulation20. Moreover, a cas8c (also known as devT) mutant exhibited delayed aggregation and reduced spore formation owing to decreased fruA transcript and FruA protein levels21. Regulation of the cas operon in M. xanthu­s is complex and involves many regulatory elements, upstream and downstream of the promoter, and negative autoregulation22. Expression of the cas genes is induced by C-signalling23 during cell aggregation and is not detectable during normal M. xanthus growth24,25. Moreover, during fruiting body formation, cas gene expression is limited to the fruiting body and peripheral cells do not express the cas operon26. Cas8c

induces the expression of fruA21, and the induction of cas gene expression probably involves the binding of FruA to one of the regulatory elements that are associated with the cas locus, resulting in a positive-feedback loop22 (FIG 1c). Although the mechanism is currently unclear, the cas genes of M. xanthu­s seem to be strongly embedded within the regulatory circuits that control fruiting-body formation. Whether the CRISPR array is also involved in development is unknown but, because Cas proteins typically function in conjunction with crRNAs, it would be interesting to determine whether there is full or partial complementarity between spacer sequences and the chromosomal sequences that are regulated by this system, as this would indicate that Cas proteins and crRNAs function together to modulate gene expression (see below). Alternatively, Cas proteins might regulate M. xanthus development independently of crRNAs. Regulation of bacterial virulence. The human pathogen Campylobacter jejuni encodes a type II CRISPR–Cas system, and expression of the Cas9 protein in a strain that lacks CRISPR loci increases virulence. Thus, this is an example of a Cas protein that functions independently of CRISPR transcripts.

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Furthermore, C. jejun­i mutants that lack cas9 show increased swarming and reduced cytotoxicity during infection of human cells27. The absence of Cas9 also led to an increase in antibody binding to cell surface structures, which suggests that Cas9 has a role in controlling the topology and/or composition of the cell envelope27. Although experimental evidence is currently lacking, the mechanism of virulence regulation in C. jejun­i might be similar to that observed in Francisella novicida strain U112. In this bacterium, the type II CRISPR–Cas system (which consists of four cas genes and an inversely oriented downstream CRISPR locus that contains 13 spacers (FIG. 2)) downregulates the expression of bacterial lipoprotein (BLP) — a virulenceassociated cell surface protein28. Downregulation of BLP is crucial for immune evasion, as it is a pathogen-associated molecular pattern that is recognized by Toll-like receptor 2 of the host immune system. Regulation of blp expression requires Cas9, the transactivating crRNA (tracrRNA) (BOX 1) and a small CRISPR–Cas-associated RNA (scaRNA), which is transcribed from a region that is immediately upstream of the inversely oriented CRISPR locus (FIG. 2). However, the CRISPR locus and the other VOLUME 12 | MAY 2014 | 319

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PROGRESS a

Fruiting body Cell mound

Myxospores

Swarming cells

Free cells

b

CRISPR locus (22 spacers) cas6

cas3

cas8c (devT)

cas7 (devR)

cas5 (devS)

cas4-cas1

cas2 Spacer

c

Repeat

Starvation

A-signal

Cas8c

Cell–cell contact fruA

C-signal

cas6

cas3

cas8c (devT)

cas7 (devR)

cas5 (devS)

cas4–cas1

cas2

FruA csgA

Activated FruA

Figure 1 | Regulation of fruiting body formation in Myxococcus xanthus.  a | Simplified schematic of the life cycle of M. xanthus1­9,101. Myxospores germinate into cells, which initiate a coordinated rippling movement in starvation conditions that results in the formation of foci of cell aggregates. The cells then form mounds and further differentiate into the fruiting body, which contains myxospores. b | The type I‑C CRISPR–Cas (clustered, regularly interspaced short palindromic repeats–CRISPRassociated proteins) system of M. xanthu­s strain DK1622 consists of seven cas genes and a downstream CRISPR locus that contains 22 spacers. c | Simplified overview of the regulatory pathways that control

cas genes are not required for the regulation of BLP expression. Tightly regulated expression of Cas9, tracrRNA and scaRNA following bacterial entry into the phagosome triggers a temporally regulated reduction in BLP expression, and mutants that lack any of these three components are severely attenuated during infection of mice. The mechanism that is proposed to underlie blp gene regulation involves the

