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ScienceDirect Gene regulation by engineered CRISPR-Cas systems Peter C Fineran and Ron L Dy The clustered regularly interspaced short palindromic repeat (CRISPR) arrays and their CRISPR associated (Cas) proteins constitute adaptive immune systems in bacteria and archaea that provide protection from bacteriophages, plasmids and other mobile genetic elements (MGEs). Recently, the ability to direct these systems to DNA in a sequence-specific manner has led to the emergence of new technologies for engineered gene regulation in bacteria and eukaryotes. These systems have the potential to enable facile high-throughput functional genomics studies aimed at identifying gene function and will be a crucial tool for synthetic biology. Here, we review the recent engineering of these systems for controlling gene expression. Addresses Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand Corresponding author: Fineran, Peter C ([email protected])

Current Opinion in Microbiology 2014, 18:83–89 This review comes from a themed issue on Cell regulation Edited by Cecilia Arraiano and Gregory M Cook

1369-5274/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mib.2014.02.007

Introduction Prokaryotes are exposed to countless infections from their viruses (bacteriophages) and also to extensive horizontal gene transfer, such as from plasmids and other MGEs. These events can yield niche-specific benefits to the bacterium, for example through acquisition of virulence factors. More commonly, MGEs pose an immediate threat (i.e. lytic viral infection) or more subtle costs associated with their replication. Bacteria have a range of mechanisms to balance these invasions, including the small RNA-based CRISPR-Cas adaptive immune systems [1–3]. CRISPR arrays are composed of short repeats of 30 bp that are separated by unique sequences of a similar size (termed spacers; see Glossary box). The spacers are derived from the foreign nucleic acids and are the basis for the sequencespecific memory of past incursions. The CRISPR-Cas mechanism has three stages: (i) adaptation, (ii) expression and (iii) interference (Figure 1). During adaptation, short nucleic acid sequences from the invader are incorporated into the CRISPR array at the promoter (leader) end, in a process requiring Cas1 and Cas2 proteins (reviewed in [4]). In the expression phase, the repeat array is transcribed as a www.sciencedirect.com

long precursor CRISPR RNA (pre-crRNA) and processed into short crRNAs that contain all or part of the spacer and flanking repeat(s). For interference, the crRNAs form a ribonucleoprotein complex with Cas protein(s) and guide this to the foreign nucleic acid (DNA or RNA), resulting in target degradation. For further details about the mechanisms of the different CRISPR-Cas systems see Box 1 and recent reviews [5–9]. The high-throughput ‘omics’ era is providing large quantities of genome sequences, transcriptomic, proteomic, metabolomic and phenomic data. These advances must be complemented by functional genomics approaches that enable the study of gene function. In bacteria, new technologies, such as random transposon insertion sequencing, are being developed [23], but it is essential to also have directed tools to analyze gene function. Recently, the ability of the CRISPR-Cas systems to specifically recognize and cleave unique DNA sequences has been exploited for rapid genome editing in diverse organisms, including bacteria [24,25]. To study gene function (especially of essential genes), and to engineer regulatory circuits for systems biology applications, it is desirable to up-regulate or down-regulate particular genes and this can be achieved by co-opted CRISPR-Cas systems. This review focuses on how CRISPR-Cas systems can be been harnessed to enable the controlled regulation of gene expression.

Transcriptional activation and repression by Cas9 The Type II CRISPR-Cas system is suitable for most genetic engineering applications, since only a single protein, Cas9, in complex with trans-activating crRNA (tracrRNA) and the crRNA, is required for specific targeting (see Box 1 and Figure 2a). The ability to specifically target the binding of Cas9 to different genomic regions, by virtue of a short crRNA, has led to the development of transcriptional control strategies in bacteria and eukaryotes [26,27,28,29,30,31,32,33,34]. These approaches typically rely on mutant forms of Streptococcus pyogenes Cas9 (termed dCas9) that have mutations in the RuvC and HNH domains (see Box 1) that abolish the nuclease activity [17]. Below, we first discuss the use of Cas9 and derivatives for transcriptional regulation in bacteria and then we highlight studies showing similar methods are possible in eukaryotes. In bacteria, three recent studies demonstrated that plasmid or chromosomal gene expression can be repressed or activated in Escherichia coli by different Cas9 proteins from S. pyogenes, Streptococcus thermophilus, Neisseria meningitidis Current Opinion in Microbiology 2014, 18:83–89

