CRISPR-Cas Systems and RNA-Guided Interference Rodolphe Barrangou∗

Advanced Review CRISPR-Cas systems and RNA-guided interference Rodolphe Barrangou∗

Clustered regularly interspaced short palindromic repeats (CRISPR) together with associated sequences (cas) form the CRISPR-Cas system, which provides adaptive immunity against viruses and plasmids in bacteria and archaea. Immunity is built through acquisition of short stretches of invasive nucleic acids into CRISPR loci as ‘spacers’. These immune markers are transcribed and processed into small noncoding interfering CRISPR RNAs (crRNAs) that guide Cas proteins toward target nucleic acids for speciﬁc cleavage of homologous sequences. Mechanistically, CRISPR-Cas systems function in three distinct stages, namely: (1) adaptation, where new spacers are acquired from invasive elements for immunization; (2) crRNA biogenesis, where CRISPR loci are transcribed and processed into small interfering crRNAs; and (3) interference, where crRNAs guide the Cas machinery to speciﬁcally cleave homologous invasive nucleic acids. A number of studies have shown that CRISPR-mediated immunity can readily increase the breadth and depth of virus resistance in bacteria and archaea. CRISPR interference can also target plasmid sequences and provide a barrier against the uptake of undesirable mobile genetic elements. These inheritable hypervariable loci provide phylogenetic information that can be insightful for typing purposes, epidemiological studies, and ecological surveys of natural habitats and environmental samples. More recently, the ability to reprogram CRISPR-directed endonuclease activity using customizable small noncoding interfering RNAs has set the stage for novel genome editing and engineering avenues. This review highlights recent studies that revealed the molecular basis of CRISPR-mediated immunity, and discusses applications of crRNA-guided interference.  2013 John Wiley & Sons, Ltd.

How to cite this article: WIREs RNA 2013, 4:267–278. doi: 10.1002/wrna.1159

INTRODUCTION CRISPR acronym was coined in 2002, following the observation of similar structures in a number lustered regularly interspaced short palindromic of bacterial and archaeal genomes.2 These loci were repeats (CRISPR) are a relatively novel family C subsequently identiﬁed in a variety of bacterial of DNA repeats that was ﬁrst discovered in 1987, and archaeal organisms, as genome drafts were in an intergenic region adjacent to the iap gene generated and became publicly available. In addition in the Escherichia coli K12 genome.1 This locus to coining the acronym, Jansen et al. established included an array of short identical 29-bp direct that these peculiar DNA repeat arrays were often repeats that were interspaced at regular intervals 2 by stretches of 32 nucleotides. Such loci seemed of associated with CRISPR-associated (cas) sequences. little interest for approximately 15 years, until the Over time, studies have shown that CRISPR-Cas systems generally occur in approximately 45%

∗ of bacterial and approximately 90% of archaeal Correspondence to: [email protected] genomes, as tracked by the CRISPRdb.3 Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, USA In 2005, three studies nearly simultaneously Conﬂict of interest: RB is a co-inventor on several patents related established that seemingly random spacer sequences to CRISPR use and applications. actually showed homology to foreign genetic elements

