CRISPR-Cas

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1 Background
- 1.1 CRISPR-Cas defense
- 1.2 CRISPR-Cas diversity, classification, and evolution
2 Summary of the most extensively characterized CRISPR endoribonucleases^[5]^[7]
3 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

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 ^[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. 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]. 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 ^[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 (39). However, analysis of the recently solved structure of Cpf1 complexed with the crRNA and target DNA 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 novel nuclease domain in Cpf1 is inserted into the RuvC domain, and it is responsible for cleavage of the target strand (40).

Summary of the most extensively characterized CRISPR endoribonucleases^[5]^[7]

Representatives of class 1 and class 2

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.^[7]

Class 1:

(4qyz).

(3x1l).

Class 2:

(5f9r).

5b43.

Class 1

CRISPR subtype I-A (Cascade)

PhoCas6nc (Cas6a) from Pyrococcus horikoshii (3qjl). . Other representatives: 3qjj, 3qjp. ^[19]
SsoCas6-1A (Sso2004, Cas6-1 family, SsCas6) from Sulfolobus solfataricus (4ill). . Other representatives: 4ilm, 4ilr. ^[20]
SsoCas6-1B (Sso1437, Cas6-1 family, SsoCas6) from Sulfolobus solfataricus (3zfv). . ^[21].

CRISPR subtype I-B (Cascade)

MmaCas6b (Mm Cas6b) from Methanococcus maripaludis. (4z7k).

CRISPR subtype I-B?

TthCas6B (TTHB231) from Thermus thermophilus. (4c9d). ^[22]. Other representative: 4c98.

CRISPR subtype I-C (Cascade)

Cas5c (Cas5d): 4f3m (Bacillus halodurans), 3kg4 (Mannheimia succiniciproducens), 3vzh (Streptococcus pyogenes), 3vzi (Xanthomonas oryzae)^[5].

CRISPR subtype I-E (Cascade)

TthCas6e (TTHB192, Cse3) from Thermus thermophilus. (2y8w). Other representatives: 1wj9, 2y8y, 2y9h, 3qrp, 3qrq, 3qrr.

EcoCas6e (CasE) from Escherichia coli. 4dzd (monomer).

Whole Cascade/I-E from Escherichia coli: 4tvx, 4u7u, 4qyz, 5h9f, 5h9e, 5cd4; see below.

Crystal structure of a CRISPR RNA-guided surveillance complex, Cascade, bound to a ssDNA target^[23]

The . 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.

The 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 (ribbon representation of the crRNA and ssDNA). . Disrupted RNA and DNA nucleotides are colored red and blue, respectively.

Structure of the Cas7 subunit.

Within Cascade, the , with a pitch of ~135Å, around the guide target hybrid. The filament is arranged such that the . .

Stabilization of the guide-target hybrid by Cas7, Cse1, and Cse2.

. 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. . The DNA target has been removed for clarity. Intercalation by Met166 from Cas7 is highlighted. , while the . Of note, the . . Each displaced RNA nucleotide adopts the syn conformation, is similarly positioned above the backbone of the downstream RNA, and is contacted by . Overview of the . 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

. 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. . Close-up view of the .

Crystal structure of E. coli Cascade bound to a PAM-containing dsDNA target at 2.45 angstrom resolution^[24]

CRISPR subtype I-F (Cascade)

PaeCas6f (Csy4) from Pseudomonas aeruginosa. (2xlj). Other representatives: 2xlk, 4al5, 4al6, 4al7.

CRISPR subtype I-U

CRISPR subtype III-A (Csm complex)

in the type III-A CRISPR-Cas interference complex from Methanocaldococcus jannaschii.

CRISPR subtype III-B (Cmr complex)

from Pyrococcus furiosus and Archaeoglobus fulgidus (3x1l).
PfuCas6-1 (PfCas6) from Pyrococcus furiosus. (3pkm). Other representative: 3i4h.

CRISPR subtype Orphan

TthCas6A (TTHB78) from T. thermophilus. (4c8z). Other representatives: 4c8y, 4c97.

CRISPR subtype IV (Csf1)

Class 2

CRISPR subtype II - SEE CRISPR-Cas9

CRISPR subtype V (Cpf1)

from Acidaminococcus sp. BV3L6 5b43.

CRISPR subtype VI (C2c2)