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Box 1 The three Types of CRISPR-Cas systems

Figure 1

Cas genes

leader

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Adaptation Phage DNA

leader CRISPR array transcription pre-crRNA

RNA [Type III-B]

Processing/ Maturation

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Targeting

degraded

Current Opinion in Microbiology

Model for CRISPR-Cas adaptive immunity. Short sequences are acquired from invaders (i.e. phages and plasmids) and incorporated into CRISPR arrays. CRISPR arrays are transcribed into precursor CRISPR RNAs (pre-crRNAs). Cas proteins process the pre-crRNAs into crRNAs, which contain a spacer flanked by repeat remnants. The crRNAs guide Cas interference complexes (gray) to their DNA targets and elicit degradation. The Type III-B systems are the exception, where RNA is targeted and degraded.

and Treponema denticola [27,32,33]. Furthermore, Bikard et al. demonstrated wider host applicability, by repressing transcription in the gram-positive bacterium, Streptococcus pneumoniae [33]. To repress transcription, two studies used dCas9 and a single guide RNA (sgRNA) [27,32], which is a functional fusion of the tracrRNA and crRNA [17] (Figure 2b). The third study utilized tracrRNA and engineered crRNAs [33]. The dCas9:sgRNA (or dCas9:tracrRNA:crRNA) complexes, when directed to different sites, reversibly inhibited transcription initiation, elongation and the binding of transcription factors [27,32,33] (Figure 2c). Transcription initiation was decreased with guide RNAs that targeted either DNA strand near the promoter, with strongest effects at the -35 sequence [32,33]. By contrast, transcriptional elongation was inhibited when dCas9 targeted the nontemplate strand of either the 50 untranslated region (UTR) or the coding sequence, whereas binding the template strand had a limited effect [27,32,33]. Native elongating transcript sequencing was used to map the 30 transcript ends associated with RNA polymerase (RNAP), which showed that RNAP paused upstream of the Cas9:sgRNA target, supporting a collision model [32]. Interestingly, the position of targeting relative to the start codon correlated with the level of repression, with protospacers closest to the ATG most effective [32,33]. Current Opinion in Microbiology 2014, 18:83–89

CRISPR-Cas systems are diverse and are classified into three major Types (I–III) and further into subtypes, based on their cas genes [10]. In the Type I systems the pre-crRNA is processed by Cas6-family endonucleases and a ribonucleoprotein complex is formed with the crRNA, Cas6 and other subtype-specific proteins [11,12]. These complexes are termed Cascade (CRISPR associated complex for antiviral defence) and bind to DNA targets (termed protospacers) that are complementary to the guide crRNA [11]. The presence of short sequences flanking the protospacer (termed protospacer adjacent motifs; PAMs) and a seed sequence matching the 8 nt at the 50 end of the spacer portion of the crRNA are required for proficient interference [13]. When these requirements are met, Cas3, a nuclease/helicase unique to Type I systems, is recruited to the protospacer and degrades the invader [14]. Type II systems generate their crRNAs differently (Figure 2a). A small RNA encoded in the vicinity of the cas genes, termed tracrRNA (trans-activating crRNA), is complementary to the CRISPR repeats and forms a tracrRNA:crRNA hybrid that is cleaved in the presence of host RNase III and Cas9 [15]. The resulting crRNA contains at its 50 , 20 nt of the 30 end of the spacer followed by 19-22 nt of the 50 end of the repeat [15]. The Cas9:tracrRNA:crRNA complex then identifies the DNA with correct PAM and protospacer [16] and through two separate single stranded DNA nicks, mediated by HNH and RuvC nuclease domains, generates a double strand break and inactivates the invader [17]. Finally, the Type III systems, like Type I, generate crRNAs with 8 nt 50 repeat-derived ‘handles’ and 30 truncated spacer regions through the processing activity of Cas6 homologues and an unidentified nuclease [18]. By contrast, the crRNA forms part of protein complexes that lack Cas6 [19,20]. The crRNAs guide these complexes to targets, resulting in cleavage of DNA (III-A) [21] or RNA (III-B) [20] (Figure 3a). A recent study has shown a novel CRISPR type that lacks cas genes and requires host polynucleotide phosphorylase (PNPase) for CRISPR transcript processing [22].