Volume 4, May/June 2013  2013 John Wiley & Sons, Ltd. 267 Advanced Review wires.wiley.com/rna such as viruses and plasmids.4–6 This subsequently with the ability to form hairpin structures. Repeats led to the hypothesis that CRISPR-Cas systems may are separated by stretches of unique sequences of be an RNA-mediated immune system, based on comparable length, called ‘spacers’.2 These sequences the presence of several key elements that would are derived from foreign genetic elements such as carry out the necessary functionalities.7 In 2007, plasmids and viruses, and these proto-spacers23 are the first experimental evidence of CRISPR-Cas typically adjacent to a short (2–5 nt) conserved proto- adaptive immunity was provided in Streptococcus spacer adjacent motif (PAM).24–25 While most loci are thermophilus, with evidence that novel spacers were relatively modest in size (∼30 repeats, representing acquired in a polarized manner following phage ∼1.6 kb), they can be large in some systems (up to exposure, with the ability to provide phage resistance nearly 500).3 Although most chromosomes carry 1–2 in a Cas-dependent manner.8 Shortly thereafter, it loci, there are extremophiles that carry 20 CRISPR was shown in E. coli that CRISPR immunity is loci belonging to different CRISPR-Cas systems.3,22 mediated by small noncoding interfering CRISPR Cas proteins constitute a highly genetically poly- RNAs (crRNAs) that guide the CRISPR-associated morphic and functionally diverse family which is complex for antiviral defense (Cascade).9 It was also involved in the various steps of CRISPR-mediated shown in Staphylococcus epidermidis that CRISPR immunity.11–22 There are several distinct Cas protein can provide immunity against plasmid DNA.10 families that have been established, and their number, These milestone studies established that CRISPR-Cas occurrence, distribution, and organization are highly systems provide adaptive immunity against phages variable. Recently, three CRISPR-Cas types have been and plasmids via crRNA-guided interference, and established,22 based on cas phylogeny and differences set the stage for the characterization of the CRISPR in the molecular mechanism of action. Although the molecular mechanism of action in functional CRISPR- most widely distributed functional domain character- Cas model systems. In the recent past, several extensive istic of Cas proteins is the RNA recognition motif or focused reviews have summarized the rapidly (RRM), several Cas families include DNA binding, expanding literature on CRISPR-Cas systems.11–21 RNA binding, helicase and nuclease motifs, reflecting Here, the core elements of CRISPR-Cas systems are their involvement in nucleic acid transactions. presented, the main system types are compared and Cas1 and Cas2, which are universally distributed contrasted, and applications of these RNA-driven across the three types, have been implicated in molecular machines are discussed. the spacer acquisition process.26–29 Cas1 specifically exhibits nuclease activity against single stranded and branched DNA such as Holiday junctions and CRISPR-Cas SYSTEMS replication forks, and may be implicated in the 26 While CRISPR-Cas systems occur in nearly half addition of novel repeats and/or spacers. It is of bacteria and most of archaea, there are vast generally believed that both Cas1 and Cas2 are differences in their distribution, number, size, and involved in novel spacer acquisition (through PAM- components across systems and organisms.11–21 associated foreign DNA sampling), novel repeat Several studies have investigated the many elements synthesis and repeat-spacer insertion at the leader 29 that constitute CRISPR-Cas systems, leading to a new end, as recently reviewed. In addition to these two classification which established three main types and universal cas genes, signature genes define each of the 10 subtypes, based on genetic content, and differences three main CRISPR-Cas types, namely cas3, cas9 and 22 in their crRNA processing and interference molecular cas10 for types I, II, and III, respectively. underpinnings.22 The three main CRISPR-Cas types Overall, CRISPR-Cas systems function in three reflect the origin and evolutionary paths that cas genes distinct stages: (1) adaptation, where new spacers and the proteins they encode have experienced, and are acquired from invasive elements and integrated illustrate their structural and functional differences. into the CRISPR locus for immunization; (2) crRNA biogenesis, where CRISPR loci are transcribed and processed into small interfering crRNAs; and Core Elements of CRISPR-Cas Systems (3) interference, where crRNAs guide the Cas The most peculiar feature of CRISPR-Cas systems machinery to specifically cleave homologous invasive is the array of identical DNA repeats that are nucleic acids. regularly separated by spacers (Figure 1). The CRISPR The leader sequence, generally defined as an repeat sequence is typically short (27–50 nt), with A/T-rich, noncoding stretch of nucleotides located most systems carrying repeats around 30 bp.11–22 upstream of the first CRISPR repeat, and often down- The repeat sequence is often partially palindromic, stream of the last cas gene in the CRISPR operon,

