Box 1: Cas protein families and functional modules
The Cas proteins can be divided into four distinct functional modules: adaptation (spacer acquisition); expression (crRNA processing and target binding); interference (target cleavage); and ancillary (regulatory and other CRISPR-associated functions) (Fig. 1). In recent years, a wealth of structural and functional information has accumulated for the core Cas proteins (Cas1–Cas10) (see Supplementary information S1 (table)), which allows them to be classified into these modules.
The adaptation module is largely uniform across CRISPR–Cas systems and consists of the Cas1 and Cas2 proteins, with possible additional involvement of the restriction endonuclease superfamily enzyme Cas4 (Ref. 91) and, in type II systems, Cas9 (Refs 63,64). Cas1, which adopts a unique α-helical fold, is an integrase that mediates the insertion of new spacers into CRISPR arrays by cleaving specific sites within the repeats17, 89, 92. The role of Cas2, which is a homologue of the mRNA interferase toxins of numerous toxin–antitoxin systems, is less well understood3, 72, 93, 94. Cas2 has been shown to form a complex with Cas1 in the Escherichia coli type I CRISPR–Cas system and is required for adaptation. However, although Cas2 has RNase95 and DNase activities96, its catalytic residues are dispensable for adaptation17, indicating that these activities are not directly involved in this process, at least in this species.
The expression and interference modules are represented by multisubunit CRISPR RNA (crRNA)–effector complexes36, 38, 39, 43, 44, 45, 46, 97, 98 (Box 2) or, in type II systems, by a single large protein, Cas9 (Refs 24,25,99). In the expression stage, pre-crRNA is bound to the multisubunit crRNA–effector complex, or to Cas9, and processed into a mature crRNA in a step catalysed by an RNA endonuclease23 (typically Cas6; in type I and type III systems) or an alternative mechanism that involves RNase III and a transactivating CRISPR RNA (tracrRNA)24 (in type II systems). However, in at least one type II CRISPR–Cas system, that of Neisseria meningitidis, crRNAs with mature 5′ ends are directly transcribed from internal promoters, and crRNA processing does not occur69.
In the interference module, the crRNA–effector complex (in type I and type III systems) or Cas9 (in type II systems) combines nuclease activity with dedicated RNA-binding domains. Target binding relies on base pair formation with the spacer region of the crRNA. Cleavage of the target is catalysed by the HD family nuclease (Cas3′′ or a domain in Cas3) in type I systems52, 100, by the combined action of the Cas7 and Cas10 proteins in type III systems26, 39, 46, 101, 102, 103, 104 or by Cas9 in type II systems25. In type I systems, the HD nuclease domain is either fused to the superfamily 2 helicase Cas3′ (Refs 50,51,52) or is encoded by a separate gene, cas3′′, whereas in type III systems a distinct HD nuclease domain is fused to Cas10 and is thought to cleave single-stranded DNA during interference105. In type II systems, the RuvC-like nuclease (RNase H fold) domain and the HNH (McrA-like) nuclease domain of Cas9 each cleave one of the strands of the target DNA25, 106. Remarkably, the large (~950–1,400 amino acids) multidomain Cas9 protein is required for all three of the functional steps of CRISPR-based immunity (adaptation, expression and interference) in type II systems and thus concentrates much of the CRISPR–Cas system's function in a single protein.
The ancillary module is a combination of various proteins and domains that, with the exception of Cas4, are much less common than the core Cas proteins in CRISPR–Cas systems. Aside from its putative role in adaptation, Cas4 is thought to contribute to CRISPR–Cas-coupled programmed cell death3, 94. Other notable components of the ancillary module include: a diverse set of proteins containing the CRISPR-associated Rossmann fold (CARF) domain35, 107, which have been hypothesized to regulate CRISPR–Cas activity107 (in many type I and type III systems); and the inactivated P-loop ATPase Csn2, which forms a homotetrameric ring that accommodates linear double-stranded DNA in the central hole (in type II systems)108, 109, 110, 111. Csn2 is not required for interference but apparently has a role in spacer integration, possibly preventing damage from the double-strand break in the chromosomal DNA6, 110. Ancillary module genes are often found outside of CRISPR–cas loci, but the functions of these stand-alone genes have not been characterized in depth72, 94.
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894, USA.
- Kira S. Makarova,
- Yuri I. Wolf,
- Daniel H. Haft &
- Eugene V. Koonin
Bioinformatics group, Department of Computer Science, University of Freiberg, Georges-Kohler-Allee 106, 79110 Freiberg, Germany.
- Omer S. Alkhnbashi,
- Fabrizio Costa,
- Sita J. Saunders &
- Rolf Backofen
Archaea Centre, Department of Biology, Copenhagen University, Ole Maaløes Vej 5, DK2200 Copenhagen N, Denmark.
- Shiraz A. Shah &
- Roger A. Garrett
Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, North Carolina 27606, USA.
- Rodolphe Barrangou
Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703HB Wageningen, Netherlands.
- Stan J. J. Brouns &
- John van der Oost
Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany.
- Emmanuelle Charpentier
DuPont Nutrition and Health, BP10, Dangé-Saint-Romain 86220, France.
- Philippe Horvath
Département de Biochimie, de Microbiologie et de Bio-informatique, Faculté des Sciences et de Génie, Groupe de Recherche en Écologie Buccale, Félix d'Hérelle Reference Center for Bacterial Viruses, Faculté de médecine dentaire, Université Laval, Québec City, Québec, Canada.
- Sylvain Moineau
Departamento de Fisiología, Genética y Microbiología. Universidad de Alicante. 03080-Alicante, Spain.
- Francisco J. M. Mojica
Biochemistry and Molecular Biology, Genetics and Microbiology, University of Georgia, Davison Life Sciences Complex, Green Street, Athens, Georgia 30602, USA.
- Rebecca M. Terns &
- Michael P. Terns
Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews, KY16 9TZ, UK.
- Malcolm F. White
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, M5S 3E5, Canada.
- Alexander F. Yakunin
BIOSS Centre for Biological Signaling Studies, Cluster of Excellence, University of Freiburg, Germany.
- Rolf Backofen
Competing interests statement
The authors declare no competing interests.
Kira S. Makarova
Yuri I. Wolf
Omer S. Alkhnbashi
Shiraz A. Shah
Sita J. Saunders
Stan J. J. Brouns
Daniel H. Haft
Francisco J. M. Mojica
Rebecca M. Terns
Michael P. Terns
Malcolm F. White
Alexander F. Yakunin
Roger A. Garrett
John van der Oost
Eugene V. Koonin