Furthermore, repression was increased with two sgRNAs targeting the same gene and multiple genes could be regulated by using different sgRNAs [32]. Specificity is crucial if these technologies are to be widely implemented. Qi et al. tested Cas9:sgRNA specificity in E. coli using RNA-sequencing and revealed that only the target gene was altered [32]. Specific guide RNA features required for repression were also examined. The optimal length of complementarity for repression was 20 nt, but 12 nt of the 30 end of the spacer was sufficient [32,33]. Point mutations also led to weaker effects, which depended on the position and number of mutations, with the 7 nt protospacer adjacent motif (PAM)-proximal part of the spacer the most crucial [32,33]. This is in agreement with a 30 seed region required for interference in Type II systems [17,24]. There was also a strict requirement for the NGG PAM sequence recognized by dCas9 for repression [32]. Use of appropriate tools, such as CRISPRTarget [35] and Cas-OFFinder [36], will enable identification of potential secondary off-target sites. Fine-tuning expression with mismatches might be feasible; however, the effects differed between protospacers [32,33,37]. Interestingly, wild-type Cas9 containing a crRNA with sufficient mismatches to abolish degradation was proficient for repression [33]. In an elegant proof-of-principle experiment, Qi et al. www.sciencedirect.com

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Figure 2

(a) CRISPR Array

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Gene repression and activation by engineered Type II CRISPR-Cas systems. (a) The mechanism of the Type II CRISPR-Cas mediated interference (for details see Box 1). (b) An engineered single guide RNA (sgRNA) is created as a fusion between tracrRNA and crRNA. (c) Transcription repression: nuclease-deficient Cas9 (dCas9) in complex with specific sgRNAs (or tracRNA and crRNAs) can bind target DNA to inhibit transcriptional initiation, elongation and the binding of transcription factors. (d) Transcription activation: dCas9 fused to domains that assist activation, such as the omega subunit of RNAP or multiples of VP16 in eukaryotes, (e) can promote the upregulation of target genes.

used dCas9:sgRNA repression (termed CRISPRi, CRISPR interference) of the E. coli lactose regulatory pathway to classify activators, repressors and transcription factor binding sites [32]. CRISPRi design strategies have now been developed and are provided in detail elsewhere [37]. By contrast to repression, activation of gene expression, requires the fusion of Cas9 to proteins that promote www.sciencedirect.com

transcription, such as those that help recruit RNAP. To achieve activation in bacteria, dCas9 was fused to the omega (v) subunit of RNAP and delivered, using engineered crRNAs, to sequences upstream of test promoters [33] (Figure 2d,e). In an E. coli rpoZ (v) mutant, activation of up to 23-fold was achieved, demonstrating the feasibility of targeted gene activation in bacteria. The system proved most effective on weak promoters and Current Opinion in Microbiology 2014, 18:83–89

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when dCas9-v was directed to between -80 and -100 relative to the transcriptional start site. Multiple reports have now shown specific activation and repression of recombinant or endogenous genes in eukaryotic cells, including mouse, human, yeast and also in mouse embryos [26,27,28,29,30,31,32]. For example, one Cas9-based system positively or negatively affected genes that influence the developmental pathways of human stem cells [34]. As observed in bacteria, simultaneous regulation of multiple genes was possible [26,28]. In these studies, dCas9 was fused to nuclear localization signals and to multiple copies of the VP16 activation domain (Figure 2d). VP16 is a viral protein that interacts with other host eukaryotic proteins to promote transcription. Targeting modified dCas9:sgRNAs to upstream regions led to activation, and multiple dCas9:sgRNA complexes caused synergistic upregulation [29,30,31] (Figure 2e). Instead of fusing VP16 domains directly to dCas9, sgRNAs containing an additional MS2-binding RNA stem-loop, enabled the recruitment of MS2-VP16 hybrid proteins [30]. This should allow greater flexibility to ‘mix-and-match’ regulatory modules. Repression was also achieved with dCas9 in eukaryotes [32] and the addition of the KRAB (Kru¨ppel associated box) silencing domain enhanced repression (up to 115-fold) [28]. In summary, these studies indicate that engineered activation or repression of specific genes in bacteria and eukaryotes is now a tractable proposition.