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FIGURE 1| CRISPR-Cas elements and mechanism of action. The various elements that constitute CRISPR-Cas systems are graphically depicted, including cas genes (with the universal cas1 and cas2), the leader (L), spacers (boxes), repeats (diamonds). Adaptive immunity is build through acquisition of new spacers at the leader end, which are derived from invasive DNA (here, phage DNA). In the expression stage, the repeat-spacer array is then transcribed as a full-length pre-crRNA, which is processed by cleavage into mature crRNAs. The mature RNAs then form a ribonucleoprotein complex with Cas proteins to mediate interference by guiding endonucleases toward homologous nucleic acid sequences. acts as a promoter for the transcription of the pre- of target DNA.9 Another key protein in the mechanism crRNA.30–35 The CRISPR leader has been shown to of action of type I systems is Cas6, an endoribonucle- include promoter elements, and binding sites for regu- ase which cleaves the pre-crRNA within the CRISPR latory proteins potentially involved in transcriptional repeat sequence during the crRNA maturation pro- 30 control of the repeat-spacer array. Overall, the bio- cess. Studies of model type I systems have documented genesis of mature crRNAs can be functionally and the involvement of Cascade in CRISPR-mediated sequentially divided into three general steps (Figure 1) interference, whereby the Cascade:crRNA ribonucle- that vary across the CRISPR-Cas types and subtypes. oprotein complex targets homologous invasive DNA First, the repeat-spacer array is transcribed into a and triggers R-loop formation for subsequent cleav- long primary transcript, the precursor CRISPR RNA age by the signature protein Cas3.36–44 An outline 9 (pre-crRNA). Second, the pre-crRNA is processed to of CRISPR-mediated immunity in type I systems is yield mature crRNAs that typically comprise a full provided in Figure 2. spacer. Finally, the mature crRNA is loaded into the The molecular processes responsible for crRNA Cas machinery to serve as a guide toward the target biogenesis and maturation are highly precise process- complementary nucleic acid. ing events. The CRISPR repeat-spacer array is ﬁrst transcribed as a long transcript deﬁned as the precur- Type I Systems sor CRISPR RNA (pre-crRNA).9,30–35 The pre-crRNA The signature gene for type I CRISPR-Cas systems is subsequently undergoes a single maturation step, cas3, which encodes a nuclease involved in the cleav- where the Cas6 endonuclease cleaves the transcript age of the target DNA.22 A signature feature of type I within the CRISPR repeat sequence, typically as part systems (Figure 2) is the CRISPR-associated complex of the Cascade complex. Generally, cleavage occurs for antiviral defense (Cascade) which is responsible for at the base of the stem-loop structure formed by the the processing of crRNA and involved in recognition palindromic CRISPR repeat sequence,38,45 generating