Sporulation

development in M. xanthu­s and the role of the cas operon. Solid arrows Nature Reviews | Microbiology indicate direct activation of the target, whereas dashed arrows indicate indirect activation. Starvation induces synthesis of the A‑signal (which consists of amino acids and peptides), which activates the transcription of fruA. Likewise, the cas (also known as dev) operon activates the expression of fruA via Cas8c. The C‑signal, which is encoded by csgA, is induced following cell–cell contact and activates FruA, which then promotes the expression of the cas (dev) genes. Thus, the cas genes form part of a positive-feedback loop with FruA, which together determine the developmental transitions of the bacterium.

formation of a ribonucleoprotein complex that consists of Cas9, the scaRNA and the tracrRNA (BOX 1). This complex is predicted to bind to the blp transcript, which would result in degradation of blp by an unknown mechanism (see below). The tracrRNA– scaRN­A duplex is predicted to function as a guide in a similar manner to the tracrRNA– crRNA complex that is formed during CRISPR-interference29,30 (FIG. 2). However,

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in contrast to its structural role during CRISPR-interference29, the tracrRNA is partially complementary to the blp transcript and has been suggested to functionally substitute for the crRNA during gene regulation28 (FIG. 2; Supplementary information S1 (figure)). Importantly, this study suggests that RNA duplexes other than tracrRNA–crRNA can function as guides for Cas proteins. The precise mechanism www.nature.com/reviews/micro

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PROGRESS of interference is currently unclear, but if Cas9 is responsible for blp mRNA degradation, this suggests that the target specificity of Cas9 (which usually targets DNA) can be changed by the guide. Alternatively, the complex might recruit RNases that degrade the mRNA; however, the involvement of other RNases was not detected28. Interestingly, the same study showed that Cas9 also promotes the invasion and replication of Neisseria meningitidis in human cell lines, which suggests that type II CRISPR–Cas systems of other bacterial species may also have a role in virulence28. In addition to the studies described above, the type II CRISPR–Cas system of Legionella pneumophila — the causative agent of Legionnaires’ disease — seems to be involved in virulence, as it is required for intracellular infection of amoebae31. Surprisingly, only the cas2 gene is needed, and the cas9, cas1 and cas4 genes and the CRISPR array are all dispensable for infection. The mechanism by which Cas2 regulates bacterial virulence in this organism is unknown, but it has been proposed that the RNase activity of Cas2 (REF. 32) might somehow regulate the mRNA levels of virulence genes. Future research is needed to address the suggested involvement of Cas2 in virulence in this species. Regulation by antisense CRISPR RNA. The Gram-positive intracellular bacterium Listeria monocytogenes expresses many regulatory small non-coding RNAs (ncRNAs), some of which are involved in virulence33. An in silico screen, followed by experimental verification, identified nine novel ncRNAs, including a CRISPR transcript (known as rliB)34. The rliB CRISPR transcript forms a strong secondary structure with multiple stem–loops that encompass both the spacer and repeat sequences35. Unlike canonical Cas-dependent processing of CRISPR transcripts (BOX 1), which results in a mature crRNA that contains a single spacer sequence, processing of the 400 nucleotide precursor rliB CRISPR transcript is polynucleotide phosphorylase-dependent and results in the formation of a mature rliB CRISPR-derived RNA of 280 nucleotides, which is composed of multiple spacers and repeats35. Some of the spacers are complementary to sequences of known virulent or temperate L. monocytogene­s phages35. Indeed, the spacers were shown to provide protection against invading DNA but only if cas genes were supplied in trans by a type I‑A CRISPR–Cas locus with CRISPR repeats identical to those of the rliB transcript35. However, of the 40 strains that