Post-transcriptional control by CRISPR-Cas Control of gene expression in bacteria can also be elicited post-transcriptionally, such as by affecting RNA stability and translation. CRISPR-Cas systems can alter RNA stability, which will affect translation of the target protein. The Type III-B (Cmr) systems are unique in that they degrade RNA [20,38,39,40] (Figure 3a). Three different Type III-B systems have been studied in detail and all require crRNAs with 8 nt 50 repeat handles for targeting RNA complementary to the spacer [20,38,39,40]. The Type III-B archaeal complex from Pyrococcus furiosus cleaves protospacer RNA via a ruler mechanism by cutting at sites 14 nt from the 30 end of the two major 45 and 39 nt crRNA species [20,41]. By contrast, the Sulfolobus solfataricus archaeal III-B complex cleaves target RNA site-specifically at UA dinucleotides [40]. Finally, the bacterial III-B complex from Thermus thermophilus carries 40 and 46 nt crRNAs and cuts target RNA at 6 nt intervals in 50 ruler mechanism [39]. The sequence-specific RNA cleavage properties were exploited by Hale et al. to demonstrate that engineered crRNAs could direct the P. furiosus III-B complex to degrade complementary RNAs in vitro [38] (Figure 3a). In the same study, a naturally-occurring crRNA target, generated by antisense transcription within a CRISPR array, was degraded by the Type III-B complex. Taken together, these studies indicate the great potential to engineer the RNA-targetCurrent Opinion in Microbiology 2014, 18:83–89

ing III-B complexes for post-transcriptional silencing. However, thus far, engineered RNA silencing by these systems in vivo has yet to be demonstrated. Other engineered solutions for post-transcriptional control can be envisioned. For example, it is possible that nuclease-deficient Type III-B systems (analogous to dCas9 discussed earlier) can be developed (Figure 3b). By guiding these nuclease deficient III-B (Cmr) complexes to specific RNA sequences, such as ribosome binding sites (RBSs) or coding regions, it might be possible to occlude ribosome binding or block elongation, and thus inhibit translation (Figure 3c). The ribonuclease responsible for protospacer cleavage by Type III systems is unclear, but has been proposed to be Cmr4 [10,39]. Potentially, RNA-targeting systems could be generated based on systems that usually target DNA, such as the Type II systems. Indeed, the Type II Cas9 protein in the pathogen Francisella novicida uses its tracrRNA with a unique RNA (termed scaRNA; small CRISPR-Casassociated RNA) to target and trigger the degradation of a virulence transcript [42]. Synthetic biology involves the generation of biological systems and networks for various uses. A unique approach to reliably control gene expression in synthetic biology has been developed by Arkin and colleagues [43]. The combinatorial ‘plug and play’ approach of synthetic biology involves the joining of different genes and regulatory elements, which affects the sequence context of particular elements, such as promoters or RBSs, making prediction of expression unreliable [44]. The reproducibility of synthetic systems can be improved by physically removing neighboring sequences (e.g. 50 UTRs and other genes) from the RNA following transcription, by using the Cas6f (a.k.a. Csy4) endoribonuclease [43]. In the Type I-F CRISPR-Cas systems, the pre-crRNA is processed by Cas6f, through recognition of the 28 nt repeat, yielding stable crRNAs [45–48,49]. Pseudomonas aeruginosa Cas6f and the 28 bp repeat were exploited to generate transcripts with discrete ends, irrespective of their original context. When the repeat was placed between a random library of variable 50 UTRs and the RBS, cleavage by Cas6f significantly improved the consistency of expression [43] (Figure 3d). Similar results were observed when different promoters, regulatory elements, natural UTRs, RBSs and reporter genes were used. Many genes involved in metabolic and biosynthetic processes are operonic. So, for engineering applications, their robust and reproducible expression is desired. By cleaving bicistrons into monocistrons, the Cas6f-repeat platform allowed reliable gene expression from synthetic operons [43]. Interestingly, ribozymes that cleave 50 UTRs were shown to have the same effect of ‘insulating’ the effects of variable flanking sequence on expression in gene circuits [50]. In theory, CRISPR-Cas endonuclease-repeat pairs with different specificities could be combined into more complex circuits www.sciencedirect.com

