CRISPR-Cas

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PART I

1 Background
- 1.1 CRISPR-Cas defense
- 1.2 CRISPR-Cas diversity, classification, and evolution
2 See aslo

Background

Highlights

CRISPR-Cas9 is a powerful tool to modulate transcription in wide range of cell types.
An expanding set of CRISPR-based transcription effectors is available.
Gene networks can be efficiently probed and modified for biotechnology applications.^[1]

CRISPR-Cas9 has recently emerged as a promising system for multiplexed genome editing as well as epigenome and transcriptome perturbation. Due to its specificity, ease of use and highly modular programmable nature, it has been widely adopted for a variety of applications such as genome editing, transcriptional inhibition and activation, genetic screening, DNA localization imaging, and many more. In this review, we will discuss non-editing applications of CRISPR-Cas9 for transcriptome perturbation, metabolic engineering, and synthetic biology.^[1]

Since the early days of genetic engineering there has been a need for control of gene expression. Naturally occurring transcription factors (TFs) have traditionally been used to achieve this goal (reviewed in ^[2]). However, their limited DNA binding sequence space required installing specific sequences within the transcription regulatory elements of the target genes. This can be technically difficult and may have unintended consequences on gene expression. Zinc fingers (ZFs) and transcription activator-like effectors (TALEs) were developed to overcome the fixed binding sequence requirements of native TFs. However, both ZFs and TALEs have significant limitations. ZFs have complicated design criteria and large highly repetitive TALE genes are difficult to synthesize and clone (reviewed in ^[3]^[4]). These challenges have recently been overcome using CRISPR-Cas9 based TFs. The biochemical properties of CRISPR-Cas9 based TFs that enable such flexibility and describe their applications to synthetic gene circuit design and multi-plexed perturbation of native gene networks.^[1]

Many bacteria and archaea possess an adaptive immune system consisting of repetitive genetic elements known as clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Similar to RNAi pathways in eukaryotes, CRISPR–Cas systems require small RNAs for sequence-specific detection and degradation of complementary nucleic acids. Cas5 and Cas6 enzymes have evolved to specifically recognize and process CRISPR-derived transcripts into functional small RNAs used as guides by interference complexes. Our detailed understanding of these proteins has led to the development of several useful Cas6-based biotechnological methods. The structures, functions, mechanisms, and applications of the enzymes responsible for CRISPR RNA (crRNA) processing, highlighting a fascinating family of endonucleases with exquisite RNA recognition and cleavage activities are reviewed.^[5]

CRISPR-Cas defense

The CRISPR-Cas systems provide protection against mobile genetic elements (MGEs) — in particular, viruses and plasmids— by sequence-specific targeting of foreign DNA or RNA ^[6]. A CRISPR-cas locus generally consists of an operon of CRISPR-associated (cas) genes and a CRISPR array composed of a series of direct repeats interspaced by variable DNA sequences (known as spacers) (Fig. 1A). The repeat sequences and lengths as well as the number of repeats in CRISPR arrays vary broadly, but all arrays possess the characteristic arrangement of alternating repeat and spacer sequences. The spacers are key elements of adaptive immunity, as they store the “memory” of an organism’s encounters with specific MGEs acquired as a result of a previous unsuccessful infection. This memory enables the recognition and neutralization of the invaders upon subsequent infections ^[6]^[7]. CRISPR loci are flanked by a diverse set of cas genes that define major CRISPR types based on gene conservation and locus organization^[5]. Despite minimal sequence homology, Cas6s have several conserved structural features that facilitate binding of both the pre-crRNA and their crRNA product with high affinity. In most CRISPR systems, due to the pseudo-palindromic nature of the repeat sequence,the pre-crRNA adopts a stem loop structure that is bound sequence- and shape-specifically and cleaved at its base.^[5] For example, PaeCas6f (Csy4) from Pseudomonas aeruginosa (2xli) in the active site. Some pre-crRNAs are predicted to be unstructured in solution and thus may be bound differently, although base pairing may be stabilized by protein interactions ^[8]^[5].

CRISPR-mediated adaptive immunity involves three steps: adaptation, expression, and interference (Fig. 1B). During the adaptation step, fragments of foreign DNA (known as protospacers) from invading elements are processed and incorporated as new spacers into the CRISPR array. The expression step involves the transcription of the CRISPR array, which is followed by processing of the precursor transcript into mature CRISPR RNAs (crRNAs)^[9]^[7]:

Examples of 3D structures of CRISPR RNA (crRNA)

from Acidaminococcus sp. BV3L6 (5kk5).
(5b43).
(5h9f).
(4u7u).

The crRNAs are assembled with one or more Cas proteins into CRISPR ribonucleoprotein (crRNP) complexes^[7].

Example of crRNP complex with one Cas protein: (5f9r).
Example of crRNP complex with several Cas proteins: (4qyz).

The interference step involves crRNA-directed cleavage of invading cognate virus or plasmid nucleic acids by Cas nucleases within the crRNP complex ^[7]. An interference complex of CRISPR-associated (Cas) proteins uses the mature crRNA as a guide to target and destroy foreign nucleic acids bearing sequence complementarity ^[10]^[9].

Fig. 1 Overview of the CRISPR-Cas systems. (A) Architecture of class 1 (multiprotein effector complexes) and class 2 (single-protein effector complexes) CRISPR-Cas systems. (B) CRISPR-Cas adaptive immunity is mediated by CRISPR RNAs (crRNAs) and Cas proteins, which form multicomponent CRISPR ribonucleoprotein (crRNP) complexes. The first stage is adaptation, which occurs upon entry of an invading mobile genetic element (in this case, a viral genome). Cas1 (blue) and Cas2 (yellow) proteins select and process the invading DNA, and thereafter, a protospacer (orange) is integrated as a new spacer at the leader end of the CRISPR array [repeat sequences (gray) that separate similar-sized, invader-derived spacers (multiple colors)]. During the second stage, expression, the CRISPR locus is transcribed and the pre-crRNA is processed into mature crRNA guides by Cas (e.g., Cas6) or non-Cas proteins (e.g., RNase III). During the final interference stage, the Cas-crRNA complex scans invading DNA for a complementary nucleic acid target, after which the target is degraded by a Cas nuclease. From ^[7]

CRISPR-Cas diversity, classification, and evolution

Classification according to the Wikipedia page CRISPR [1] with additions

CRISPR Class 1 uses a complex of multiple Cas proteins

CRISPR type I (Cas3)

CRISPR type I-A (Cascade) - see CRISPR subtype I-A

CRISPR type I-B (Cascade) - see CRISPR subtype I-B

CRISPR type I-C (Cascade) - see CRISPR subtype I-C

CRISPR type I-D (Cas10d)

CRISPR type I-E (Cascade) - see CRISPR subtype I-E

CRISPR type I-F (Csy1, Csy2, Csy3) - see CRISPR subtype I-F

CRISPR type I-U (GSU0054)

CRISPR type III (Cas10)

CRISPR type III-A (Csm complex) - see CRISPR subtype III-A (Csm complex)

CRISPR type III-B (Cmr complex)

CRISPR type III-C (Cas10 or Csx11)

CRISPR type III-D (Csx10)

CRISPR type Orphan

CRISPR type IV (Csf1)

CRISPR type IV-A

CRISPR type IV-B

CRISPR Class 2 uses a single large Cas protein

CRISPR type II-A - see CRISPR-Cas9

CRISPR type II-B (Cas4)

CRISPR type II-C

CRISPR type V (Cpf1, C2c1, C2c3) - see CRISPR type V

CRISPR type VI (Cas13a (previously known as C2c2), Cas13b, Cas13c, Cas13d) - see CRISPR type VI

The rapid evolution of highly diverse CRISPR-Cas systems is thought to be driven by the continuous arms race with the invading MGEs. The latest classification scheme for CRISPR-Cas systems, which takes into account the repertoire of cas genes and the sequence similarity between Cas proteins and the locus architecture, includes two classes that are currently subdivided into six types and 19 subtypes ^[7]^[11]^[12]. The key feature of the organization and evolution of the CRISPR-Cas loci is their pronounced modularity. The module responsible for the adaptation step is largely uniform among the diverse CRISPR-Cas systems and consists of the cas1 and cas2 genes, both of which are essential for the acquisition of spacers. In many CRISPR-Cas variants, the adaptation module also includes the cas4 gene. By contrast, the CRISPR-Cas effector module, which is involved in the maturation of the crRNAs as well as in target recognition and cleavage, shows a far greater versatility (Fig. 2A) ^[7]^[11].