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FIGURE 2| Type I CRISPR-Cas systems. Details are provided for the prototypical Escherichia coli K12 type I-E system (ygcB-ygbF). The universal cas1 and cas2 genes are shown in black. The type I signature gene, cas3, is shown in red, while the crRNA maturation nuclease cas6 is shown in green. Repeats are represented as black diamonds, and spacers as white boxes. a crRNA consisting of a full spacer flanked by a short cleavage54–59 (Figure 3). Another peculiar molecular 5 -handle (∼8nt)anda3-stem loop derived from the element unique to type II systems is the tracrRNA,49 CRISPR repeat (∼17–21 nt). The beauty of the system which includes a sequence (∼25 nt) complementary to is illustrated by the seahorse-like structure of the Cas- the CRISPR repeat, which specifically trans-activates cade complex, which includes a Cas7 hexameric back- pre-crRNA cleavage within the CRISPR repeat, and bone, a Cse1 tail and a Cas6e head38,39,46,47 (Figure 2). sets the stage for crRNA maturation and processing by Once processed, the mature crRNA guides the the housekeeping endoribonuclease RNAse III, which Cascade complex for interference, where the ribonu- acts as a dicing effector.55 The three component system cleoprotein complex specifically recognizes target tracrRNA-Cas9-RNAse III is responsible for the mat- DNA sequences homologous to the loaded crRNA, uration of an approximately 40 nt crRNA which then and provides interference through nicking of each forms a ribonucleoprotein complex to mediate inter- strand of the invasive nucleic acid after R-loop for- ference through dsDNA cleavage.56 TracrRNA base mation. Once cleaved, Cas3 further degrades the pairs with each CRISPR repeat sequence within the target DNA by 3 → 5 endonuclease activity.41,43,44 pre-crRNA to form a double-stranded RNA template Mechanistically, interference involves recognition of which is specifically cleaved by RNAse III in the pres- a PAM upstream of the target proto-spacer, and ence of Cas9.55 Target dsDNA cleavage is mediated by is enthalpically driven by a 7–8 nt seed sequence at the 5-end of the spacer, which directs target sequence homologous pairing and triggers R-loop formation.48,49 Once the target DNA strands in an R-loop are separated, interference can occur through DNA cleavage. Cas3 is recruited for DNA degradation through ATP-dependent helicase activity (DExH domain) and ssDNA cleavage driven by an HD nuclease domain.22,36,37,41 Cas3 is a multifunctional protein which possesses nuclease (N-terminus HD-type domain) activity that is magnesium-dependent, helicase activity (DExD/H box and HelC domains) that is ATP-dependent, and 3 → 5-oriented processivity of the proto-spacer ssDNA.40,41,43,44 Type I model systems include the well- characterized E. coli type I-E9,28,50–52 and Pseu- domonas aeruginosa type I-F42,45,53 systems.

FIGURE 3| Type II CRISPR-Cas systems. Details are provided for the Type II Systems prototypical Streptococcus thermophilus DGCC7710 type II-A system. For type II systems, the signature gene is the The universal cas1 and cas2 genes are shown in black. The type II idiosyncratic cas9,22 which encodes a large protein signature gene, cas9, is shown in purple. Repeats are represented as involved in both crRNA biogenesis and target DNA black diamonds, and spacers as white boxes.