Gene regulation by engineered CRISPR-Cas systems Fineran and Dy 87

Figure 3

(a)

CRISPR Array Cas6

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Post-transcriptional control by engineered CRISPR-Cas systems. (a) Interference model for the Type III-B RNA targeting systems (for details see Box 1), which can be engineered to target and degrade specific RNAs. (b) Theoretically, nuclease-deficient III-B system can be loaded with engineered target crRNAs and (c) be guided to mRNAs to inhibit translation by occluding ribosome binding or blocking elongation. (d) In synthetic biology, inconsistency in protein expression, caused by variable flanking sequences, can be reduced by generating defined RNA ends by using a Cas6f-repeat platform that cleaves at an engineered repeat.

to enable separately controllable processing platforms. Finally, the endonuclease-repeat platforms could potentially activate transcripts through removal of RNA secondary structures that inhibit translation. www.sciencedirect.com

Conclusion Research into phage biology has a reputation of providing game-changing tools for biotechnology, such as restriction enzymes and phage display [51]. CRISPR-Cas systems, in Current Opinion in Microbiology 2014, 18:83–89

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particular those derived from the Type II/Cas9 systems, are the latest addition, and are revolutionizing genetic manipulation [25,52]. The ability to now use Cas9-based systems for controlling gene expression provides further possibilities, such as the use of oligonucleotide libraries for genome-wide regulatory screens [32] and the ability to combine both editing/interference and regulatory functions in a single experiment [27]. These technologies still need optimization and this will require a better understanding of how these systems work at a molecular level. It will also be desirable to tune Cas9 and the guide RNAs to more delicately and precisely manipulate expression profiles and genetic circuits. Finally, we foresee an untapped potential for developing post-transcriptional RNA-targeting technologies, such as those based on the Type III-B systems. These will complement the Cas9 transcriptional toolkit and open up options for controlling and assessing post-transcriptional processes.

Conflict of interests The authors have no conflicting financial interests.

Acknowledgements P.C.F. is supported by a Rutherford Discovery Fellowship from the Royal Society of New Zealand and by the University of Otago through the Dean’s Strategic Research Fund. P.C.F. thanks past and present members of his laboratory for helpful discussions and their contribution to CRISPR-Cas research. We thank Max Wilkinson and Raymond Staals for critically reading the manuscript.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Labrie SJ, Samson JE, Moineau S: Bacteriophage resistance mechanisms. Nat Rev Microbiol 2010, 8:317-327.

2.

Samson JE, Magadan AH, Sabri M, Moineau S: Revenge of the phages: defeating bacterial defences. Nat Rev Microbiol 2013, 11:675-687.

3.

4.

5.

Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P: CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007, 315:1709-1712. Fineran PC, Charpentier E: Memory of viral infections by CRISPR-Cas adaptive immune systems: acquisition of new information. Virology 2012, 434:202-209. Sorek R, Lawrence CM, Wiedenheft B: CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu Rev Biochem 2013, 82:237-266.

6.

Barrangou R: CRISPR-Cas systems and RNA-guided interference. Wiley Interdiscip Rev RNA 2013, 4:267-278.

7.

Wiedenheft B, Sternberg SH, Doudna JA: RNA-guided genetic silencing systems in bacteria and archaea. Nature 2012, 482:331-338.

8.

Westra ER, Swarts DC, Staals RH, Jore MM, Brouns SJ, van der Oost J: The CRISPRs, they are A-Changin’: how prokaryotes generate adaptive immunity. Annu Rev Genet 2012, 46:311-339.

9.

Richter C, Chang JT, Fineran PC: Function and regulation of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems. Viruses 2012, 4:2291-2311.