Figure. 2. CRISPR diversity and evolution. (A) Modular organization of the CRISPR-Cas systems. LS, large subunit; SS, small subunit. A putative small subunit that might be fused to the large subunit in several type I subtypes is indicated by an asterisk. Cas3 is shown as fusion of two distinct genes encoding the helicase Cas3′ and the nuclease HD Cas3′′; in some type I systems, these domains are encoded by separate genes. Functionally dispensable components are indicated by dashed outlines. Cas6 is shown with a thin solid outline for type I because it is dispensable in some systems, and by a dashed line for type III because most systems lack this gene and use the Cas6 provided in trans by other CRISPR-Cas loci. The two colors for Cas4 and C2c2 and three colors for Cas9 and Cpf1 reflect the contributions of these proteins to different stages of the CRISPR-Cas response (see text). The question marks indicate currently unknown components. From ^[7]^[11] (B) Evolutionary scenario for the CRISPR-Cas systems. TR, terminal repeats; TS, terminal sequences; HD, HD-family endonuclease; HNH, HNH-family endonuclease; RuvC, RuvC-family endonuclease; HEPN, putative endoribonuclease of HEPN superfamily. Genes and portions of genes shown in gray denote sequences that are thought to have been encoded in the respective mobile elements but were eliminated in the course of evolution of CRISPR-Cas systems. From ^[7]^[12]

The 2 classes of CRISPR-Cas systems differ fundamentally with respect to the organization of the effector module ^[11]. Class 1 systems (including types I, III, and IV) are present in bacteria and archaea, and encompass effector complexes composed of 4-7 Cas protein subunits [e.g., the (CRISPR-associated complex for antiviral defense) (Cascade) of type I systems, and the Csm/Cmr complexes of type III systems]. Most of the subunits of the class 1 effector complexes — in particular, Cas5, Cas6, and Cas7—contain variants of the RNA-binding RRM (RNA recognition motif) domain.^[7]

Examples of RRM fold

(2i0x).
(3kg4).
(3i4h).
(1wj9).
(2xlj).
(3ps0).

Although the sequence similarity between the individual subunits of type I and type III effector complexes is generally low, the complexes share strikingly similar overall architectures that suggest a common origin ^[12]. The ancestral CRISPR-Cas effector complex most likely resembled the extant type III complexes, as indicated by the presence of the archetypal type III protein, the large Cas10 subunit, which appears to be an active enzyme of the DNA polymerase–nucleotide cyclase superfamily, unlike its inactive type I counterpart (Cas8) ^[12]^[7]. The cas6 gene family encodes a set of RNA endonucleases responsible for crRNA processing in Type I and Type III CRISPR systems. Type II systems use a trans-activating RNA (tracrRNA) together with endogenous RNase III for crRNA maturation. In Type I-B, I-C, I-E, and I-F systems, the endoRNase stays bound to the crRNA and assembles into a complex with other Cas proteins for downstream targeting ^[9], while in Type I-A and III systems, the crRNA alone is loaded into the targeting complex and Cas6 dissociates ^[5].

In the less common class 2 CRISPR-Cas systems (types II, V, and VI), which are almost completely restricted to bacteria, the effector complex is represented by a single multidomain protein ^[11]. The best-characterized class 2 effector is Cas9 (type II), the RNA-dependent endonuclease that contains two unrelated nuclease domains, HNH and RuvC, that are responsible for the cleavage of the target and the displaced strand, respectively, in the crRNA–target DNA complex (, 4zt0). The type II loci also encode a trans-acting CRISPR RNA (tracrRNA) that evolved from the corresponding CRISPR repeat and is essential for pre-crRNA processing and target recognition in type II systems. Cas9 is directed to its DNA targets by forming a ribonucleoprotein complex with these 2 small non-coding RNAs: crRNA and tracrRNA. By elegant engineering, (4zt9^[13]) that too efficiently directs Cas9 protein to DNA targets encoded within the guide sequence of sgRNA ^[14]:

Examples of 3D structures of single guide RNA (sgRNA)

(5fw2).
(5b2s).
(5b2p).
(4axw).

The , termed the guide sequence, adjacent to a ^[14]^[15]. Despite this, a ^[14]^[16]^[17]^[18], more so within the 5’ proximal position of the guide sequence.

The prototype type V effector Cpf1 (subtype V-A) contains only one nuclease domain (RuvC-like) that is identifiable by sequence analysis. However, analysis of the recently solved structure of has revealed a second nuclease domain, the fold of which is unrelated to HNH or any other known nucleases. In analogy to the HNH domain in Cas9, the , and it is responsible for cleavage of the target strand.^[7]^[19]

Screening of microbial genomes and metagenomes for undiscovered class 2 systems has resulted in the identification of three novel CRISPR-Cas variants. These include subtypes V-B and V-C, which resemble Cpf1 in that their predicted effector proteins contain a single, RuvC-like nuclease domain. Cleavage of target DNA by the type V-B effector, denoted C2c1, has been experimentally demonstrated. Type VI is unique in that its effector protein contains two conserved HEPN domains that possess ribonuclease (RNase) activity (Fig. 2A).^[7]^[12]

Recent comparative genomic analyses of variant CRISPR-Cas systems (Fig. 2B) ^[12] have revealed a strong modular evolution with multiple combinations of adaptation modules and effector modules, as well as a pivotal contribution of mobile genetic elements to the origin and diversification of the CRISPR-Cas systems. The ancestral prokaryotic adaptive immune system could have emerged via the insertion of a casposon (a recently discovered distinct class of self-synthesizing transposons that appear to encode a Cas1 homolog) next to an innate immunity locus (probably consisting of genes encoding a Cas10 nuclease and possibly one or more RNA binding proteins). Apart from providing the Cas1 nuclease/integrase that is required for recombination during spacer acquisition, the casposon may also have contributed the prototype CRISPR repeat unit that could have evolved from one of the inverted terminal repeats of the casposon. An additional toxin-antitoxin module that inserted either in the ancestral casposon or in the evolving adaptive immunity locus probably provided the cas2 gene, thus completing the adaptation module. The Cas10 nuclease and one or more additional proteins with an RRM fold (the ultimate origin of which could be a polymerase or cyclase that gave rise to Cas10) of the hybrid locus could have subsequently evolved to become the ancestral CRISPR-Cas effector module ^[12]^[7].

The widespread occurrence of class 1 systems in archaea and bacteria, together with the proliferation of the ancient RRM domain in class 1 effector proteins, strongly suggests that the ancestral CRISPR-Cas belonged to class 1. Most likely, the multiple class 2 variants then evolved via several independent replacements of the class 1 effector locus with nuclease genes that were derived from distinct MGEs (Fig. 2B). In particular, type V effector variants (Cpf1) seem to have evolved from different families of the TnpB transposase genes that are widespread in transposons ^[12], whereas the type II effector (Cas9) may have evolved from IscB, a protein with two nuclease domains that belongs to a recently identified distinct transposon family. Notably, class 2 CRISPR-Cas systems, in their entirety, appear to have been derived from different MGEs: Cas1 from a casposon, Cas2 from a toxin-antitoxin module, and the different effector proteins (such as Cas9 and Cpf1) from respective transposable elements ^[12]^[7].