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processing, which occurs through a ruler mechanism anchored at the primary crRNA processing site, is mediated by additional Cas genes.62 The approximately 67 nt Cas6-generated crRNA is further trimmed at the 3 to generate a 39–45 nt mature crRNA which drives subsequent interference. In type III-B systems, the Cas RAMP module (CMR) effector complex specifically targets RNAs.34,61 Csm and Cmr Cas proteins are implicated in crRNA maturation in type III-A and type III-B CRISPR-Cas systems, respectively. Similar to type I systems, crRNA guides Cascade-like complexes toward the cognate nucleic acid for cleavage. Likewise, similar to type I systems, the endoribonuclease Cas6 is involved in cleavage of pre-crRNA within the CRISPR repeat sequence. Type III model systems include the S. epidermidis type III-A10,62 and Pyrococcus furiosus type FIGURE 4| Type III CRISPR-Cas systems. Details are provided for III-B,34,60,61 CRISPR-Cas systems. The former tar- the prototypical Pyrococcus furiosus type III-B system. The universal gets DNA through the Csm complex, while the cas1 and cas2 genes are shown in black. The type III signature gene, latter has been shown to specifically target RNA cas10, is shown in orange, while the crRNA maturation nuclease cas6 is through the Cmr complex,10,61 which reflects the shown in green. Repeats are represented as black diamonds, and broad target range of these fantastic and flexible spacers as white boxes. molecular machines. Type III systems frequently occur in archaea.15,22 HNH and RuvC domains in Cas9 (Figures 3 and 5), which cleave the complementary and noncomplementary strands, respectively, exactly 3 nt away from the CRISPR-Cas ROLES AND APPLICATIONS 3 -end of the proto-spacer, which is 2 bp away from The genetic polymorphism of CRISPR-Cas types 54,57,58 the PAM. Interestingly, two type II-A systems and subtypes reflects their functional diversity and in S. thermophilus cleave target phage and plasmid illustrate the range of potential applications. The DNA at the same position, though they recognize dif- primary application avenues to date include leveraging ferent PAMS, namely NNAGAAW for CRISPR1 and crRNA-mediated interference for phage resistance 54,58,59 NGGNG for CRISPR3. in industrially relevant organisms, and exploiting Type II model systems include the well- CRISPR hypervariability for genotyping purposes, 8,23,24,54,56,57 characterized S. thermophilus and Strep- primarily in pathogenic species. ,55,57 tococcus pyogenes type II-A CRISPR-Cas The transcriptional regulation of CRISPR-Cas systems. Interestingly, it appears to date that type systems has been thoroughly investigated in the E. coli 22 II systems occur perhaps exclusively in bacteria. type I-E system, and studies have revealed that there Sequence details are provided in Figure 5. is extensive transcriptional control by DNA-binding global regulators such as H-NS, LeuO, BaeSR, and 31,32,64,65 Type III Systems cAMP-CRP. Additional elements have been implicated in CRISPR transcript regulation, including The signature gene for type III CRISPR-Cas systems stress and environmental factors, which reflect the is cas10,22 which encodes a nuclease implicated functional diversity of these systems. Noteworthy, in target nucleic acid interference. In type III CRISPR-Cas loci can be constitutively expressed, and systems (Figure 4), once transcribed, the pre-crRNA generate crRNA levels that account for the majority subsequently undergoes two maturation steps: first, of cellular small noncoding RNAs.55 Transcriptional the Cas6 endonuclease cleaves within the CRISPR control and high constitutive expression can be repeat; second, protein complexes (analogous to the leveraged specifically or selectively, for exploitation type I Cascade) further trim the cleaved pre-crRNA to of these systems for various applications, as needed. generate a shorter mature crRNA.16,34,60–63 For type III systems, crRNA processing and maturation operate in a ruler mechanism manner, whereby Cas6 cleaves crRNA-Mediated Interference within the 5-end of the transcribed CRISPR repeat The primary use of crRNA-mediated interference to generate a 8-nt 5-handle.16,60,62 The secondary by CRISPR-Cas systems has been immunization

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(a)

(b)

(c)

FIGURE 5| crRNA-mediated DNA cleavage. (a) Sequences and details are provided for the Streptococcus thermophilus CRISPR3-Cas system, which contains 36nt CRISPR repeats and 30nt spacers. crRNA biogenesis requires Cas9, RNaseIII and tracrRNA. (b) dsDNA cleavage is driven by RuvC and HNH strand nicking within a R-loop, 3 nt away from the 3 terminus of the protospacer, which is flanked by the 5-NGGNG-3 proto-spacer adjacent motif (PAM). Rather than used the natural Cas9-crRNA-tracrRNA system, it is possible to generate a chimeric RNA which mimics the crRNA-tracrRNA duplex for a two-component flexible system. (c) Because RuvC and HNH each nick one strand of the target DNA, it is possible to reprogram the Cas9 endonuclease to either generate a dsDNA break, or a nick on either strand exactly 3 bp upstream of the PAM. Using the WT Cas9 endonuclease, dsDNA cleavage is generated (top). Using a HNH- mutant, the (−) strand is cleaved (bottom left), whereas using a RuvC- mutant, the (+) strand is nicked (bottom right). Likewise, any crRNA targeting a sequence flanked by the PAM can be used to reprogram the Cas9 nuclease. of bacteria against lytic phages.17 The original a bias in viral sequences sampling, and that the substantiation of CRISPR-encoded adaptive immunity wild-type spacer content may ‘prime’ subsequent against phages in the dairy starter culture species acquisition of the immune markers.50,51 The presence S. thermophilus has set the stage for phage resistance of CRISPR-Cas systems in many industrially relevant improvement in industrial cultures.8 In order to bacteria and model organisms sets the stage for many extend the industrial lifespan of highly valuable potential applications in the biotechnology and food dairy cultures, the most desirable and efficient industries.17,66 strains are exposed to problematic phages, and In addition to natural selection of CRISPR CRISPR bacteriophage insensitive mutants (BIMs) are variants,8,54,59 it is possible to artificially engineer selected for further improvement through iterative spacers and integrate them into organisms of inter- phage challenges. Ultimately, this ‘CRISPeRization’ est for enhanced virus immunity. The ability to process generates isogenic variants that have acquired design spacers in silico and generate them artificially resistance against a broad range of problematic through DNA synthesis sets the stage for customized phages. One advantage of this natural process is immunization, and opens avenues for providing resis- that such mutants are not considered genetically tance against the uptake and/or dissemination of modified microorganisms. The iterative acquisition of plasmids or genetic material that carry undesirable spacers provides increased resistance both in depth determinants such as antibiotic markers, pathogenic and range. It was recently shown that there is traits, or mobile elements. Indeed, it has been shown