Pro-inflammatory response

BLP

BLP expression downregulated F. novicida cell Degradation of blp transcript

scaRNA

Unknown nuclease or Cas9 tracrRNA

Cas9

blp mRNA

tracrRNA cas9

cas1

cas2

CRISPR locus (13 spacers)

scaRNA

cas4

Figure 2 | Virulence regulation by Cas9–tracrRNA–scaRNA in Francisella novicida.  The type II‑B CRISPR–Cas (clustered, regularly interspaced short palindromic repeats–CRISPR-associated proteins) Nature Reviews | Microbiology system of F. novicid­a strain U112 consists of four cas genes and an inversely oriented downstream CRISPR locus that contains 13 spacers. The region upstream of the CRISPR locus encodes a small CRISPR–Cas-associated RNA (scaRNA) and the downstream region encodes a transactivating CRISPR RNA (tracrRNA). Promoters of CRISPR and tracrRNA are indicated as previously reported30. In the previously proposed model28, expression of the immunostimulatory bacterial lipoprotein (BLP) is controlled by this CRISPR–Cas system. The blp mRNA transcript is bound by the Cas9–tracrRNA– scaRNA ribonucleoprotein complex, as the tracrRNA transcript has complementarity to the blp mRNA and is thought to facilitate recognition and binding (Supplemetary information S1 (figure)). This triggers degradation of blp either by Cas9 and/or by another nuclease. Ultimately, this results in reduced expression of BLP on the cell surface and promotes virulence owing to a downregulated immune response.

were analysed (all of which contain the rliB CRISPR locus), only 12 carry a type I‑A CRISPR–Cas system. Thus, the ubiquitous nature of the rliB locus in all sequenced L. monocytogene­s strains strongly suggests that it has an alternative function35 as, in the absence of type I‑A cas genes, the transcript does not provide immunity. One such function could be the regulation of partially matching genes. In accordance with such a gene regulatory function, it was found that overexpression of the rliB transcript results in a twofold increase in the mRNA levels of an operon (known as feoAB) that encodes a ferrous iron transporter34. A follow‑up study showed that a strain lacking rliB colonizes the liver of infected mice more efficiently

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than the wild-type strain, which indicates that the rliB-mediated increase in feoAB transcript levels is involved in controlling Listeria spp. virulence33. The regulatory effect of rliB is cas-independent, as the L. monocytogene­s strain that was studied does not have cas genes. Together with the studies described above, this finding supports the suggestion that the CRISPR transcripts and Cas components of adaptive immune systems can have independent roles in the control of bacterial virulence, potentially via gene regulation. Putative mechanisms of gene regulation The molecular details of the mechanisms underlying gene regulation by CRISPR VOLUME 12 | MAY 2014 | 321