SEE CRISPR-Cas Part II

See aslo

References

↑ ^1.0 ^1.1 ^1.2 Didovyk A, Borek B, Tsimring L, Hasty J. Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications. Curr Opin Biotechnol. 2016 Aug;40:177-84. doi: 10.1016/j.copbio.2016.06.003. Epub, 2016 Jun 23. PMID:27344519 doi:http://dx.doi.org/10.1016/j.copbio.2016.06.003
↑ Brophy JA, Voigt CA. Principles of genetic circuit design. Nat Methods. 2014 May;11(5):508-20. doi: 10.1038/nmeth.2926. PMID:24781324 doi:http://dx.doi.org/10.1038/nmeth.2926
↑ Straubeta A, Lahaye T. Zinc fingers, TAL effectors, or Cas9-based DNA binding proteins: what's best for targeting desired genome loci? Mol Plant. 2013 Sep;6(5):1384-7. doi: 10.1093/mp/sst075. Epub 2013 May 29. PMID:23718948 doi:http://dx.doi.org/10.1093/mp/sst075
↑ Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014 Apr;32(4):347-55. doi: 10.1038/nbt.2842. Epub 2014 Mar 2. PMID:24584096 doi:http://dx.doi.org/10.1038/nbt.2842
↑ ^5.0 ^5.1 ^5.2 ^5.3 ^5.4 Hochstrasser ML, Doudna JA. Cutting it close: CRISPR-associated endoribonuclease structure and function. Trends Biochem Sci. 2015 Jan;40(1):58-66. doi: 10.1016/j.tibs.2014.10.007. Epub, 2014 Nov 18. PMID:25468820 doi:http://dx.doi.org/10.1016/j.tibs.2014.10.007
↑ ^6.0 ^6.1 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 Mar 23;315(5819):1709-12. PMID:17379808 doi:http://dx.doi.org/10.1126/science.1138140
↑ ^7.00 ^7.01 ^7.02 ^7.03 ^7.04 ^7.05 ^7.06 ^7.07 ^7.08 ^7.09 ^7.10 ^7.11 ^7.12 ^7.13 ^7.14 Mohanraju P, Makarova KS, Zetsche B, Zhang F, Koonin EV, van der Oost J. Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems. Science. 2016 Aug 5;353(6299):aad5147. doi: 10.1126/science.aad5147. PMID:27493190 doi:http://dx.doi.org/10.1126/science.aad5147
↑ Kunin V, Sorek R, Hugenholtz P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol. 2007;8(4):R61. PMID:17442114 doi:http://dx.doi.org/10.1186/gb-2007-8-4-r61
↑ ^9.0 ^9.1 ^9.2 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 Aug 15;321(5891):960-4. doi: 10.1126/science.1159689. PMID:18703739 doi:http://dx.doi.org/10.1126/science.1159689
↑ Garneau JE, Dupuis ME, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadan AH, Moineau S. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 2010 Nov 4;468(7320):67-71. doi: 10.1038/nature09523. PMID:21048762 doi:http://dx.doi.org/10.1038/nature09523
↑ ^11.0 ^11.1 ^11.2 ^11.3 ^11.4 Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ, Charpentier E, Haft DH, Horvath P, Moineau S, Mojica FJ, Terns RM, Terns MP, White MF, Yakunin AF, Garrett RA, van der Oost J, Backofen R, Koonin EV. An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol. 2015 Nov;13(11):722-36. doi: 10.1038/nrmicro3569. Epub 2015, Sep 28. PMID:26411297 doi:http://dx.doi.org/10.1038/nrmicro3569
↑ ^12.0 ^12.1 ^12.2 ^12.3 ^12.4 ^12.5 ^12.6 ^12.7 ^12.8 Shmakov S, Abudayyeh OO, Makarova KS, Wolf YI, Gootenberg JS, Semenova E, Minakhin L, Joung J, Konermann S, Severinov K, Zhang F, Koonin EV. Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Mol Cell. 2015 Nov 5;60(3):385-97. doi: 10.1016/j.molcel.2015.10.008. Epub 2015, Oct 22. PMID:26593719 doi:http://dx.doi.org/10.1016/j.molcel.2015.10.008
↑ Jiang F, Zhou K, Ma L, Gressel S, Doudna JA. STRUCTURAL BIOLOGY. A Cas9-guide RNA complex preorganized for target DNA recognition. Science. 2015 Jun 26;348(6242):1477-81. doi: 10.1126/science.aab1452. PMID:26113724 doi:http://dx.doi.org/10.1126/science.aab1452
↑ ^14.0 ^14.1 ^14.2 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 Aug 17;337(6096):816-21. doi: 10.1126/science.1225829. Epub 2012, Jun 28. PMID:22745249 doi:http://dx.doi.org/10.1126/science.1225829
↑ Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):E2579-86. Epub 2012 Sep 4. PMID:22949671 doi:http://dx.doi.org/10.1073/pnas.1208507109
↑ 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 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022. PMID:23452860 doi:http://dx.doi.org/10.1016/j.cell.2013.02.022
↑ 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 Aug;41(15):7429-37. doi: 10.1093/nar/gkt520. Epub 2013, Jun 12. PMID:23761437 doi:http://dx.doi.org/10.1093/nar/gkt520
↑ Kuscu C, Arslan S, Singh R, Thorpe J, Adli M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Biotechnol. 2014 Jul;32(7):677-83. doi: 10.1038/nbt.2916. Epub 2014 May 18. PMID:24837660 doi:http://dx.doi.org/10.1038/nbt.2916
↑ Krupovic M, Makarova KS, Forterre P, Prangishvili D, Koonin EV. Casposons: a new superfamily of self-synthesizing DNA transposons at the origin of prokaryotic CRISPR-Cas immunity. BMC Biol. 2014 May 19;12:36. doi: 10.1186/1741-7007-12-36. PMID:24884953 doi:http://dx.doi.org/10.1186/1741-7007-12-36

Proteopedia Page Contributors and Editors (what is this?)

Alexander Berchansky, Michal Harel

Retrieved from "http://52.214.119.220/wiki/index.php/CRISPR-Cas"