272  2013 John Wiley & Sons, Ltd. Volume 4, May/June 2013 WIREs RNA CRISPR-Cas systems and RNA-guided interference that spacers targeting antibiotic resistance genes can can be used for genome editing, stacking, shuffling, prevent the uptake of plasmids that carry these and engineering, potentially in any organism.71 genes.54 In addition, CRISPR-Cas systems can be engi- Currently, genome editing methods rely primar- neered to prevent the horizontal transfer of virulence ily on zinc finger nucleases (ZFNs) and transcription factors.67 A negative correlation has been observed activator-like effector nucleases (TALENs) that trig- between the occurrence of CRISPR-Cas systems and ger nucleotide modifications by DNA-repair systems pathogenicity of Streptococcus,67 and Enterococcus.68 at the cleavage site. Given challenges and limitations Likewise, CRISPR-encoded immunity can influence inherent to ZFNs and TALENs, such as nonspecific the occurrence of mobile genetic elements such as cleavage and low target frequencies, crRNA-guided prophages.69,70 nucleases offer tremendous potential in terms of The ability to transfer functional CRISPR-Cas specificity and flexibility for next generation genome systems across phylogenetically distant genera and editing.71 In some ways, crRNA-guided nucleases species provides flexibility in the use of crRNA- combine the flexibility of RNA interference (RNAi) mediated interference.56 Initial work in Staphylo- with the efficiency of restriction enzymes. coccus epidermidis established that CRISPR-encoded Moving forward, the challenge is to assess spacers can reduce plasmid uptake,10 and several whether this flexible two-component system can subsequent studies have shown that CRISPR-Cas sys- be readily transferred to eukaryotic cells of inter- tems can interfere with plasmid transfer in several est to generate ssDNA and dsDNA breaks in vivo organisms.50,51,54,56,58 for generating mutations at desired locations with In summary, studies have shown that unprecedented flexibility and accuracy. Adapting this CRISPR-Cas systems can block the horizontal trans- system to organisms of interest such as plants for fer of conjugative plasmids, which can actually limit crop protection and agricultural seed enhancement, the uptake and dissemination of undesirable genetic or human and mammalian cells for medical and material such as pathogenic islands and antibiotic biotechnological purposes will be a milestone step resistance cassettes. Likewise, interference can be for next-generation genome engineering. Moreover, provided against elements that migrate to the host the ability to reprogram the cleavage machinery has chromosome such as lysogenic prophages.69,70 opened new avenues for customized DNA restriction, nicking, and genome engineering and editing. The availability of short PAMs (notably dinucleotides) ∼ crRNA-Mediated Cleavage that would randomly occur approximately every 8 bp provides an opportunity to develop customized Following the establishment that crRNA-mediated restriction enzymes that could target nearly any spe- interference results in cleavage of the target DNA,48,53 cific position in any genome. Indeed, two recent two reports have shows that type II systems can milestone studies show that Cas9 nucleases can be readily be reprogrammed using customized spacers reprogrammed using custom guide RNAs to direct to generate double-stranded DNA cleavage.57,58 genome editing in human cells.72,73 These revolu- Actually, a versatile system was engineered, based tionary studies specifically showed that the type II on a chimeric RNA effector, which can readily direct Cas9 nuclease can generate precise mutations in the Cas9 nuclease toward sequences of interest, to human and mouse cell lines, and can target sev- generate single-stranded breaks or double-stranded eral sequences concurrently through guide RNA breaks in nearly any sequence of interest.57 This multiplexing.72,73 This sets the stage for exploita- versatile chimeric RNA consists of portions of crRNA tion of Cas nucleases for genome editing and engi- and tracrRNA, linked together, which can guide neering with unprecedented flexibility and accuracy, Cas9 and form a ribonucleoprotein complex that opening up possibilities for next generation genome mediates specific DNA cleavage in bacteria. Because surgery. the HNH and RuvC domains of Cas9 nick the crRNA-complementary and noncomplementary DNA strands, respectively, variants of Cas9 that contain an CRISPR-Based Typing inactivating mutation at either of these sites can readily Early on, CRISPR loci were primarily used as generate strand-specific nicks at an exact location of templates for genotyping given their hypervariable interest58 (Figure 5). An advantage of nicks over blunt nature.17 While studies have specifically documented breaks is the precision of the error-free homologous hypervariability in pathogenic species such as Yersinia recombination repair machinery, as opposed to the pestis,6 E. coli74,75 and Salmonella enterica,76,77 there error-prone nonhomologous end-joining machinery. is potential for CRISPR-based genotyping in numer- 17 Accordingly, this flexible and reprogrammable system ous genera and species. Because spacers are typically

