Review Article | Published:

Unravelling the structural and mechanistic basis of CRISPR–Cas systems

Nature Reviews Microbiology volume 12, pages 479492 (2014) | Download Citation


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.

Key points

  • CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) is an adaptive immune system in bacteria and archaea, which is categorized into three distinct types (known as type I, type II and type III) that differ in their compositions and mechanisms of action. Adaptive immunity occurs in three distinct stages: acquisition, expression and interference.

  • In all three types of CRISPR–Cas system, spacer acquisition relies on a pair of conserved Cas proteins (Cas1 and Cas2), which are probably assisted by a variable set of additional (potentially both Cas and non-Cas) enzymes.

  • Primary processing of CRISPR RNAs (crRNAs) is catalysed either by Cas6-like ribonucleases (in the type I and type III systems) or by RNase III (which targets the crRNA and transactivating crRNA (tracrRNA) duplex in type II systems).

  • Mature crRNAs form CRISPR ribonucleoprotein (crRNP) complexes by associating with either Cascade-like multiprotein complexes (in type I and type III systems) or the multidomain Cas9 protein (in type II systems).

  • Discrimination of 'self' nucleic acid from 'non-self' nucleic acid enables crRNP complexes to specifically target invading nucleic acid (usually DNA). Some complexes recruit additional nucleases (for example, Cas3 in type I systems), whereas other crRNPs have intrinsic nuclease domains (for example, Cas9 in type II systems).

  • Fundamental studies have elucidated many mechanistic features of CRISPR–Cas functionality by integrating genetics, biochemistry and structural biology. This has provided an excellent basis for developing a wide range of applications, from the manipulation of gene expression in bacteria to genome editing in eukaryotes.

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The authors thank members of the van der Oost laboratory (R. Staals and S. Brouns) and the Wiedenheft laboratory (specifically M.-C. Rollins and S. Golden) for discussions and critical reading of this manuscript. This work was supported by a Netherlands Organisation for Scientific Research (NWO) Earth and Life Sciences (ALW-TOP) grant (grant number 854.10.003) and NWO ALW Open competition to J.O. (grant number 820.02.003). E.R.W. received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement number 327606. Postdoctoral training for R.N.J. is supported by the US National Institutes of Health (grant number R01GM108888). B.W. is supported by the US National Institutes of Health (grant numbers P20GM103500 and R01GM108888), US National Science Foundation EPSCoR (grant number EPS-110134), the M.J. Murdock Charitable Trust and the Montana State University Agricultural Experimental Station.

Author information


  1. Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands.

    • John van der Oost
    •  & Edze R. Westra
  2. Environment and Sustainability Institute, University of Exeter, Penryn, Cornwall, TR10 9FE, UK.

    • Edze R. Westra
  3. Department of Microbiology and Immunology, Montana State University, PO Box 173520, Bozeman, Montana 59717, USA.

    • Ryan N. Jackson
    •  & Blake Wiedenheft


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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to John van der Oost.

Supplementary information

PDF files

  1. 1.

    Supplementary information S1 (table)

    Overview of Cas proteins.

  2. 2.

    Supplementary information S2 (figure)

    Crystal structure of target-bound Cas9.

  3. 3.

    Supplementary information S3 (figure)

    Homology model of Cas3.

  4. 4.

    Supplementary information S4 (figure)

    RNA–DNA hybrid in Cascade/I-E.

  5. 5.

    Supplementary information S5 (figure)

    Nucleoprotein filaments with distinct physiological functions have conserved architectures.


Restriction–modification systems

(R–M systems). Innate defence systems in bacteria and archaea that enable the discrimination of 'non-self' DNA from 'self' DNA. These systems typically consist of an endonuclease that specifically recognizes and cleaves a short palindromic sequence motif in invading DNA and a methyltransferase that methylates a nucleotide within the same motif in the genomic DNA of the host cell, thereby protecting self DNA from degradation.


(CRISPR-associated complex for antiviral defence). A multisubunit Cas (CRISPR-associated protein) complex that associates with a CRISPR RNA (crRNA) in type I CRISPR–Cas systems. Recent insights have revealed that the Cascade core is conserved in type III CRISPR ribonucleoprotein (crRNP) complexes.


The DNA sequence upstream of a CRISPR locus; it contains the promoter and sequence elements that drive polarized repeat duplication and spacer acquisition.

Protospacer adjacent motif

(PAM). A short signature sequence (of 2–5 nucleotides) that flanks the protospacer in invading DNA. Recognition of the PAM by type I and type II CRISPR–Cas systems triggers interference.


A protein fold that resembles the widely distributed RNA-recognition motif (RRM); it is also referred to as a ferredoxin fold.


The target DNA sequence that is complementary to the spacer of the CRISPR RNA.


A dimeric endoribonuclease that cleaves double-stranded RNA; it typically generates products that have a 2 nucleotide overhang at the 3′ end.


One of two nucleolytic domains in Cas9; it is related to the nucleolytic domain of McrA-like restriction endonucleases.


One of two nucleolytic domains in Cas9; it is homologous to nucleases that are involved in recombination.


A structure that is formed by the hybridization of an RNA strand with double-stranded DNA. The RNA base pairs with a complementary sequence in one of the DNA strands, which causes the displaced DNA strand to form a loop.


A short sequence within the CRISPR RNA that is required for perfect base pairing with the target sequence. This short stretch of 7–8 nucleotides is most probably the site of initial hybridization with the complementary target strand, resulting in R-loop formation and CRISPR interference.

HD-nuclease domain

The domain of Cas3 that is responsible for the nucleolytic degradation of double-stranded DNA targets. In many Cas3 proteins, this nuclease domain is fused to a helicase domain.

SF2-helicase domain

(Superfamily 2-helicase domain). The multidomain component of Cas3 that is responsible for unwinding double-stranded DNA targets. Proteins of the SF2-type helicase superfamily consist of two RecA-like domains (with an ATP-binding site at their interface) and a flexible carboxy-terminal domain.


The classic right-handed DNA double helix (established by Watson and Crick), which is the predominant DNA conformation under physiological conditions.


A nuclease that is involved in eukaryotic RNA interference and bacterial and archaeal DNA interference. Argonaute contains an RNaseH nucleolytic domain that is homologous to RuvC.


A conformation of the DNA helix that is more compact than the B-form; it is often present in double-stranded RNA and in DNA–RNA hybrids.

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