@@ Line 1: / Line 1: @@
-<StructureSection load='4qyz' size='450' side='right' scene='74/742625/Cv/4' caption=''>
+<StructureSection load='4qyz' size='350' side='right' scene='74/742625/Cv/4' caption='E. coli CRISPR complex with RNA and DNA (PDB code [[4qyz]])'>
+'''PART I'''
 =Background=
 Highlights
@@ Line 7: / Line 8: @@
 *Gene networks can be efficiently probed and modified for biotechnology applications.<ref name="Did">PMID:27344519</ref>
-CRISPR-Cas9 has recently emerged as a promising system for multiplexed genome editing as well as epigenome and transcriptome perturbation. Due to its specificity, ease of use and highly modular programmable nature, it has been widely adopted for a variety of applications such as genome editing, transcriptional inhibition and activation, genetic screening, DNA localization imaging, and many more. In this review, we will discuss non-editing applications of CRISPR-Cas9 for transcriptome perturbation, metabolic engineering, and synthetic biology.<ref name="Did">PMID:27344519</ref>
+'''CRISPR-Cas9''' has recently emerged as a promising system for multiplexed genome editing as well as epigenome and transcriptome perturbation. Due to its specificity, ease of use and highly modular programmable nature, it has been widely adopted for a variety of applications such as genome editing, transcriptional inhibition and activation, genetic screening, DNA localization imaging, and many more. In this review, we will discuss non-editing applications of CRISPR-Cas9 for transcriptome perturbation, metabolic engineering, and synthetic biology.<ref name="Did">PMID:27344519</ref>
 Since the early days of genetic engineering there has been a need for control of gene expression. Naturally occurring transcription factors (TFs) have traditionally been used to achieve this goal (reviewed in <ref name="Prin1">PMID:24781324</ref>). However, their limited DNA binding sequence space required installing specific sequences within the transcription regulatory elements of the target genes. This can be technically difficult and may have unintended consequences on gene expression. Zinc fingers (ZFs) and transcription activator-like effectors (TALEs) were developed to overcome the fixed binding sequence requirements of native TFs. However, both ZFs and TALEs have significant limitations. ZFs have complicated design criteria and large highly repetitive TALE genes are difficult to synthesize and clone (reviewed in <ref name="Prin2">PMID:23718948</ref><ref name="Prin3">PMID:24584096</ref>). These challenges have recently been overcome using CRISPR-Cas9 based TFs. The biochemical properties of CRISPR-Cas9 based TFs that enable such flexibility and describe their applications to synthetic gene circuit design and multi-plexed perturbation of native gene networks.<ref name="Did">PMID:27344519</ref>
@@ Line 35: / Line 36: @@
 ==CRISPR-Cas diversity, classification, and evolution==
-The rapid evolution of highly diverse CRISPRCas systems is thought to be driven by the continuous arms race with the invading MGEs. The latest classification scheme for CRISPR-Cas systems, which takes into account the repertoire of ''cas'' genes and the sequence similarity between Cas proteins and the locus architecture, includes two classes that are currently subdivided into six types and 19 subtypes <ref name="Rev4">doi:10.1126/science.aad5147</ref><ref name="Rev430">doi:10.1038/nrmicro3569</ref><ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref>. The key feature of the organization and evolution of the CRISPR-Cas loci is their pronounced modularity. The module responsible for the adaptation step is largely uniform among the diverse CRISPR-Cas systems and consists of the ''cas1'' and ''cas2'' genes, both of which are essential for the acquisition of spacers. In many CRISPR-Cas variants, the adaptation module also includes the ''cas4'' gene. By contrast, the CRISPR-Cas effector module, which is involved in the maturation of the crRNAs as well as in target recognition and cleavage, shows a far greater versatility (Fig. 2A) <ref name="Rev4">doi:10.1126/science.aad5147</ref><ref name="Rev430">doi:10.1038/nrmicro3569</ref>.
+'''Classification according to the Wikipedia page CRISPR [https://en.wikipedia.org/wiki/CRISPR] with additions'''
-[[Image:F2r.jpg|left|450px|thumb|Figure. 2. CRISPR diversity and evolution. (A) Modular organization of the CRISPR-Cas systems. LS, large subunit; SS, small subunit. A putative small subunit that might be fused to the large subunit in several type I subtypes is indicated by an asterisk. Cas3 is shown as fusion of two distinct genes encoding the helicase Cas3′ and the nuclease HD Cas3′′; in some type I systems, these domains are encoded by separate genes. Functionally dispensable components are indicated by dashed outlines. Cas6 is shown with a thin solid outline for type I because it is dispensable in some systems, and by a dashed line for type III because most systems lack this gene and use the Cas6 provided in trans by other CRISPR-Cas loci. The two colors for Cas4 and C2c2 and three colors for Cas9 and Cpf1 reflect the contributions of these proteins to different stages of the CRISPR-Cas response (see text). The question marks indicate currently unknown components. From <ref name="Rev4">doi:10.1126/science.aad5147</ref><ref name="Rev430">doi:10.1038/nrmicro3569</ref> (B) Evolutionary scenario for the CRISPR-Cas systems. TR, terminal repeats; TS, terminal sequences; HD, HD-family endonuclease; HNH, HNH-family endonuclease; RuvC, RuvC-family endonuclease; HEPN, putative endoribonuclease of HEPN superfamily. Genes and portions of genes shown in gray denote sequences that are thought to have been encoded in the respective mobile elements but were eliminated in the course of evolution of CRISPR-Cas systems. From <ref name="Rev4">doi:10.1126/science.aad5147</ref><ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref>]]
+'''CRISPR Class 1 uses a complex of multiple Cas proteins'''
-{{Clear}}
-The 2 classes of CRISPR-Cas systems differ fundamentally with respect to the organization of the effector module <ref name="Rev430">doi:10.1038/nrmicro3569</ref>. Class 1 systems (including types I, III, and IV) are present in bacteria and archaea, and encompass effector complexes composed of 4-7 Cas protein subunits [''e.g.'', the ('''C'''RISPR-'''as'''sociated '''c'''omplex for '''a'''ntiviral '''de'''fense) ('''Cascade''') of type I systems, and the Csm/Cmr complexes of type III systems]. Most of the subunits of the class 1 effector complexes — in particular, Cas5, Cas6, and Cas7—contain variants of the RNA-binding RRM (RNA recognition motif) domain. Although the sequence similarity between the individual subunits of type I and type III effector complexes is generally low, the complexes share strikingly similar overall architectures that suggest a common origin <ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref>. The ancestral CRISPR-Cas effector complex most likely resembled the extant type III complexes, as indicated by the presence of the archetypal type III protein, the large Cas10 subunit, which appears to be an active enzyme of the DNA polymerase–nucleotide cyclase superfamily, unlike its inactive type I counterpart (Cas8) <ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref>. The ''cas6'' gene family encodes a set of RNA endonucleases responsible for crRNA processing in Type I and Type III CRISPR systems. Type II systems use a trans-activating RNA (tracrRNA) together with endogenous RNase III for crRNA maturation (Figure 2).In Type I-B, I-C, I-E, and I-F systems, the endoRNase stays bound to the crRNA and assembles into a complex with other Cas proteins for downstream targeting <ref name="Rev312">doi:10.1126/science.1159689</ref>, while in Type I-A and III systems, the crRNA alone is loaded into the targeting complex and Cas6 dissociates <ref name="Rev3">PMID:25468820</ref>.