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Accordingly, CRISPR can provide valuable insights BOX 1 into phage-host coevolutionary dynamics. Further studies across a broad range of CRISPR=RNAI + ADAPTIVE IMMUNITY pathogenic genera and species will determine whether Although CRISPR and RNAi are both mediated by these hypervariable loci are broadly useful for epi- small noncoding RNAs which guide a ribonucleo- demiological surveys, clinical analyses, and food safety protein complex toward complementary nucleic monitoring. Also, novel bioinformatic tools are neces- acid for sequence-specific cleavage, there are sary for processing of the high-throughput sequencing fundamental differences between CRISPR and data, and the visualization of complex datasets. RNAi. Common denominators include common- In addition to CRISPR-mediated interference alities between the protein, structural and enzy- against viruses and plasmids, as well as programmable matic machinery involved in the interfering RNA DNA cleavage or typing, CRISPR-Cas systems have biogenesis, namely the eukaryotic RNA-induced been documented to play roles in physiological silencing complex (RISC), Dicer and Argonaute processes including biofilm formation, regulation of (ARO), and the prokaryotic Cas and CRISPR- intracellular RNA levels,87 andresistancetostress associated complex for antiviral defense (Cas- in various organisms. An intriguing potential is the cade). In contrast, significant differences include ability to use RNA-targeting systems (such a type III- the composition of the interfering RNAs (length, B systems) to control transcript levels,34,61,87. Perhaps sequence, content) and the physical nature of the other ancillary roles remain to be discovered for these target nucleic acid (DNA for most CRISPR systems, multifunctional systems (Box 1). versus RNA for RNAi). In some ways, CRISPR- Cas systems are arguably more analogous to the eukaryotic adaptive immune system, based CONCLUSION on the fact that spacers capture encounters with particular invasive elements and ‘vaccinate’ CRISPR-Cas systems constitute an adaptive immune the host for subsequent exposures, akin to the system in bacteria and archaea which provides antigen-generating adaptive immune response RNA-guided interference against invasive nucleic of mammals. acids through sequence-specific cleavage. Overall, adaptive and inheritable immunity is built through acquisition of foreign genetic material into CRISPR sequentially inserted in a polarized manner at the loci as spacers that are subsequently transcribed and leader end of the locus,8 they provide a genetic processed into small interfering crRNAs that guide basis for tracking the historical path of a strain, the Cas machinery to cleave homologous nucleic and establishing phylogenetic relationships and shared acids. The intrinsic features of CRISPR-Cas systems ancestry between strains. provide several different avenues for molecular An under-appreciated potential use of CRISPR- biology-based applications centered around RNA- Cas systems is their ability to provide insights into guided interference. Industrial applications include the genetic content and diversity of mixed and com- bacterial starter culture enhancement through iterative plex microbial populations. Indeed, several studies phage resistance build up. Interference against DNA have shown that CRISPR spacer content can be inves- acquisition may also be useful for vaccination tigated to probe ecological diversity and host-viral of bacteria against uptake and dissemination of interactions in natural samples and complex biological undesirable genetic content such as antibiotic systems such as microbial mats, acid mine drainage, resistance cassettes or pathogenic and virulence traits. and human gut microbiome.78–85 Noteworthy, these The many recent and on-going studies on the CRISPR loci can provide time-resolved information about the mechanism of action highlight the potential of these type and sequence of foreign genetic elements to which systems, and will provide a molecular basis for bacteria and archaea have been exposed. Because the exploitation of the versatile Cas proteins. While phage arms race between hosts and their viruses impacts resistance and genotyping have received significant the evolutionary trajectory that both sets of genome attention until now, the ability to readily generate undergo, CRISPR-encoded immunity gives rise to programmable RNA-guided nucleases opens up broad escape viral populations that specifically mutate their avenues for next-generation genome editing, notably genetic content in response to the acquired CRISPR in eukaryotic cell lines. spacers. Mutations patterns typically include alter- A key contribution of CRISPR-Cas systems ations (single nucleotide polymorphisms or insertions moving forward is their unique ability to provide and deletions) of the proto-spacer or the PAM.15,23,86 insights into the genetic biodiversity of complex