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PROGRESS transcripts and/or Cas proteins seem to be diverse, but are poorly understood. The best studied examples of gene regulation (which are outlined above) suggest that CRISPR transcripts and Cas proteins function independently of each other in this context. Thus far, M. xanthu­s development seems to be the only exception, as it is currently unclear whether CRISPR functions together with cas genes during developmental regulation. Nevertheless, some studies indicate that CRISPR transcripts and Cas proteins can potentially function together to regulate the expression of genes that have only partial complementarity (in the case of CRISPR–Cas systems that target DNA) and genes that are fully complementary (in the case of type III‑B CRISPR–Cas systems that target RNA). Gene regulation by a partial match. Most CRISPR–Cas systems cleave complementary DNA, with the exception of type III‑B systems, which cleave complementary RNA (BOX 1). Therefore, if crRNAs are complementary to genomic sequences, this usually results in cell death owing to endonucleolytic cleavage of the genome36,37 (see below). However, DNA cleavage by CRISPR–Cas requires near-complete complementarity, and targets that have only partial complementary can escape cleavage, depending on the number and position of the mismatches38,39. Similarly to RNAi, in which microRNAs regulate the expression of partially complementary genes, crRNAs might regulate the expression of partially matching target genes. CRISPR–Cas-mediated gene regulation by partial complementarity is supported by studies on the regulation of swarming and biofilm formation in P. aeruginosa4­0,41. An unanticipated link between these group behaviours and the P. aeruginos­a type I‑F CRISPR–Cas system was observed when studying a lysogen of phage DMS3, which is a Mu‑like phage41. Unexpectedly, DMS3 lysogens lose the ability to swarm and to form biofilms, but this phenotype is reversed in the presence of cas mutations41. Only those cas genes that are required for interference42 (and not the cas1 gene, which is specifically involved in adaptation; BOX 1) were essential for altering group behaviour. In addition, a spacer was required that is partially complementary to gene 42 of the DMS3 prophage40. Owing to the mismatches, this spacer does not provide resistance against the phage42. Although the mechanism is unclear, it is tempting to speculate that this CRISPR–Cas system regulates the expression of gene 42 via partial base-pairing of the crRNA with the

target DNA or mRNA (the crRNA would be antisense to the mRNA). However, how this leads to biofilm inhibition is unclear, as deletion of gene 42 from the DMS3 genome has no effect on biofilm formation40. It remains possible that targeting gene 42 alters the expression of other genes in the same operon (which contains 20 genes in total)41 that potentially have a role in controlling group behaviour. The mechanistic details of CRISPR–Cas-dependent gene regulation by partial complementarity between the crRNA and the target nucleic acid remain mostly obscure; systematic analyses are needed to obtain a full appreciation of this apparent CRISPR–Cas function. Gene regulation by RNA cleavage. Although most CRISPR–Cas systems target DNA, Cmr complexes that are encoded by type III‑B systems are an exception, and instead cleave complementary RNA in vitro43–45 and in vivo46,47. The ability to degrade RNA raises the possibility that type III‑B systems regulate endogenous gene expression. However, only cleavage of an antisense CRISPR transcript (that was generated from a promoter within one of the spacer sequences) has been observed thus far46. Acquisition of a spacer that contains a promoter element is most probably an accidental event and, despite the potential, no examples of Cmr-mediated endogenous gene regulation have thus far been reported. Genome evolution by self-targeting Besides the regulation of gene expression, recent studies suggest that self-targeting by CRISPR–Cas can contribute to genome evolution. However, as most CRISPR–Cas systems cleave DNA, the targeting of chromo­ somal sequences results in cytotoxicity36,37,48. Self-targeting of the chromosome can involve targeting of the CRISPR locus (which, by definition, is complementary to the CRISPR transcript), or it can occur when spacers that are derived from genomic sequences are incorporated into the CRISPR locus, both of which result in genomic cleavage. The cytotoxicity that is associated with self-targeting has led to the selection of avoidance mechanisms that enable discrimination between CRISPR spacers (self) and protospacers (non-self)49–51. Chromosomally derived spacer sequences still pose a severe risk to cell survival36, but bioinformatic48,52,53 and experimental analyses54 have shown that such self-derived spacer-acquisition events occur repeatedly. Although self-targeting events are generally lethal, cells survive in some cases by acquiring mutations that