+CRISPR type I (Cas3)
-In the less common class 2 CRISPR-Cas systems (types II, V, and VI), which are almost completely restricted to bacteria, the effector complex is represented by a single multidomain protein <ref name="Rev430">doi:10.1038/nrmicro3569</ref>. The best-characterized class 2 effector is Cas9 (type II), the RNA-dependent endonuclease that contains two unrelated nuclease domains, HNH and RuvC, that are responsible for the cleavage of the target and the displaced strand, respectively, in the crRNA–target DNA complex (<scene name='74/746096/Cv3/1'>Domain organization of nuclease lobe of Cas9 from S. pyogenes</scene>, [[4zt0]]). The type II loci also encode a trans-acting CRISPR RNA (tracrRNA) that evolved from the corresponding CRISPR repeat and is essential for pre-crRNA processing and target recognition in type II systems. Cas9 is directed to its DNA targets by forming a ribonucleoprotein complex with these 2 small non-coding RNAs: crRNA and tracrRNA. By elegant engineering, <scene name='74/742625/Cv3/8'>crRNA and tracrRNA can be joined end-to-end and transcribed as a single guide RNA (sgRNA)</scene> ([[4zt9]]<ref name="dCAS9">PMID:26113724</ref>) that too efficiently directs Cas9 protein to DNA targets encoded within the guide sequence of sgRNA <ref name="Jinek">PMID:22745249</ref>:
+CRISPR type I-A (Cascade) - see [[CRISPR subtype I-A]]
-''Examples of 3D structures of single guide RNA (sgRNA)''
-*<scene name='74/742625/Cv/42'>Cas9-sgRNA-target DNA complex from Streptococcus pyogenes</scene> ([[5fw2]]).
-*<scene name='74/742625/Cv2/12'>Cas9-sgRNA-target DNA complex from Streptococcus pyogenes</scene> ([[5b2s]]).
-*<scene name='74/742625/Cv2/13'>Cas9-sgRNA-target DNA complex from Francisella tularensis</scene> ([[5b2p]]).
-*<scene name='74/742625/Cv3/2'>Cas9-sgRNA-target DNA complex from Staphylococcus aureus</scene> ([[4axw]]).
-The <scene name='74/742625/Cv3/4'>optimal DNA target of the complex is determined by a Watson–Crick base pairing of a short ∼20-nt sequence within sgRNA (within the crRNA in wild-type)</scene>, termed the guide sequence, adjacent to a <scene name='74/742625/Cv3/10'>few nucleotide long conserved motif recognized directly by Cas9 protein (protospacer adjacent motif, PAM)</scene> <ref name="Jinek">PMID:22745249</ref><ref name="Prin6">PMID:22949671</ref>. Despite this, a <scene name='74/742625/Cv/44'>few mismatches between guide sequence and target DNA can be tolerated</scene> <ref name="Jinek">PMID:22745249</ref><ref name="Prin7">PMID:23452860</ref><ref name="Prin8">PMID:23761437</ref><ref name="Prin9">PMID:24837660</ref>, more so within the 5’ proximal position of the guide sequence.
-The prototype type V effector Cpf1 (subtype V-A) contains only one nuclease domain (RuvC-like) that is identifiable by sequence analysis. However, analysis of the recently solved structure of <scene name='74/746096/Cv5/1'>Cpf1 complexed with the crRNA and target DNA</scene> has revealed a second nuclease domain, the fold of which is unrelated to HNH or any other known nucleases. In analogy to the HNH domain in Cas9, the <scene name='74/746096/Cv5/2'>novel nuclease domain (labeled Nuc) in Cpf1 is inserted into the RuvC domain</scene>, and it is responsible for cleavage of the target strand.<ref name="Rev4">doi:10.1126/science.aad5147</ref><ref name="Cpf1">PMID:24884953</ref>
+CRISPR type I-B (Cascade) - see [[CRISPR subtype I-B]]
-Screening of microbial genomes and metagenomes for undiscovered class 2 systems has resulted in the identification of three novel CRISPR-Cas variants. These include subtypes V-B and V-C, which resemble Cpf1 in that their predicted effector proteins contain a single, RuvC-like
+CRISPR type I-C (Cascade) - see [[CRISPR subtype I-C]]
-nuclease domain. Cleavage of target DNA by the type V-B effector, denoted C2c1, has been experimentally demonstrated. Type VI is unique in that its effector protein contains two conserved HEPN domains that possess ribonuclease (RNase) activity (Fig. 2A).<ref name="Rev4">doi:10.1126/science.aad5147</ref><ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref>
-Recent comparative genomic analyses of variant CRISPR-Cas systems (Fig. 2B) <ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref> have revealed a strong modular evolution with multiple combinations of adaptation modules and effector modules, as well as a pivotal contribution of mobile genetic elements to the origin and diversification of the CRISPR-Cas systems. The ancestral prokaryotic adaptive immune system could have emerged via the insertion of a casposon (a recently discovered distinct class of self-synthesizing transposons that appear to encode a Cas1 homolog) next to an innate immunity locus (probably consisting of genes encoding a Cas10 nuclease and possibly one or more RNA binding proteins). Apart from providing the Cas1 nuclease/integrase that is required for recombination during spacer acquisition, the casposon may also have contributed the prototype CRISPR repeat unit that could have evolved from one of the inverted terminal repeats of the casposon. An additional toxin-antitoxin module that inserted either in the ancestral casposon or in the evolving adaptive immunity locus probably provided the ''cas2'' gene, thus completing the adaptation module. The Cas10 nuclease and one or more additional proteins with an RRM fold (the ultimate origin of which could be a polymerase or cyclase that gave rise to Cas10) of the hybrid locus could have subsequently evolved to become the ancestral CRISPR-Cas effector module <ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref><ref name="Rev4">doi:10.1126/science.aad5147</ref>.
+CRISPR type I-D (Cas10d)
-The widespread occurrence of class 1 systems in archaea and bacteria, together with the proliferation of the ancient RRM domain in class 1 effector proteins, strongly suggests that the ancestral CRISPR-Cas belonged to class 1. Most likely, the multiple class 2 variants then evolved via several independent replacements of the class 1 effector locus with nuclease genes that were derived from distinct MGEs (Fig. 2B). In particular, type V effector variants (Cpf1) seem to have evolved from different families of the TnpB transposase genes that are widespread in transposons <ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref>, whereas the type II effector (Cas9) may have evolved from IscB, a protein with two nuclease domains that belongs to a recently identified distinct transposon family. Notably, class 2 CRISPR-Cas systems, in their entirety, appear to have been derived from different MGEs: Cas1 from a casposon, Cas2 from a toxin-antitoxin module, and the different effector proteins (such as Cas9 and Cpf1) from respective transposable elements <ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref><ref name="Rev4">doi:10.1126/science.aad5147</ref>.
+CRISPR type I-E (Cascade) - see [[CRISPR type I-E (Cascade)|CRISPR subtype I-E]]
-==CRISPR adaptation==
+CRISPR type I-F (Csy1, Csy2, Csy3) - see [[CRISPR subtype I-F]]
-The spacers of a CRISPR array represent a chronological archive of previous invader encounters. The captured spacer sequences are integrated into the CRISPR loci after exposure to MGEs, at the leader end of the array that contains the start site of CRISPR transcription. Analysis of invader target sequences (also called protospacers) has revealed a short motif directly adjacent to the target sequence, called the protospacer adjacent motif (PAM). This PAM motif allows self/nonself discrimination by the host in two ways: (i) because its presence in alien targets is required for nonself interference, and (ii) because its absence in the host’s CRISPR array avoids self-targeting. In class 1–type I and class 2–type II systems, the PAM is not only involved in interference, but also plays a role in spacer selection during the adaptation stage, implying the acquisition of functional spacers only. The PAM is a short [2 to 7 nucleotides (nt)], partially redundant sequence that in itself cannot preclude incorporation of spacers from the host DNA because of the low information content of the motif. The short PAM appears to be the result of an evolutionary trade-off between efficient incorporation of spacers from nonself DNA and preventing an autoimmune reaction.<ref name="Rev4">doi:10.1126/science.aad5147</ref>
