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, 12 (7), 479-92

Unravelling the Structural and Mechanistic Basis of CRISPR-Cas Systems


Unravelling the Structural and Mechanistic Basis of CRISPR-Cas Systems

John van der Oost et al. Nat Rev Microbiol.


Bacteria and archaea have evolved sophisticated adaptive immune systems, known as CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems, which target and inactivate invading viruses and plasmids. Immunity is acquired by integrating short fragments of foreign DNA into CRISPR loci, and following transcription and processing of these loci, the CRISPR RNAs (crRNAs) guide the Cas proteins to complementary invading nucleic acid, which results in target interference. In this Review, we summarize the recent structural and biochemical insights that have been gained for the three major types of CRISPR-Cas systems, which together provide a detailed molecular understanding of the unique and conserved mechanisms of RNA-guided adaptive immunity in bacteria and archaea.

Conflict of interest statement

Competing interests statement

The authors declare no competing interests.


Figure 1
Figure 1. Overview of the CRISPR–Cas system
Adaptive immunity by CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) systems is mediated by CRISPR RNAs (crRNAs) and Cas proteins, which form multicomponent CRISPR ribonucleoprotein (crRNP) complexes. The cas genes are coloured according to function, as indicated by the four functional categories in coloured boxes: spacer acquisition (yellow); crRNA processing (pink); crRNA assembly and surveillance (blue); and target degradation (purple). Involvement of non-Cas components (grey) is indicated, either when experimentally demonstrated (for example, RNase III processing in type II systems) or when anticipated (for example, the potential involvement of housekeeping repair and/or recombination enzymes). The first stage is known as acquisition, which occurs following the entry of an invading mobile genetic element (in this case, a viral genome). The invading DNA is fragmented and a new protospacer (green) is selected, processed and integrated as a new spacer at the leader end of the CRISPR array. During the second stage, which is known as expression, the CRISPR locus is transcribed and the pre-crRNA is processed into small crRNAs by CRISPR-associated (Cas6) and/or housekeeping ribonucleases (such as RNase III). The mature crRNAs and Cas proteins assemble to form a crRNP complex. During the final stage of interference, the crRNP scans invading DNA for a complementary nucleic acid target and on successful recognition, the target is eventually degraded by Cas nucleases.
Figure 2
Figure 2. Diversity of CRISPR–Cas systems
The CRISPR-associated (Cas) proteins can be divided into distinct functional categories as shown. The three types of CRISPR–Cas systems are defined on the basis of a type-specific signature Cas protein (indicated by an asterisk) and are further subdivided into subtypes. The CRISPR ribonucleoprotein (crRNP) complexes of type I and type III systems contain multiple Cas subunits, whereas the type II system contains a single Cas9 protein. Boxes indicate components of the crRNP complexes for each system. The type III-B system is unique in that it targets RNA, rather than DNA, for degradation.
Figure 3
Figure 3. CRISPR spacer acquisition
a | Proposed stages of CRISPR spacer acquisition: fragmentation of invading DNA (in this case, phage DNA), selection of the protospacer by recognition of the protospacer adjacent motif (PAM), processing of the pre-spacer, nicking of the leader-end repeat in the CRISPR locus, integration of the new spacer and duplication of the flanking repeat. Both type I and type II systems rely on PAM recognition for spacer integration, whereas the type III systems do not. b | Crystal structures of Cas1 (from Pseudomonas aeruginosa; Protein Databank (PDB) accession 3GOD) and Cas2 (from Desulfovibrio vulgaris, PDB accession 3OQ2), which are the two main endonucleases that are involved in spacer acquisition. Cas1 is a metal-dependent, dimeric endonuclease (DNase) with a unique three-dimensional fold that consists of an amino-terminal β-strand domain and a carboxy-terminal α-helical domain. Sequence conservation (indicated by colour intensity) of Cas1 shows that the metal ion-binding site is highly conserved among Cas1 family proteins. Cas2 is a metal-dependent, dimeric endonuclease (RNase and/or DNase), with a metal-binding site at the interface of the two subunits (which is composed of RAMP domains). The conservation model was generated using Consurf and the figure was made using PyMol.
Figure 4
Figure 4. Biogenesis of crRNAs