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inactivate self-targeting. These mutations can occur in cas genes, spacers, repeats and protospacer targets, and they can involve large-scale genome rearrangements, presumably owing to recombinational DNA repair following CRISPR–Cas-mediated genomic cleavage36. For example, crRNAs that target a chromosomal horizontally acquired island in Pectobacteriu­m atrosepticum are generally lethal, but surviving mutants contain various chromosomal deletions, including complete removal of the targeted ~100 kb island that is involved in plant pathogenicity36. Similar large chromosomal deletions were detected when other core genes (such as lacZ) were targeted by CRISPR–Cas36. In those rare cases in which the genome rearrangement confers a fitness benefit, self-targeting events could actually be advantageous. Owing to the repetitive nature of CRISPR loci, they have also been suggested to have roles in genomic shuffling. For example, in strains of Thermotogales neapolitana and Thermotogales maritima, CRISPR loci are found at the junctions of major chromosomal rearrangements55, and in E. col­i, CRISPR repeats were found to have a role in the duplication of a chromosomal region that contains rpoS (which is the stress-response sigma factor), resulting in increased fitness during adaptation to high temperatures56. As some spacers in CRISPR arrays match host genes, other studies have proposed that self-targeting might lead to CRISPR-mediated gene regulation57,58. DNA repair CRISPR–Cas systems not only cause damage to microbial genomes but can also function in DNA repair. For example, Cas1 of the E. col­i type I‑E system physically and genetically interacts with DNA-recombination and DNA-repair enzymes (for example, RecB, RecC, RuvB and UvrC). Deletion of cas1 or the associated CRISPR loci results in increased sensitivity to DNA-damaging agents and defects in chromosome segregation59. In vitro, Cas1 has endodeoxyribo­ nuclease activity on DNA structures that are common intermediates of recombination and repair59,60. These data reveal an unexpected role for CRISPR–Cas in DNA repair, which may function in parallel with its function in immunity. Interestingly, although CRISPR arrays were initially thought to function in replicon partitioning61, Cas proteins were initially proposed to function as part of a novel DNA-repair system62. Consistent with a role in DNA repair, cas gene expression in Pyrococcus furiosus increases following exposure to γ-radiation63. www.nature.com/reviews/micro

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PROGRESS Novel immune mechanisms and hijacking Competition between MGEs. Many MGEs encode CRISPR–Cas systems of their own, which probably contributes to the horizontal spread of these systems between bacteria and archaea64. Recent studies have shown that these MGE-encoded systems are used to compete with other MGEs. For example, phage-encoded CRISPR–Cas systems have been shown to target other phages, and this might enable them to compete with each other65–67. Another striking example of a CRISPR–Cas system that functions in competition between MGEs is provided by a phage-encoded system that targets a phageinducible chromosomal island (PICI)-like element (PLE) in Vibrio cholera­e68. This PLE resembles a Staphylococcus aureus pathogenicity island, which excises from the genome following phage infection and recruits phage capsids from the invading phage for its own transduction69. The type I‑F CRISPR–Cas system that is carried on the genome of phage ICP1 (International Centre for Diarrhoeal Disease Research, Bangladesh cholera phage 1) contains a spacer that targets a PLE on the V. cholera­e serogroup O1 genome. In the absence of the ICP1 CRISPR–Cas system, the PLE blocks phage proliferation; however, in the presence of the phage-encoded CRISPR–Cas system, ICP1 overcomes the anti-phage activity of PLE68. Thus, the phage has evolved, or has acquired, a CRISPR–Cas system to counteract the PLE defence system of its host. Interestingly, some orphan chromosomal CRISPR loci have been found to contain spacers that target cas genes (the products of which they would normally associate with10,16,48), which suggests that these loci might provide protection against highly similar CRISPR–Cas systems that are encoded by MGEs. In theory, crRNA from the orphan chromosomal CRISPR locus could ‘hijack’ and guide the Cas of the MGE to cleave the cas operon that is encoded by the MGE. These cas-targeting orphan CRISPR loci16 do not include cas genes, most probably because they have been deleted from the chromosome. This provides further bioinformatic support for the cytotoxic effects and genomic changes that are elicited by self-targeting36,48.