Current Opinion in Microbiology 2014, 18:83–89

10. Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF et al.: Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 2011, 9:467-477. 11. Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J: Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 2008, 321:960-964. 12. Wiedenheft B, Lander GC, Zhou K, Jore MM, Brouns SJ, van der Oost J, Doudna JA, Nogales E: Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature 2011, 477:486-489. 13. Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, van der Oost J, Brouns SJ, Severinov K: Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci U S A 2011, 108:10098-10103. 14. Westra ER, van Erp PB, Kunne T, Wong SP, Staals RH, Seegers CL, Bollen S, Jore MM, Semenova E, Severinov K et al.: CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by cascade and Cas3. Mol Cell 2012, 46:595-605. 15. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E: CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 2011, 471:602-607. 16. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA: DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 2014 http://dx.doi.org/10.1038/nature130. 17. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA,  Charpentier E: A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012, 337:816-821. This study demonstrated that Cas9 contains two RNAs (tracrRNA and crRNA) and targets and cleaves DNA via the HNH and RuvC domains. Furthermore, tracrRNA and crRNA could be fused into a functional sgRNA. 18. Carte J, Wang R, Li H, Terns RM, Terns MP: Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev 2008, 22:3489-3496. 19. Heidrich N, Vogel J: Same same but different: new structural insight into CRISPR-Cas complexes. Mol Cell 2013, 52:4-7. 20. Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, Terns RM, Terns MP: RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 2009, 139:945-956. 21. Marraffini LA, Sontheimer EJ: CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 2008, 322:1843-1845. 22. Sesto N, Touchon M, Andrade JM, Kondo J, Rocha EP, Arraiano CM, Archambaud C, Westhof E, Romby P, Cossart P: A PNPase dependent CRISPR system in Listeria. PLoS Genet 2014, 10:e1004065. 23. van Opijnen T, Camilli A: Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat Rev Microbiol 2013, 11:435-442. 24. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA: RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat  Biotechnol 2013, 31:233-239. The authors demonstrated that Cas9 could be used as an effective method to specifically delete genes in bacterial genomes. 25. Pennisi E: The CRISPR craze. Science 2013, 341:833-836. 26. Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan S, Shivalila CS, Dadon DB, Jaenisch R: Multiplexed activation of endogenous genes by CRISPR-on, an RNAguided transcriptional activator system. Cell Res 2013, 23:1163-1171. 27. Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM:  Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods 2013, 10:1116-1121. www.sciencedirect.com

Gene regulation by engineered CRISPR-Cas systems Fineran and Dy 89

This study demonstrated the ability to use evolutionarily divergent Cas9 proteins in bacterial and eukaryotic cells to separately and simultaneously control gene regulation and phage infection (DNA cleavage).

42. Sampson TR, Saroj SD, Llewellyn AC, Tzeng YL, Weiss DS: A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature 2013, 497:254-257.

28. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, SternGinossar N, Brandman O, Whitehead EH, Doudna JA et al.: CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 2013, 154:442-451.

43. Qi L, Haurwitz RE, Shao W, Doudna JA, Arkin AP: RNA processing  enables predictable programming of gene expression. Nat Biotechnol 2012, 30:1002-1006. This study highlighted a novel use of a Cas6f-repeat platform to selectively cleave synthetic transcripts in vivo to increase the reproducibility of gene expression irrespective of the flanking sequence context.

29. Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK: CRISPR RNA-guided activation of endogenous human genes. Nat Methods 2013, 10:977-979. 30. Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S,  Yang L, Church GM: CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 2013, 31:833-838. This paper demonstrated that aptamer RNAs could be fused to sgRNAs for the recruitment of fusion proteins containing transcriptional activation domains. The specificity of Cas9:sgRNA regulation was assessed using a unique method involving a barcoded diverse library of binding sites and RNA-seq to determine expression. 31. Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW et al.: RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods 2013, 10:973-976. 32. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA: Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013, 152:1173-1183. 33. Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA:  Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res 2013, 41:7429-7437. The authors were the first to show that bacterial genes could be activated by dCas9 when fused to the omega subunit of RNAP. Wild-type Cas9 was also able to repress genes when sufficient crRNA:protospacer mismatches abrogated the nuclease activity. 34. Kearns NA, Genga RM, Enuameh MS, Garber M, Wolfe SA, Maehr R: Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells. Development 2014, 141:219-223. 35. Biswas A, Gagnon JN, Brouns SJ, Fineran PC, Brown CM: CRISPRTarget: bioinformatic prediction and analysis of crRNA targets. RNA Biol 2013, 10:817-827. 36. Bae S, Park J, Kim JS: Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 2014 http:// dx.doi.org/10.1093/bioinformatics/btu048. 37. Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS: CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat Protoc 2013, 8:2180-2196. 38. Hale CR, Majumdar S, Elmore J, Pfister N, Compton M, Olson S,  Resch AM, Glover CV 3rd, Graveley BR, Terns RM et al.: Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs. Mol Cell 2012, 45:292-302. The authors demonstrated that a Type III-B (Cmr) system could be loaded with engineered crRNAs to direct it to target complementary RNAs in vitro. They also provide the first in vivo evidence of RNA degradation by these systems. 39. Staals RH, Agari Y, Maki-Yonekura S, Zhu Y, Taylor DW, van Duijn E, Barendregt A, Vlot M, Koehorst JJ, Sakamoto K et al.: Structure and activity of the RNA-targeting type III-B CRISPRCas complex of Thermus thermophilus. Mol Cell 2013, 52:135145. 40. Zhang J, Rouillon C, Kerou M, Reeks J, Brugger K, Graham S, Reimann J, Cannone G, Liu H, Albers SV et al.: Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. Mol Cell 2012, 45:303-313. 41. Hale C, Kleppe K, Terns RM, Terns MP: Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA 2008, 14:2572-2579.

