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<StructureSection load='4qyz' size='450' side='right' scene='74/742625/Cv/4' caption=''>
<StructureSection load='' size='350' side='right' scene='74/742625/Cv3/13' caption='Cas9 complex with RNA (PDB code [[4zt0]])'>
==Crystal structure of a CRISPR RNA-guided surveillance complex, Cascade, bound to a ssDNA target<ref>PMID:25123481</ref>==
'''Part I'''
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.
*<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.
SEE ALSO
*[[CRISPR-Cas9 Part II]]
*[[CRISPR-Cas]]
'''Background'''


===Structure of the Cas7 subunit.===
Highlights


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>.
*'''CRISPR-Cas9 (CRISPR type II-A)''' 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.<ref name="Did">PMID:27344519</ref>


===Stabilization of the guide-target hybrid by Cas7, Cse1, and Cse2.===
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>
<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. Cas7.5) 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/33'>residues from both the Cas7 palm (e.g. Cas7.4 palm; Ser43 and Arg46) and thumb (e.g. Cas7.3 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.
 
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>
 
====Transcriptional regulation with CRISPR-Cas9====
 
[[Image:1-s2.0-S0958166916301537-gr1.jpg|left|450px|thumb|'''Figure 1.''' 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>). 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 1a). 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>:
 
''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.
*<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).
 
In the type II-A system, the Cas9-tracrRNA complex and Csn2 are involved in spacer acquisition along with the Cas1-Cas2 complex <ref name="Rev453">doi:10.1101/gad.257550.114</ref><ref name="Rev471">doi:10.1038/nature14245</ref>; the involvement of Cas9 in adaptation is likely to be a general feature of type II systems. Although the key residues of Cas9 involved in PAM recognition are dispensable for spacer acquisition, they are essential for the incorporation of new spacers with the correct PAM sequence <ref name="Rev471">doi:10.1038/nature14245</ref>. The involvement of Cas9 in PAM recognition and protospacer selection <ref name="Rev471">doi:10.1038/nature14245</ref> suggests that in type II systems Cas1 may have lost this role.
 
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 1b) <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>.
 
'''Cas9-sgRNA-target DNA complexes from Streptococcus pyogenes:'''
*<scene name='74/746096/Cv3/1'>Domain organization of nuclease lobe of Cas9 from S. pyogenes</scene> ([[4zt0]])
*<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/Cv4/5'>CAS9 with sgRNA and tagret DNA</scene> ([[4oo8]]).
*<scene name='74/742625/Cv4/11'>Cas9-sgRNA-dsDNA complex</scene> ([[5f9r]]).
Other representatives: [[5y36]], [[4un3]].
 
'''For continuation please see [[CRISPR-Cas9 Part II]]'''
=See aslo=
*[[Cas9]]
*[[Endonuclease]]
*[[Cas9 (hebrew)]]


===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>.
</StructureSection>
</StructureSection>
== References ==
== References ==
<references/>
<references/>
[[Category:Topic Page]]
[[Category:Crispr]]
[[Category: Crispr-associated]]
[[Category: endonuclease]]
[[Category: Cas9]]

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

Alexander Berchansky, Michal Harel
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