Cell dormancy and abortive infection. It has recently been proposed that Cas1 and Cas2 may have an additional function in defence against phage by inducing cell dormancy following phage infection70. According to this hypothesis, Cas1 and Cas2 function analogously to toxin–antitoxin (TA) systems71. TA systems can provide phage

resistance and are composed of a toxin that induces dormancy and eventual cell death unless it is neutralized by its corresponding antitoxin. The best characterized example is the ToxIN system from P. atrosepticum, in which the endoribonuclease toxin (ToxN) is released from the inactive ToxIN complex following phage infection, resulting in the degradation of phage and host transcripts, which elicits cell suicide71–73. This process is also known as abortive infection, as phage infection is aborted by the death of the host bacterium. The prediction that Cas2 might function as an mRNA-cleaving toxin that is activated following phage infection is based on the cleavage activity of Cas2 proteins from several organisms (including Sulfolobus solfataricus32) and owing to its structural homology with VapD74, a toxin component of a TA system in Haemophilus influenzae75. In the proposed model, Cas1 is predicted to function as the antitoxin and neutralizes Cas2 via a Cas1–Cas2 interaction. Indeed, an interaction occurs between Cas1 and the Cas2–Cas3 fusion protein that is present in type I‑F systems76. It is proposed that, following phage infection, Cas2 is released from Cas1 and induces host cell dormancy, thereby giving the CRISPR–Cas system sufficient time to acquire spacers from the invading phage. However, if spacer acquisition is unsuccessful, it is predicted that Cas2 mediated cleavage of mRNA transcripts results in cell suicide, analogously to the activity of the ToxN endoribonuclease of P. atrosepticum7­2. The observation that other Cas2 proteins do not cleave single-stranded RNA (ssRNA)77,78 and the fact that Cas1 and Cas2 can be overexpressed independently of each other54, argue against this hypothesis. Thus, further studies are required to elucidate whether Cas2 has a role in inducing cell dormancy and abortive infection. Evolutionary context As outlined in this Progress article, it is now emerging that CRISPR–Cas systems have diverse roles in a wide range of processes. However, whether these additional roles have been selected or are mere by‑products of the established role of the system in defence is more difficult to reconcile. If a novel function of a CRISPR–Cas system or its individual components confers a clear fitness advantage (that is, it is adaptive) in the environment in which the organism evolved, it is logical to assume that this function is a direct result of natural selection79. In this section, we speculate on how selection acts on, and has acted on, the different CRISPR– Cas functions that are discussed above.

NATURE REVIEWS | MICROBIOLOGY

Selected CRISPR–Cas functions. In addition to the combined function of CRISPR and Cas in prokaryotic adaptive immunity (and the exploitation of this immune function by MGEs), it is now emerging that individual components of these systems could have been selected for their roles in regulating gene expression. For example, cas gene expression in M. xanthu­s is tightly regulated during development and strongly increases spore formation — a key determinant of M. xanthu­s fitness80. The regulation of genes that control virulence by the F. novicid­a Cas9–tracrRNA– scaRNA ribonucleoprotein complex is also probably a directly selected function of these components, as cas genes are specifically induced when F. novicid­a enters the phagosome28, leading to BLP downregulation and escape from the host immune system. Moreover, degenerated CRISPR–Cas modules in several Francisella species are associated with loss of the blp gene, which would argue in favour of a selected function of the system in regulating BLP expression81. It is important to note that selection can act on both their role in gene regulation and their role in immunity: the CRISPR–Cas systems of M. xanthu­s, F. novicid­a and C. jejuni also seem to function in defence, as their CRISPR arrays contain spacers that are complementary to known phage sequences22,27,82. It is parsimonious to assume that CRISPR and cas genes originally had separate functions, and there are indications that cas genes may have been directly selected for gene regulation before the association with CRISPR. In M. xanthu­s, it is unclear whether CRISPR transcripts are involved in cas-dependent regulation of development; in L. pneumophil­a, Cas2 alone regulates virulence; and, expression of C. jejun­i cas9 (lacking the flanking regions that potentially encode tracrRNA and scaRNA) in a strain that lacks all other components of the system still results in increased virulence27. More­ over, the over-representation of pathogens as hosts of type II CRISPR–Cas systems28,83, together with the emerging role of Cas9 in regulating virulence in several species27,28, suggests that Cas9 might have had a virulencerelated (or gene-regulatory) function before its association with CRISPR arrays. This is compatible with the finding that, in 31 of 103 type II CRISPR–Cas systems, Cas9 is not associated with Cas1 (which is universally present in all CRISPR–Cas systems)4, suggesting that these are either degenerated CRISPR–Cas systems or that Cas9 proteins can function as stand-alone enzymes. Another putative ancient (and potentially directly selected) function is the role VOLUME 12 | MAY 2014 | 323

