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Review
. 2015 May;39(3):442-63.
doi: 10.1093/femsre/fuv019. Epub 2015 Apr 30.

DNA and RNA Interference Mechanisms by CRISPR-Cas Surveillance Complexes

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Free PMC article
Review

DNA and RNA Interference Mechanisms by CRISPR-Cas Surveillance Complexes

André Plagens et al. FEMS Microbiol Rev. .
Free PMC article

Abstract

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) adaptive immune systems use small guide RNAs, the CRISPR RNAs (crRNAs), to mark foreign genetic material, e.g. viral nucleic acids, for degradation. Archaea and bacteria encode a large variety of Cas proteins that bind crRNA molecules and build active ribonucleoprotein surveillance complexes. The evolution of CRISPR-Cas systems has resulted in a diversification of cas genes and a classification of the systems into three types and additional subtypes characterized by distinct surveillance and interfering complexes. Recent crystallographic and biochemical advances have revealed detailed insights into the assembly and DNA/RNA targeting mechanisms of the various complexes. Here, we review our knowledge on the molecular mechanism involved in the DNA and RNA interference stages of type I (Cascade: CRISPR-associated complex for antiviral defense), type II (Cas9) and type III (Csm, Cmr) CRISPR-Cas systems. We further highlight recently reported structural and mechanistic themes shared among these systems.

Keywords: CRISPR; Cas9; Cascade; DNA interference; guide crRNAs; ribonucleoprotein complexes; tracrRNA; viruses.

Figures

Figure 1.
Figure 1.
CRISPR-Cas systems and conserved stages of CRISPR-Cas activity. The general organization of a CRISPR-Cas locus is indicated. In the first stage of CRISPR-Cas activity—acquisition—the universal proteins Cas1 and Cas2 recognize viral DNA that is flanked by a PAM. The protospacer is excised and integrated as a spacer sequence into the extending CRISPR array. The CRISPR array is transcribed from the leader sequence and processed into mature crRNAs that are incorporated into crRNP surveillance complexes. The Cas protein composition of the complexes is schematically depicted for the three different CRISPR-Cas types. Nucleases are indicated by scissors and proteins proposed to fulfill similar roles are colored accordingly.
Figure 2.
Figure 2.
Assembly of the type I-E Cascade structure. The I-E Cascade complex has a seahorse-shaped structure and consists of 11 protein subunits ((Cse1)1-(Cse2)2-(Cas5)1-(Cas7)6-(Cas6e)1) and a single 61-nt crRNA (pdb: 4TVX). Cas6e is tightly bound to the 3 stem-loop structure of the mature crRNA and positioned at the head of the complex. Cas5e directly caps the 5 handle of the crRNA, which leads to the hook-like structure of the crRNA. The structure of Cas5 and Cas7 displays a conserved palm-thumb domain arrangement, highlighting the intertwined assembly of the Cascade backbone. The thumb of either Cas5 or each of the six Cas7 subunits (Cas7.1-Cas7.6) kinks the crRNA at position −1 in the 5 handle and every sixth position in the spacer sequence, and buries the base between the thumb and the palm of the adjacent Cas7 subunit. The two small Cse2 subunits (Cse2.1–Cse2.2) are connected to the crRNA backbone via protein:protein interactions to the Cas7 subunits. The large subunit Cse1 is positioned at the Cascade tail and interacts with Cas5, Cas7 and Cse2.
Figure 3.
Figure 3.
Structures of S. pyogenes (Spy) type II-A Cas9. (A) Crystal structure of the apoenzyme SpyCas9 resolved at 2.6 Å (pdb: 4CMP) (Jinek et al.2014). (B) Structure of SpyCas9 bound to sgRNA and target DNA resolved at 2.5 Å (pdb: 4UN3) (Anders et al.2014). While the CTD and the RuvC domains remain in their positions, the REC lobe of Cas9 accommodates its position to facilitate sgRNA and target DNA binding. At the same time, the disordered HNH domain of inactive Cas9 (A) undergoes a conformational change for cleavage of the targeted strand.
Figure 4.
Figure 4.
Structure of the DNA nuclease Cas3. The type I-E Cas3 crystal structure of Th. fusca (pdb: 4QQW) reveals two tandem RecA-like domains, one HD-type nuclease domain and a CTD located at the top of the ensemble. The core helicase, containing two RecA-like domains, forms a cleft that locates the residues for the binding of NTP, Mg2+ ions and the ssDNA substrate. Two Fe(II) ions are located at the catalytic center's HD motif. The 5 end of the ssDNA enters Cas3 from the RecA2 side and is further threaded to RecA1 and the HD-type nuclease domain (indicated by a scissor). The CTD is proposed to close the ssDNA channel and to contact the Cascade complex.
Figure 5.
Figure 5.
Mechanism of type I Cascade-mediated DNA interference. After the assembly of the crRNA-loaded Cascade, the surveillance complex (SSU: small subunits, LSU large subunit) scans DNA sequences. Potential DNA targets are identified via PAM recognition. This triggers the destabilization of the DNA duplex and allows the crRNA to pair with the target strand, while the non-target strand is displaced and spanned via the large and small subunit. Following R-loop formation, interaction sites at the base of the large subunit enable a stable interaction with Cas3. The HD domain of Cas3 nicks the DNA strand downstream of the PAM and the duplex is further unwound in 3–5 direction and degraded. The remaining single-stranded target DNA is proposed to be cleaved by the stand-alone Cas3 enzyme.
Figure 6.
Figure 6.
Mechanism of type II Cas9-mediated DNA interference. In the absence of type II specific dual-RNA, Cas9 is in an inactive state. Upon binding to tracrRNA and crRNA, Cas9 undergoes conformational changes, subsequently enabling dual-RNA guided binding to the target DNA. After successful DNA interrogation for a PAM and subsequent nucleation, the guide RNA pairs with the seed sequence on the DNA. This is followed by activation of the Cas9 nuclease domains HNH and RuvC for cleavage of the target and non-target DNA strand, respectively.
Figure 7.
Figure 7.
Comparison of type III crRNP-mediated RNA interference. (A) The type III-A Csm complex of Su. solfataricus is composed of 13 subunits that are arranged as a basal body and two intertwined filaments. Located at the base of the Csm complex is the large subunit Cas10, serving as an anchor for the major and the minor filament. The major filament consists of Csm4 and six Csm3 subunits and binds the crRNA. The minor filament consists of three Csm2 subunits and two additional Csm3 subunits. The Csm3 units that form the backbone of the Csm complex were shown to act as target RNA nucleases (indicated by a scissor) in S. thermophilus. (B) The type III-B Cmr complex of P. furiosus contains an extended helical backbone composed of Cmr4 and Cmr5 subunits. The tail is composed of the stable heterodimer Cas10 and Cmr3, while the curled head contains Cmr1 and Cmr6. The crRNA-binding backbone is formed by several Cmr4 subunits, while the Cas10-Cmr3 heterodimer plays a role in the recognition of the crRNA 5 handle. A second helical structure is formed by three Cmr5 subunits and is inserted alongside the helical Cmr4 filament. Cleavage of target RNA by Cmr4 was observed in 6-nt intervals (indicated by a scissor).

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