274  2013 John Wiley & Sons, Ltd. Volume 4, May/June 2013 WIREs RNA CRISPR-Cas systems and RNA-guided interference communities, and reveal the evolutionary and highly the angles through which the scientific community dynamic genomic interplay between viruses and their is investigating these fascinating systems, including hosts. With ever-increasing sequencing throughput, phylogenetic studies, genetic analyses, mathemati- these hypervariable loci will provide a solid basis for cal modelling, evolutionary surveys, epidemiological data analysis and interpretation of population com- studies, and now genome editing. Between emerging position and dynamics in complex systems. While applications in eukaryotic genome engineering, and publication and citation rates reflect scientific interest rapidly increasing interest in the field, the intellectual and activity in the CRISPR field, the emergence of property landscape will rapidly become competitive. pending patent applications (12 published filings to It is important to highlight the frenetic pace at date) illustrate the potential industrial and commer- which the CRISPR literature is evolving, which illus- cial applications of these fantastic systems. Certainly, trates the speed at which the topic is expanding and the diversity of CRISPR-related topics and foci reflect evolving.

ACKNOWLEDGMENT I would like to acknowledge my former DuPont colleagues and many academic collaborators with whom I have had the pleasure to explore CRISPR-Cas systems.

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FURTHER READING Barrangou R, van der Oost J, eds. CRISPR-Cas Systems: RNA-Mediated Adaptive Immunity in Bacteria and Archaea. Heidelberg: Springer; 2013. ISBN: 978-3-642-34656-9.

278  2013 John Wiley & Sons, Ltd. Volume 4, May/June 2013