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 ^5.5 ^5.6 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 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 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
↑ Wang R, Zheng H, Preamplume G, Shao Y, Li H. The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA complex. Protein Sci. 2012 Mar;21(3):405-17. doi: 10.1002/pro.2028. Epub 2012 Feb 9. PMID:22238224 doi:http://dx.doi.org/10.1002/pro.2028
↑ Shao Y, Li H. Recognition and Cleavage of a Nonstructured CRISPR RNA by Its Processing Endoribonuclease Cas6. Structure. 2013 Feb 27. pii: S0969-2126(13)00017-8. doi:, 10.1016/j.str.2013.01.010. PMID:23454186 doi:http://dx.doi.org/10.1016/j.str.2013.01.010
↑ Reeks J, Sokolowski RD, Graham S, Liu H, Naismith JH, White MF. Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site for CRISPR RNA processing. Biochem J. 2013 Mar 25. PMID:23527601 doi:10.1042/BJ20130269
↑ Niewoehner O, Jinek M, Doudna JA. Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases. Nucleic Acids Res. 2013 Oct 22. PMID:24150936 doi:http://dx.doi.org/10.1093/nar/gkt922
↑ Mulepati S, Heroux A, Bailey S. Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssDNA target. Science. 2014 Aug 14. pii: 1256996. PMID:25123481 doi:http://dx.doi.org/10.1126/science.1256996
↑ Hayes RP, Xiao Y, Ding F, van Erp PB, Rajashankar K, Bailey S, Wiedenheft B, Ke A. Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli. Nature. 2016 Feb 25;530(7591):499-503. doi: 10.1038/nature16995. Epub 2016 Feb, 10. PMID:26863189 doi:http://dx.doi.org/10.1038/nature16995

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Alexander Berchansky, Michal Harel

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

Categories: Topic Page | Crispr | Crispr-associated | Endonuclease

@@ Line 48: / Line 48: @@
 *<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 (39). However, analysis of the recently solved structure of Cpf1 complexed with the crRNA and target DNA 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 novel nuclease domain in Cpf1 is inserted into the RuvC domain, and it is responsible for cleavage of the target strand (40).
-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.
 =Summary of the most extensively characterized CRISPR endoribonucleases<ref name="Rev3">PMID:25468820</ref><ref name="Rev4">doi:10.1126/science.aad5147</ref>=
@@ Line 142: / Line 141: @@
 ==Class 2==
-===CRISPR subtype II===
+===CRISPR subtype II - SEE [[CRISPR-Cas9]]===
-====Transcriptional regulation with CRISPR-Cas9====
-[[Image:1-s2.0-S0958166916301537-gr1.jpg|left|450px|thumb|'''Figure 3.''' Overview of Cas9 nuclease and dCas9-based transcription factors. (a) Wild-type Cas9 endonuclease guided by crRNA:tracrRNA to a specific site in DNA creates a double-stranded DNA break. (b) dCas9, nuclease deactivated mutant of Cas9, is an RNA programmable DNA binding protein. It can act as a steric repressor of transcription in prokaryotes and eukaryotes. sgRNA is an artificial chimeric molecule consisting of crRNA and tracrRNA molecules connected with a short loop. (c) dCas9 fusion with various transcription effectors can be used to repress or activate transcription. (d) Effector domains can be recruited by sgRNA in addition to dCas9 for enhanced activity. (e) sgRNA can be modified with specific protein binding hairpins to concurrently recruit repressor or activator domains in the same cell.<ref name="Did">PMID:27344519</ref>]]
-{{Clear}}
-Cas9 is a key protein of bacterial Type II CRISPR adaptive immune system (reviewed in <ref name="Prin4">PMID:26432244</ref>). <scene name='74/742625/Cv3/9'>Domain organization of the type II-A Cas9 protein from S. pyogenes</scene> (SpyCas9; PDB entry [[4zt0]]<ref name="dCAS9">PMID:26113724</ref>). In its native context, Cas9 is an RNA-guided endonuclease that is responsible for targeted degradation of the invading foreign DNA–plasmids and phages. Cas9 is directed to its DNA targets by forming a ribonucleoprotein complex with two small non-coding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) (Figure 3a). 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> (PDB entry [[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> (see also above ''Examples of 3D structures of single guide RNA (sgRNA)''). 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.
-*<scene name='74/742625/Cv4/3'>When Cas9 is in the apo state, its PAM-interacting cleft is largely disordered</scene><ref name="dCAS9">PMID:26113724</ref> ([[4cmp]]<ref name="CAS9">PMID:24505130</ref>).
-*<scene name='74/742625/Cv4/4'>Pre-target state of CAS9 with sgRNA</scene> ([[4zt0]])<ref name="dCAS9">PMID:26113724</ref>.
-*<scene name='74/742625/Cv4/5'>CAS9 with sgRNA and tagret DNA</scene> ([[4oo8]])<ref name="CAS9D">PMID:24529477</ref>.
-*<scene name='74/742625/Cv4/7'>Animation of these scenes</scene>.
-*<scene name='74/742625/Cv4/11'>Cas9-sgRNA-dsDNA complex</scene> ([[5f9r]])<ref name="Ski">PMID:26841432</ref>.
-*<scene name='74/742625/Cv4/12'>sgRNA-dsDNA without protein is shown</scene>.
-*<scene name='74/742625/Cv4/14'>Scissile phosphates</scene> - place of accurate, precise, and programmable DNA cleavage.
-*<scene name='74/742625/Cv4/15'>Cas9 interacts with both separated DNA strands</scene> (sgRNA is not shown).
-Cas9 nuclease can be converted into <scene name='74/742625/Cv4/8'>deactivated Cas9 (dCas9)</scene> (PDB entry [[4zt9]]), an RNA-programmable DNA-binding protein, by <scene name='74/742625/Cv4/9'>mutating two key residues within its nuclease domains</scene> (Figure 3b) <ref name="dCAS9">PMID:26113724</ref><ref name="Did">PMID:27344519</ref><ref name="Jinek">PMID:22745249</ref><ref name="Prin6">PMID:22949671</ref>.
-In the simplest case, dCas9 can repress transcription by sterically interfering with transcription initiation or elongationby being targeted to the gene of interest with a properly chosen sgRNA <ref name="Jinek">PMID:22745249</ref><ref name="Prin6">PMID:22949671</ref><ref name="Prin7">PMID:23452860</ref><ref name="Prin8">PMID:23761437</ref><ref name="Prin10">PMID:25422271</ref><ref name="Prin11">PMID:26390083</ref><ref name="Prin12">PMID:23849981</ref><ref name="Prin13">PMID:23977949</ref><ref name="Prin14">PMID:24797424</ref>. The repression strength is strongly dependent on the position with respect to the target promoter as well as the nature of promoter itself <ref name="Prin7">PMID:23452860</ref><ref name="Prin8">PMID:23761437</ref><ref name="Prin10">PMID:25422271</ref><ref name="Prin11">PMID:26390083</ref>. In prokaryotes, repression of up to 1000-fold was achieved when targeting dCas9 to either DNA strand within a promoter or to the non-template DNA strand downstream. However, in eukaryotic cells such steric repression is weaker: only up to 2-fold and 20-fold repression was observed with natural promoters in mammalian and yeast cells correspondingly<ref name="Prin7">PMID:23452860</ref><ref name="Prin12">PMID:23849981</ref><ref name="Prin13">PMID:23977949</ref>. As a notable exception, synthetic promoters specifically constructed for direct repression by dCas9 can be repressed up to 100-fold in mammalian cells<ref name="Prin14">PMID:24797424</ref>.
 ===CRISPR subtype V (Cpf1)===