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PROGRESS of Cas1 in DNA repair in E. coli5­9. This might be conserved in other organisms, as exemplified by the increase in P. furiosu­s cas gene expression following exposure to γ-radiation63. The reported nuclease activity of Cas1 (REFS 59,60) might have been adapted during evolution to function in the opening of CRISPR repeats during spacer integration. Side-effect functions of CRISPR–Cas. Other examples of CRISPR–Cas functions are probably not the direct result of selection, but are instead simply side effects of the immunity function. For example, although gene regulation might be responsible for CRISPR–Cas–prophage-dependent alter­ ations in swarming and biofilm formation in P. aeruginos­a, this specific regulatory function is unlikely to have contributed to the maintenance of the CRISPR–Cas system in this species. This is because biofilm formation and swarming are regulated by many other pathways84,85, and the proposed mechanism of CRISPR–Cas regulation (which involves a spacer that partially matches a prophage) seems to be unnecessarily complex as well as affording poor regulation — the biofilm phenotype is effectively determined by the presence or absence of the phage. The finding that some P. aeruginos­a Mu‑like phages encode CRISPR inhibitors (which are proteins that specifically block CRISPR-interference)86 strongly suggests that the primary function of the system is in adaptive immunity, and the CRISPR– Cas–prophage-dependent changes in group behaviour are probably a by‑product of the successful targeting of a similar phage. Another example of a possible side-effect function is genomic rearrangement. This is far more likely to confer detrimental, rather than beneficial, fitness effects36, which means that selection would act against any CRISPR–Cas variants that increase the probability of this occurring. That said, such genome remodelling has the potential to produce radically new genotypes that could have a large fitness advantage and hence, although this function is not subject to direct selection, this consequence of CRISPR–Cas activity might still have an important role in prokaryotic evolution. Conclusions and future perspectives From the studies that are discussed in this Progress article, it has become clear that CRISPR–Cas systems have functions beyond their canonical role in providing adaptive immunity. The most widespread of these additional functions seems to be the regulation of endogenous gene expression. The

discovery of CRISPR and/or Cas-mediated gene regulation has mainly come from unbiased screening for regulators of phenotypes of interest, such as virulence or group behaviour; hence, it is likely that components of CRISPR–Cas systems regulate many more genes than those that have been identified so far. However, predicting CRISPR–Casmediate­d gene regulation requires a much more detailed understanding of the mechanisms that are involved, in particular, the base-pairing requirements. This, together with the limitations that are imposed by self-targeting, will also aid in understanding the potential for CRISPR–Cas systems to promote the rapid evolution of gene expression. Eventually, the importance of CRISPR–Cas in the evolution of gene expression needs to be fully addressed by bioinformatics or realtime evolution experiments. More specifically, this would involve using bioinformatics to predict the gene-regulatory functions of partially matching spacers or observing the regulatory effects of newly acquired spacers on partially matching genes during laboratory evolution experiments. Whereas some CRISPR–Cas systems may have additional functions beyond adaptive immunity, other CRISPR–Cas systems are likely to function exclusively in defence. In other cases, CRISPR–Cas systems may have completely lost their immune-related function. For example, E. col­i type I‑E CRISPR– Cas systems do not confer immunity in non-engineered strains under laboratory conditions11,12, and CRISPR arrays of natural E. col­i isolates show very slow evolution rates, which seem to be incompatible with a role for CRISPR–Cas in defence15,16. Thus, the primary function of type I‑E CRISPR– Cas systems of E. col­i does not seem to be immune-related, and so the maintenance of these systems in E. col­i suggests that they are likely to have other functions87. It is possible that this system lost its adaptive immunity function because there is a net benefit to receiving MGEs (despite the obvious costs) in certain ecological contexts. E. col­i are often host-associated and, in this relatively harsh and changing environment, the beneficial effects of incorporating novel genetic material might outweigh the costs — analogous to the evolution of sexual reproduction in eukaryotes88. Indeed, several examples that are described in the literature indicate that inhibiting horizontal gene transfer by CRISPR–Cas systems can be costly in conditions in which the microorganism needs to rapidly adapt. For example, a Streptococcus pneumoniae strain that has a CRISPR–Cas system engineered to target capsule genes