www.sciencedirect.com

44. Endy D: Foundations for engineering biology. Nature 2005, 438:449-453. 45. Haurwitz RE, Jinek M, Wiedenheft B, Zhou K, Doudna JA: Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 2010, 329:1355-1358. 46. Haurwitz RE, Sternberg SH, Doudna JA: Csy4 relies on an unusual catalytic dyad to position and cleave CRISPR RNA. EMBO J 2012, 31:2824-2832. 47. Przybilski R, Richter C, Gristwood T, Clulow JS, Vercoe RB, Fineran PC: Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum. RNA Biol 2011, 8:517-528. 48. Sternberg SH, Haurwitz RE, Doudna JA: Mechanism of substrate selection by a highly specific CRISPR endoribonuclease. RNA 2012, 18:661-672. 49. Vercoe RB, Chang JT, Dy RL, Taylor C, Gristwood T, Clulow JS,  Richter C, Przybilski R, Pitman AR, Fineran PC: Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands. PLoS Genet 2013, 9:e1003454. This study demonstrated that engineered CRISPR systems can be used to induce large deletions in bacterial genomes, such as the expulsion of pathogenicity islands. 50. Lou C, Stanton B, Chen YJ, Munsky B, Voigt CA: Ribozymebased insulator parts buffer synthetic circuits from genetic context. Nat Biotechnol 2012, 30:1137-1142. 51. Petty NK, Evans TJ, Fineran PC, Salmond GP: Biotechnological exploitation of bacteriophage research. Trends Biotechnol 2007, 25:7-15. 52. Mali P, Esvelt KM, Church GM: Cas9 as a versatile tool for engineering biology. Nat Methods 2013, 10:957-963. Glossary box Cas (CRISPR associated): Proteins required for the CRISPR-Cas resistance mechanism. These are diverse and encoded in close proximity to the CRISPR arrays. CRISPR (clustered regularly interspaced short palindromic repeats): Arrays of repeat-spacer units in bacteria and archaea that form the CRISPR-Cas memory. crRNAs: The guide RNAs generated after cleavage of the pre-crRNA. The crRNA contains all or part of a single spacer and flanking repeat sequence(s). dCas9: Nuclease deficient mutant derivative of Cas9 with RuvC and HNH mutations. PAM (protospacer adjacent motif): A short sequence stretch (2–8 nt) adjacent to the protospacer that is required for adaptation and interference. pre-crRNA: The full length RNA transcript derived from the CRISPR array. Protospacer: A complementary sequence targeted by a specific crRNA. Seed sequence: A short 8–13 nt region of the spacer that is required for complementary base pairing for interference. sgRNA (single guide RNA): an engineered RNA that combines both tracrRNA and crRNA into a single transcript for gene targeting by Cas9. Spacer: Unique sequences typically derived from foreign DNA that are located between repeats in CRISPR array. tracrRNA (trans-activating crRNA): An antisense RNA encoded near the Type II cas genes, which binds to the pre-crRNA and is required for maturation of crRNAs, in combination with Cas9 and host RNase III.

Current Opinion in Microbiology 2014, 18:83–89