CRISPR-Cas

From Proteopedia

Revision as of 14:18, 27 November 2016

Contents

Background

CRISPR-Cas defense

CRISPR-Cas diversity, classification, and evolution

Summary of the most extensively characterized CRISPR endoribonucleases^[5]^[7]

Representatives of class 1 and class 2

Class 1

CRISPR subtype I-A (Cascade)

CRISPR subtype I-B (Cascade)

CRISPR subtype I-B?

CRISPR subtype I-C (Cascade)

CRISPR subtype I-E (Cascade)

Crystal structure of a CRISPR RNA-guided surveillance complex, Cascade, bound to a ssDNA target^[23]

Structure of the Cas7 subunit.

Stabilization of the guide-target hybrid by Cas7, Cse1, and Cse2.

Interactions capping the tail of Cascade

Crystal structure of E. coli Cascade bound to a PAM-containing dsDNA target at 2.45 angstrom resolution^[24]

CRISPR subtype I-F (Cascade)

CRISPR subtype I-U

CRISPR subtype III-A (Csm complex)

CRISPR subtype III-B (Cmr complex)

CRISPR subtype Orphan

CRISPR subtype IV (Csf1)

Class 2

CRISPR subtype II - SEE CRISPR-Cas9

CRISPR subtype V (Cpf1)

CRISPR subtype VI (C2c2)

See aslo

References

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CRISPR-Cas

From Proteopedia

Revision as of 14:18, 27 November 2016

Contents

Background

CRISPR-Cas defense

CRISPR-Cas diversity, classification, and evolution

Summary of the most extensively characterized CRISPR endoribonucleases[5][7]

Representatives of class 1 and class 2

Class 1

CRISPR subtype I-A (Cascade)

CRISPR subtype I-B (Cascade)

CRISPR subtype I-B?

CRISPR subtype I-C (Cascade)

CRISPR subtype I-E (Cascade)

Crystal structure of a CRISPR RNA-guided surveillance complex, Cascade, bound to a ssDNA target[23]

Structure of the Cas7 subunit.

Stabilization of the guide-target hybrid by Cas7, Cse1, and Cse2.

Interactions capping the tail of Cascade

Crystal structure of E. coli Cascade bound to a PAM-containing dsDNA target at 2.45 angstrom resolution[24]

CRISPR subtype I-F (Cascade)

CRISPR subtype I-U

CRISPR subtype III-A (Csm complex)

CRISPR subtype III-B (Cmr complex)

CRISPR subtype Orphan

CRISPR subtype IV (Csf1)

Class 2

CRISPR subtype II - SEE CRISPR-Cas9

CRISPR subtype V (Cpf1)

CRISPR subtype VI (C2c2)

See aslo

References

Proteopedia Page Contributors and Editors (what is this?)

Views

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Summary of the most extensively characterized CRISPR endoribonucleases^[5]^[7]

Crystal structure of a CRISPR RNA-guided surveillance complex, Cascade, bound to a ssDNA target^[23]

Crystal structure of E. coli Cascade bound to a PAM-containing dsDNA target at 2.45 angstrom resolution^[24]