+CRISPR type I-U (GSU0054)
-=Summary of the most extensively characterized CRISPR endoribonucleases<ref name="Rev3">PMID:25468820</ref><ref name="Rev4">doi:10.1126/science.aad5147</ref>=
+CRISPR type III (Cas10)
+CRISPR type III-A (Csm complex) - see [[CRISPR subtype III-A (Csm complex)]]
-==Representatives of class 1 and class 2==
+CRISPR type III-B (Cmr complex)
-Class 1 CRISPR-Cas systems are considered to be the evolutionary ancestral systems. The class 2 systems have evolved from class 1 systems via the insertion of transposable elements encoding various nucleases, and are now being used as tools for genome editing.<ref name="Rev4">doi:10.1126/science.aad5147</ref>
-*'''Class 1:'''
-<scene name='74/742625/Cv/5'>Cascade complex (Subtype I-E)</scene> ([[4qyz]]).
-<scene name='74/742625/Cv5/3'>Cmr complex (Subtype III-B)</scene> ([[3x1l]]).
+CRISPR type III-C (Cas10 or Csx11)
-*'''Class 2:'''
-<scene name='74/742625/Cv5/6'>Cas9 complex (Subtype II)</scene> ([[5f9r]]).
-<scene name='74/742625/Cv5/8'>Cpf1 complex (Subtype V-A)</scene> [[5b43]].
+CRISPR type III-D (Csx10)
-==Class 1==
-===CRISPR subtype I-A (Cascade)===
-*'''PhoCas6nc (Cas6a)''' from ''Pyrococcus horikoshii'' ([[3qjl]]). <scene name='74/742625/Cv4/16'>Pf7 RNA is able to mediate dimer formation through an exclusive RNA interface</scene>. Other representatives: [[3qjj]], [[3qjp]]. <ref name="PhoCas6nc">PMID:22238224</ref>
+CRISPR type Orphan
-*'''SsoCas6-1A (Sso2004, Cas6-1 family, SsCas6)''' from ''Sulfolobus solfataricus'' ([[4ill]]). <scene name='74/742625/Cv/45'>Dimeric structure of SsCas6 bound with the 24-mer noncleavable RNA</scene>. Other representatives: [[4ilm]], [[4ilr]]. <ref name="SsoCas6">PMID:23454186</ref>
-*'''SsoCas6-1B (Sso1437, Cas6-1 family, SsoCas6)''' from ''Sulfolobus solfataricus'' ([[3zfv]]). <scene name='74/742625/Cv4/17'>Dimeric structure of SsoCas6-1B</scene>. <ref name="SsoCas61B">PMID:23527601</ref>.
-===CRISPR subtype I-B (Cascade)===
+CRISPR type IV (Csf1)
-*'''MmaCas6b (Mm Cas6b)''' from ''Methanococcus maripaludis''. <scene name='74/742625/Cv4/18'>Crystal structure of CRISPR RNA processing endoribonuclease Cas6b</scene> ([[4z7k]]).
+CRISPR type IV-A
-===CRISPR subtype I-B?===
+CRISPR type IV-B
-*'''TthCas6B (TTHB231)''' from ''Thermus thermophilus''.  <scene name='74/742625/Cv/46'>Cas6B (TTHB231) product complex</scene> ([[4c9d]]). <ref name="Cas6B">PMID:24150936</ref>. Other representative: [[4c98]].
+'''CRISPR Class 2 uses a single large Cas protein'''
-===CRISPR subtype I-C (Cascade)===
+CRISPR type II-A - see [[CRISPR-Cas9]]
-*'''Cas5c (Cas5d)''': [[4f3m]] (''Bacillus halodurans''), [[3kg4]] (''Mannheimia succiniciproducens''), [[3vzh]] (''Streptococcus pyogenes''), [[3vzi]] (''Xanthomonas oryzae'')<ref name="Rev3">PMID:25468820</ref>.
+CRISPR type II-B (Cas4)
-===CRISPR subtype I-E (Cascade)===
+CRISPR type II-C
-*'''TthCas6e (TTHB192, Cse3)''' from ''Thermus thermophilus''. <scene name='74/742625/Cv4/21'>CRISPR endoribonuclease TthCas6e (Cse3) bound to 20 nt RNA</scene> ([[2y8w]]). Other representatives: [[1wj9]], [[2y8y]], [[2y9h]], [[3qrp]], [[3qrq]], [[3qrr]].
+CRISPR type V (Cpf1, C2c1, C2c3) - see [[CRISPR type V]]
-*'''EcoCas6e (CasE)''' from ''Escherichia coli''. [[4dzd]] (monomer).
+CRISPR type VI (Cas13a (previously known as C2c2), Cas13b, Cas13c, Cas13d) - see [[CRISPR type VI]]
-* '''Whole Cascade/I-E''' from ''Escherichia coli'': [[4tvx]], [[4u7u]], [[4qyz]], [[5h9f]], [[5h9e]], [[5cd4]]; see below.
+The rapid evolution of highly diverse CRISPR-Cas systems is thought to be driven by the continuous arms race with the invading MGEs. The latest classification scheme for CRISPR-Cas systems, which takes into account the repertoire of ''cas'' genes and the sequence similarity between Cas proteins and the locus architecture, includes two classes that are currently subdivided into six types and 19 subtypes <ref name="Rev4">doi:10.1126/science.aad5147</ref><ref name="Rev430">doi:10.1038/nrmicro3569</ref><ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref>. The key feature of the organization and evolution of the CRISPR-Cas loci is their pronounced modularity. The module responsible for the adaptation step is largely uniform among the diverse CRISPR-Cas systems and consists of the ''cas1'' and ''cas2'' genes, both of which are essential for the acquisition of spacers. In many CRISPR-Cas variants, the adaptation module also includes the ''cas4'' gene. By contrast, the CRISPR-Cas effector module, which is involved in the maturation of the crRNAs as well as in target recognition and cleavage, shows a far greater versatility (Fig. 2A) <ref name="Rev4">doi:10.1126/science.aad5147</ref><ref name="Rev430">doi:10.1038/nrmicro3569</ref>.
-====Crystal structure of a CRISPR RNA-guided surveillance complex, Cascade, bound to a ssDNA target<ref>PMID:25123481</ref>====
+[[Image:F2r.jpg|left|450px|thumb|Figure. 2. CRISPR diversity and evolution. (A) Modular organization of the CRISPR-Cas systems. LS, large subunit; SS, small subunit. A putative small subunit that might be fused to the large subunit in several type I subtypes is indicated by an asterisk. Cas3 is shown as fusion of two distinct genes encoding the helicase Cas3′ and the nuclease HD Cas3′′; in some type I systems, these domains are encoded by separate genes. Functionally dispensable components are indicated by dashed outlines. Cas6 is shown with a thin solid outline for type I because it is dispensable in some systems, and by a dashed line for type III because most systems lack this gene and use the Cas6 provided in trans by other CRISPR-Cas loci. The two colors for Cas4 and C2c2 and three colors for Cas9 and Cpf1 reflect the contributions of these proteins to different stages of the CRISPR-Cas response (see text). The question marks indicate currently unknown components. From <ref name="Rev4">doi:10.1126/science.aad5147</ref><ref name="Rev430">doi:10.1038/nrmicro3569</ref> (B) Evolutionary scenario for the CRISPR-Cas systems. TR, terminal repeats; TS, terminal sequences; HD, HD-family endonuclease; HNH, HNH-family endonuclease; RuvC, RuvC-family endonuclease; HEPN, putative endoribonuclease of HEPN superfamily. Genes and portions of genes shown in gray denote sequences that are thought to have been encoded in the respective mobile elements but were eliminated in the course of evolution of CRISPR-Cas systems. From <ref name="Rev4">doi:10.1126/science.aad5147</ref><ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref>]]
-The <scene name='74/742625/Cv/5'>crystal structure of ssDNA-bound Cascade has the seahorse architecture</scene>. The body is formed by a helical filament of six Cas7 subunits (Cas7.1 to 7.6) wrapped around the crRNA guide, with a head-to-tail dimer of Cse2 (Cse2.1 and Cse2.2) at the belly. Cas6e and the 3′ handle of crRNA cap the Cas7 filament at the head while Cas5 and the 5′ handle cap the tail. The N-terminal base of Cse1 is positioned at the tail of the filament; the C-terminal four-helix bundle contacts Cse2.2. The ssDNA target is juxtaposed to the guide region of the crRNA in a groove formed by the Cas7 filament, the four-helix bundle of Cse1, and the Cse2 dimer.
+{{Clear}}