a | The CRISPR array is transcribed to produce a pre-CRISPR RNA (pre-crRNA) transcript, the primary processing of which occurs by cleavage (red triangles) within the repeat sequences, producing crRNAs in which spacers are flanked by repeat-derived handles. b | Generation of CRISPR guide RNAs in type I and type III CRISPR–Cas systems. Primary processing of the pre-crRNA is catalysed by Cas6, which typically results in a crRNA with a 5′ handle of 8 nucleotides, a central spacer sequence and a longer 3′ handle. In some subtypes, the 3′ handle forms a stem–loop structure, in other systems, secondary processing of the 3′ end of crRNA (yellow triangles) is catalysed by unknown ribonucleases. c | In type II CRISPR–Cas systems, the repeat sequences of the pre-crRNA hybridize with complementary sequences of transactivating CRISPR RNA (tracrRNA). The double-stranded RNA is cleaved by RNase III (red triangles) and further trimming of the 5′ end of the spacer is carried out by additional nucleases (yellow triangle). d | Crystal structures of CRISPR-associated ribonucleases that catalyse primary processing of pre-crRNA. Cas6e (from the type I-E system; Protein Databank (PDB) accession 4C9D) and Cas6f (from the type I-F system; PDB accession 4AL7) are shown complexed to the hairpin of the crRNA (blue). In type I-C systems, a Cas5 variant (known as Cas5d) substitutes for Cas6 and is involved in pre-crRNA processing (PDB accession 4F3M). For all three structures, the location of the active site (which contains a catalytic histidine residue) is indicated with a circle. Sequence conservation is indicated by colour intensity. The conservation model was generated using Consurf and the figure was made using PyMol.
Figure 5
Figure 5. Architecture of crRNP complexes
a | Schematic representation of the subunit composition of different CRISPR ribonucleoprotein (crRNP) complexes from all three CRISPR–Cas types. The colours indicate homology with conserved Cas proteins or defined components of the complexes, as shown in the key. The numbers refer to protein names that are typically used for individual subunits of each subtype (for example, subunit 5 of the type I-A (Csa) complex refers to Csa5, whereas subunit 2 of the type I-E (Cse) complex refers to Cse2, and so on). The CRISPR RNA (crRNA) is shown, including the spacer (green) and the flanking repeats (grey). Truncated Cas3 domains (Cas3′ and Cas3″) have been suggested to be part of the type I-A complex, and fusions of Cas3 with Cascade subunits (for example, with Cse1 (REF. 103)) have been found in some type I-E systems (shown as a dashed Cas3 homologue). Cas9 is depicted in complex with single-guide RNA (sgRNA), with an artificial linker (light grey) between the crRNA and the tracrRNA. Subunits with a RAMP (that is, an RNA-recognition motif (RRM)) fold are shown with a bold outline. The grey subunit in the type III-A Csm complex has been proposed to be a Cas7 homologue. b | Structural comparison of crRNP complexes (colours as in part a): cryo- electron microscopy (cryo-EM) structures of Escherichia coli Cascade/I-E bound to a crRNA (two views after 90 ° rotation; Electron Microscopy Data Bank (EMDB) accession 5314; 8.8 Å), with additional double-stranded DNA (dsDNA) target (9 Å) and with additional Cas3 (20 Å). Cryo-EM structure of Streptococcus pyogenes Cas9 (of the type II-A system) bound to a single-guide RNA (sgRNA; not shown) and a 20 nucleotide target single-stranded DNA (ssDNA; not shown) (EMDB accession 5860; 21 Å), revealing a recognition lobe and a nuclease lobe, with a cleft in which the crRNA–DNA hybrid is located (see crystal structure; Supplementary information S2 (figure)). Cryo-EM structure of type III crRNP complexes: Sulfolobus solfataricus Csm complex (EMDB accession 2420; 30 Å), and Cmr complexes from Pyrococcus furiosus (EMDB accession 5740; 12 Å) and Thermus thermophilus.
Figure 6
Figure 6. Surveillance and interference by crRNP complexes
Proposed mechanisms of targeting for the three different types of CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) systems. a | In type I systems, the Cascade complex searches for a complementary protospacer in the invader DNA via target scanning. The large subunit (Cse1 or Cas8) of the complex recognizes the protospacer adjacent motif (PAM) sequence by a ‘non-self activation’ strategy (BOX 1), which is followed by hybridization between the seed sequence and the target DNA. If these initial criteria are met, complete base pairing results in R-loop formation and a simultaneous conformational change in the Cascade complex, which probably triggers Cas3 recruitment and subsequent degradation of the displaced target DNA strand (red triangles indicate endonucleolytic cleavage). The dashed arrow indicates processivity by the concerted helicase (green triangle) and exonuclease activities in the 3′ to 5′ direction. b | In type II systems, the Cas9 complex, bound to the CRISPR RNA (crRNA)–transactivating crRNA (tracrRNA) duplex, follows a similar mechanism of PAM-dependent recognition of invading DNA. However, unlike type I systems, the PAM is located upstream (at the 5′ end) of the protospacer and both target DNA strands are cleaved by Cas9-mediated nuclease activity. c | In type III-A systems, the crRNA-bound Csm complex targets DNA in a PAM-independent process, using a ‘self inactivation’ strategy (BOX 1). The stand-alone nuclease that is responsible for DNA degradation has been proposed to be Csm6 (also known as Csx1) (FIG. 2; Supplementary information S1 (table)). d | In type III-B systems, the crRNA-guided Cmr complex targets invading RNA in a PAM-independent process. After recognition and hybridization of crRNA and a complementary target RNA sequence, cleavage of this target occurs at multiple sites (red triangles). The nuclease that is responsible for RNA degradation has been proposed to be a subunit of the Cmr complex (Cmr4; Supplementary information S1 (table)). With the exception of type I systems, in which Cas3 mediates target degradation (part a), all other systems (parts bd), are thought to involve non-Cas nucleases for complete target degradation.

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