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(which therefore prevented the natural transformation of these genes) had impaired virulence in a mouse model89. Moreover, a bioinformatics study has revealed that resistance of Enterococcus faecalis to antibiotics is inversely correlated with the presence of CRISPR–Cas systems90. Furthermore, the degeneration of CRISPR–Cas type II systems is observed in highly virulent Francisella species and is associated with the concomitant loss of blp and with the acquisition of novel virulence traits, such as the genes that encode O‑antigen and duplication of the Francisella spp. pathogenicity island81. These examples illustrate that inactivation of CRISPR–Cas systems might increase adaptive potential. In this context, it is also interesting to note that, when the avian pathogen Mycoplasma gallisepticum recently adapted to a new niche by jumping from the chicken to the wild house finch, isolates from the new host showed much lower diversity in their CRISPR loci and had lost cas genes9. Perhaps the benefit of acquiring new MGEs in this new environment outweighed the costs, or alternatively, the predation pressure by MGEs was relaxed in the new host. It is also possible that this type II system of M. gallisepticu­m was required for colonization of the chicken host but was no longer needed in the finch host. Understanding the diversity of CRISPR–Cas functionality both within and between species will inevitably require knowledge of the specific selective pressures that are present in different ecological contexts. Edze R. Westra and Angus Buckling are at Biosciences, University of Exeter, Penryn, Cornwall TR10 9EZ, UK. Peter C. Fineran is at the Department of Microbiology and Immunology, University of Otago, Dunedin 9054, New Zealand. Correspondence to E.R.W. e-mail: [email protected] doi:10.1038/nrmicro3241 Published online 7 April 2014 1. Westra, E. R. et al. The CRISPRs, they are a‑changin’: how prokaryotes generate adaptive immunity. Annu. Rev. Genet. 46, 311–339 (2012). 2. Labrie, S. J., Samson, J. E. & Moineau, S. Bacteriophage resistance mechanisms. Nature Rev. Microbiol. 8, 317–327 (2010). 3. Wiedenheft, B., Sternberg, S. H. & Doudna, J. A. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338 (2012). 4. Makarova, K. S. et al. Evolution and classification of the CRISPR–Cas systems. Nature Rev. Microbiol. 9, 467–477 (2011). 5. Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007). 6. Andersson, A. F. & Banfield, J. F. Virus population dynamics and acquired virus resistance in natural microbial communities. Science 320, 1047–1050 (2008). 7. Tyson, G. W. & Banfield, J. F. Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses. Environ. Microbiol. 10, 200–207 (2008).

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Acknowledgements

E.R.W. received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007‑2013) under Research Executive Agency (REA) grant agreement number 327606. A.B. is sup‑ ported by a UK Royal Society Wolfson Research Merit Award. P.C.F. is supported by a Rutherford Discovery Fellowship from the Royal Society of New Zealand.

Competing interests statement

The authors declare no competing interests.

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