-*<scene name='74/742625/Cv/6'>90° rotation about axis Z</scene>.
-*<scene name='74/742625/Cv/7'>90° rotation about axis Y</scene>.
-The <scene name='74/742625/Cv/10'>two strands of the guide-target hybrid</scene> do not twist around one another in a helix, but instead adopt an underwound ribbon-like structure reminiscent of a ladder. The 5′ and 3′ ends of the curved target strand are ~102Å apart, roughly the length of straight B-form dsDNA with an identical sequence (~107 Å). The crRNA (green) and ssDNA target (orange) are displayed in a spheres representation. Underwinding is facilitated by <scene name='74/742625/Cv/12'>kinks that occur every sixth base pair in the backbone of both strands of the hybrid</scene> (ribbon representation of the crRNA and ssDNA). <scene name='74/742625/Cv/14'>At each kink, complementary nucleotides are rotated ~90°, in opposing directions, from the axis of the duplex</scene>.  Disrupted RNA and DNA nucleotides are colored red and blue, respectively.
+The 2 classes of CRISPR-Cas systems differ fundamentally with respect to the organization of the effector module <ref name="Rev430">doi:10.1038/nrmicro3569</ref>. Class 1 systems (including types I, III, and IV) are present in bacteria and archaea, and encompass effector complexes composed of 4-7 Cas protein subunits [''e.g.'', the ('''C'''RISPR-'''as'''sociated '''c'''omplex for '''a'''ntiviral '''de'''fense) ('''Cascade''') of type I systems, and the Csm/Cmr complexes of type III systems]. Most of the subunits of the class 1 effector complexes — in particular, Cas5, Cas6, and Cas7—contain variants of the RNA-binding RRM (RNA recognition motif) domain.<ref name="Rev4">doi:10.1126/science.aad5147</ref>
+''Examples of RRM fold''
+*<scene name='74/746096/Cv6/9'>RRM fold of Cas2</scene> ([[2i0x]]).
+*<scene name='74/746096/Cv6/13'>2 RRM folds of N- and C-terminal domains of Cas5</scene> ([[3kg4]]).
+*<scene name='74/746096/Cv6/11'>2 RRM folds of N- and C-terminal domains of Cas6</scene> ([[3i4h]]).
+*<scene name='74/746096/Cv6/12'>2 RRM folds of N- and C-terminal domains of Cas6e</scene> ([[1wj9]]).
+*<scene name='74/746096/Cv6/14'>2 RRM folds of N- and C-terminal domains of Cas6f</scene> ([[2xlj]]).
+*<scene name='74/746096/Cv6/10'>RRM fold of Cas7</scene> ([[3ps0]]).
-=====Structure of the Cas7 subunit.=====
+Although the sequence similarity between the individual subunits of type I and type III effector complexes is generally low, the complexes share strikingly similar overall architectures that suggest a common origin <ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref>. The ancestral CRISPR-Cas effector complex most likely resembled the extant type III complexes, as indicated by the presence of the archetypal type III protein, the large Cas10 subunit, which appears to be an active enzyme of the DNA polymerase–nucleotide cyclase superfamily, unlike its inactive type I counterpart (Cas8) <ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref><ref name="Rev4">doi:10.1126/science.aad5147</ref>. The ''cas6'' gene family encodes a set of RNA endonucleases responsible for crRNA processing in Type I and Type III CRISPR systems. Type II systems use a trans-activating RNA (tracrRNA) together with endogenous RNase III for crRNA maturation. In Type I-B, I-C, I-E, and I-F systems, the endoRNase stays bound to the crRNA and assembles into a complex with other Cas proteins for downstream targeting <ref name="Rev312">doi:10.1126/science.1159689</ref>, while in Type I-A and III systems, the crRNA alone is loaded into the targeting complex and Cas6 dissociates <ref name="Rev3">PMID:25468820</ref>.
-Within Cascade, the <scene name='74/742625/Cv/25'>six Cas7 subunits form a right-handed filament</scene>, with a pitch of ~135Å, around the guide target hybrid. <scene name='74/742625/Cv/16'>Rocket representation of one Cas7 colored by domain: thumb (green), fingers (blue), and palm (purple).</scene> The filament is arranged such that the <scene name='74/742625/Cv/17'>thumb of one Cas7 subunit, composed of an extended β hairpin, extends toward the fingers of the adjacent subunit</scene>. <scene name='74/742625/Cv/18'>90° rotation about axis Z</scene>.
+In the less common class 2 CRISPR-Cas systems (types II, V, and VI), which are almost completely restricted to bacteria, the effector complex is represented by a single multidomain protein <ref name="Rev430">doi:10.1038/nrmicro3569</ref>. The best-characterized class 2 effector is Cas9 (type II), the RNA-dependent endonuclease that contains two unrelated nuclease domains, HNH and RuvC, that are responsible for the cleavage of the target and the displaced strand, respectively, in the crRNA–target DNA complex (<scene name='74/746096/Cv3/1'>Domain organization of nuclease lobe of Cas9 from S. pyogenes</scene>, [[4zt0]]). The type II loci also encode a trans-acting CRISPR RNA (tracrRNA) that evolved from the corresponding CRISPR repeat and is essential for pre-crRNA processing and target recognition in type II systems. Cas9 is directed to its DNA targets by forming a ribonucleoprotein complex with these 2 small non-coding RNAs: crRNA and tracrRNA. By elegant engineering, <scene name='74/742625/Cv3/8'>crRNA and tracrRNA can be joined end-to-end and transcribed as a single guide RNA (sgRNA)</scene> ([[4zt9]]<ref name="dCAS9">PMID:26113724</ref>) that too efficiently directs Cas9 protein to DNA targets encoded within the guide sequence of sgRNA <ref name="Jinek">PMID:22745249</ref>:
-=====Stabilization of the guide-target hybrid by Cas7, Cse1, and Cse2.=====
-<scene name='74/742625/Cv/36'>Each 5-bp segment of the hybrid is situated between the palm of one Cas7 subunit (e.g. Cas7.2) and the fingers of the adjacent subunit (e.g. Cas7.3)</scene>. 5-bp segment is colored red. Extensive contacts between the guide region of the crRNA and the Cas7 filament bury a large portion of the crRNA backbone, leaving the bases solvent-exposed. The absence of direct contacts between protein side chains and bases of the crRNA explains the lack of sequence specificity by Cascade for the guide sequence. <scene name='74/742625/Cv/37'>Close-up view of the bound crRNA</scene>. The DNA target has been removed for clarity. Intercalation by Met166 from Cas7 is highlighted. <scene name='74/742625/Cv/38'>Several highly conserved polar and positively charged residues (Arg20, Lys27, Ser40, Gln42, Lys45, and Lys49 - colored in magenta) from the palm of one Cas7 (e.g. Cas7.3) contact the RNA backbone</scene>, while the <scene name='74/742625/Cv/39'>fingers from the adjacent Cas7 (e.g. Cas7.2) subunit (residues 109-111, 163-169, colored in plum) contact both strands of the hybrid across the minor groove</scene>. Of note, the <scene name='74/742625/Cv/40'>Thumb (colored in olive) of one Cas7 subunit (e.g. Cas7.4) pushes through the guide-target hybrid at the 1-bp gaps</scene>. <scene name='74/742625/Cv/32'>Representation of 5 thumbs protruding guide-target hybrid at the 1-bp gaps</scene>. Each displaced RNA nucleotide adopts the syn conformation, is similarly positioned above the backbone of the downstream RNA, and is contacted by <scene name='74/742625/Cv/41'>residues from both the Cas7 palm (e.g. Cas7.3 palm; Ser43 and Arg46) and thumb (e.g. Cas7.4 thumb; Thr201 and Val203)</scene>. Overview of the <scene name='74/742625/Cv1/2'>interactions between the ssDNA target and Cse2.1, Cse2.2, and Cse1</scene>. The proteins are represented as rockets, the DNA as a surface. The positions of the disrupted DNA nucleotides (royal blue) are indicated.
-=====Interactions capping the tail of Cascade=====
-<scene name='74/742625/Cv/34'>Cas5 caps the tail of the Cas7 filament at the 5′ end of the crRNA</scene>. The structure of Cascade reveals that Cas5 is structurally related to Cas7, as it consists of a palm(residues 1 to 78 and 115 to 224) and a thumb (residues 79 to 114) domain, but lacks a fingers domain. <scene name='74/742625/Cv1/3'>Rocket representation of Cas7 and Cas5 colored by domain: thumb (green), fingers (blue), and palm (purple)</scene>. Close-up view of the  <scene name='74/742625/Cv/35'>interaction between Cas5, Cas7.6, and the 5′ hook</scene>.
-====Crystal structure of E. coli Cascade bound to a PAM-containing dsDNA target at 2.45 angstrom resolution<ref>PMID:26863189</ref>====
-<scene name='74/742625/Cv2/4'>Crystal structure of E. coli Cascade bound to a PAM-containing dsDNA target</scene>.
-*<scene name='74/742625/Cv2/9'>crRNA-dsDNA hybrid</scene>.
-*<scene name='74/742625/Cv2/7'>crRNA-dsDNA hybrid and Cascade proteins</scene>.
-*<scene name='74/742625/Cv2/8'>Thumbs of Cas7.2-7.6 protrude crRNA-dsDNA hybrid at the 1-bp gaps</scene>.
-===CRISPR subtype I-F (Cascade)===
-*'''PaeCas6f (Csy4)''' from ''Pseudomonas aeruginosa''. <scene name='74/742625/Cv4/23'>Csy4-crRNA complex</scene> ([[2xlj]]). Other representatives:  [[2xlk]], [[4al5]], [[4al6]], [[4al7]].
-===CRISPR subtype I-U ===
-===CRISPR subtype III-A (Csm complex)===
-*<scene name='74/742625/Cv5/1'>Csm3-Csm4 subcomplex</scene> in the type III-A CRISPR-Cas interference complex from ''Methanocaldococcus jannaschii''.
-===CRISPR subtype III-B (Cmr complex)===
-*<scene name='74/742625/Cv5/3'>Cmr complex (Subtype III-B)</scene> from ''Pyrococcus furiosus'' and ''Archaeoglobus fulgidus'' ([[3x1l]]).
-*'''PfuCas6-1 (PfCas6)''' from ''Pyrococcus furiosus''. <scene name='74/742625/Cv4/24'>PfuCas6-1 bound to crRNA</scene> ([[3pkm]]). Other representative: [[3i4h]].
-===CRISPR subtype Orphan===
+''Examples of 3D structures of single guide RNA (sgRNA)''
+*<scene name='74/742625/Cv/42'>Cas9-sgRNA-target DNA complex from Streptococcus pyogenes</scene> ([[5fw2]]).
+*<scene name='74/742625/Cv2/12'>Cas9-sgRNA-target DNA complex from Streptococcus pyogenes</scene> ([[5b2s]]).
+*<scene name='74/742625/Cv2/13'>Cas9-sgRNA-target DNA complex from Francisella tularensis</scene> ([[5b2p]]).
+*<scene name='74/742625/Cv3/2'>Cas9-sgRNA-target DNA complex from Staphylococcus aureus</scene> ([[4axw]]).
-*'''TthCas6A (TTHB78)''' from ''T. thermophilus''. <scene name='74/742625/Cv4/25'>TthCas6A bound to crRNA</scene> ([[4c8z]]). Other representatives: [[4c8y]], [[4c97]].
+The <scene name='74/742625/Cv3/4'>optimal DNA target of the complex is determined by a Watson–Crick base pairing of a short ∼20-nt sequence within sgRNA (within the crRNA in wild-type)</scene>, termed the guide sequence, adjacent to a <scene name='74/742625/Cv3/10'>few nucleotide long conserved motif recognized directly by Cas9 protein (protospacer adjacent motif, PAM)</scene> <ref name="Jinek">PMID:22745249</ref><ref name="Prin6">PMID:22949671</ref>. Despite this, a <scene name='74/742625/Cv/44'>few mismatches between guide sequence and target DNA can be tolerated</scene> <ref name="Jinek">PMID:22745249</ref><ref name="Prin7">PMID:23452860</ref><ref name="Prin8">PMID:23761437</ref><ref name="Prin9">PMID:24837660</ref>, more so within the 5’ proximal position of the guide sequence.
-===CRISPR subtype IV (Csf1)===
+The prototype type V effector Cpf1 (subtype V-A) contains only one nuclease domain (RuvC-like) that is identifiable by sequence analysis. However, analysis of the recently solved structure of <scene name='74/746096/Cv5/1'>Cpf1 complexed with the crRNA and target DNA</scene> has revealed a second nuclease domain, the fold of which is unrelated to HNH or any other known nucleases. In analogy to the HNH domain in Cas9, the <scene name='74/746096/Cv5/2'>novel nuclease domain (labeled Nuc) in Cpf1 is inserted into the RuvC domain</scene>, and it is responsible for cleavage of the target strand.<ref name="Rev4">doi:10.1126/science.aad5147</ref><ref name="Cpf1">PMID:24884953</ref>
-==Class 2==
+Screening of microbial genomes and metagenomes for undiscovered class 2 systems has resulted in the identification of three novel CRISPR-Cas variants. These include subtypes V-B and V-C, which resemble Cpf1 in that their predicted effector proteins contain a single, RuvC-like nuclease domain. Cleavage of target DNA by the type V-B effector, denoted C2c1, has been experimentally demonstrated. Type VI is unique in that its effector protein contains two conserved HEPN domains that possess ribonuclease (RNase) activity (Fig. 2A).<ref name="Rev4">doi:10.1126/science.aad5147</ref><ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref>
-===CRISPR subtype II - SEE [[CRISPR-Cas9]]===
+Recent comparative genomic analyses of variant CRISPR-Cas systems (Fig. 2B) <ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref> have revealed a strong modular evolution with multiple combinations of adaptation modules and effector modules, as well as a pivotal contribution of mobile genetic elements to the origin and diversification of the CRISPR-Cas systems. The ancestral prokaryotic adaptive immune system could have emerged via the insertion of a casposon (a recently discovered distinct class of self-synthesizing transposons that appear to encode a Cas1 homolog) next to an innate immunity locus (probably consisting of genes encoding a Cas10 nuclease and possibly one or more RNA binding proteins). Apart from providing the Cas1 nuclease/integrase that is required for recombination during spacer acquisition, the casposon may also have contributed the prototype CRISPR repeat unit that could have evolved from one of the inverted terminal repeats of the casposon. An additional toxin-antitoxin module that inserted either in the ancestral casposon or in the evolving adaptive immunity locus probably provided the ''cas2'' gene, thus completing the adaptation module. The Cas10 nuclease and one or more additional proteins with an RRM fold (the ultimate origin of which could be a polymerase or cyclase that gave rise to Cas10) of the hybrid locus could have subsequently evolved to become the ancestral CRISPR-Cas effector module <ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref><ref name="Rev4">doi:10.1126/science.aad5147</ref>.
-===CRISPR subtype V (Cpf1)===
+The widespread occurrence of class 1 systems in archaea and bacteria, together with the proliferation of the ancient RRM domain in class 1 effector proteins, strongly suggests that the ancestral CRISPR-Cas belonged to class 1. Most likely, the multiple class 2 variants then evolved via several independent replacements of the class 1 effector locus with nuclease genes that were derived from distinct MGEs (Fig. 2B). In particular, type V effector variants (Cpf1) seem to have evolved from different families of the TnpB transposase genes that are widespread in transposons <ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref>, whereas the type II effector (Cas9) may have evolved from IscB, a protein with two nuclease domains that belongs to a recently identified distinct transposon family. Notably, class 2 CRISPR-Cas systems, in their entirety, appear to have been derived from different MGEs: Cas1 from a casposon, Cas2 from a toxin-antitoxin module, and the different effector proteins (such as Cas9 and Cpf1) from respective transposable elements <ref name="Rev431">doi:10.1016/j.molcel.2015.10.008</ref><ref name="Rev4">doi:10.1126/science.aad5147</ref>.
-*<scene name='74/742625/Cv5/8'>Cpf1 complex (Subtype V-A)</scene> from ''Acidaminococcus sp. BV3L6'' [[5b43]]. Other representive: [[5kk5]].
-*<scene name='74/746096/Cv6/1'>Cpf1 in complex with CRISPR RNA</scene> from ''Lachnospiraceae bacterium ND2006'' [[5id6]].
-===CRISPR subtype VI (C2c2)===
+SEE [[CRISPR-Cas Part II]]
 =See aslo=
 *[[Cas9]]

CRISPR-Cas

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CRISPR-Cas defense

CRISPR-Cas diversity, classification, and evolution

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