Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

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Science  05 Aug 2016:
Vol. 353, Issue 6299, aad5147
DOI: 10.1126/science.aad5147
  • Evolution of CRISPR-Cas systems resulted in incredible structural and functional diversity.

    Class 1 CRISPR-Cas systems are considered to be the evolutionary ancestral systems. The class 2 systems have evolved from class 1 systems via the insertion of transposable elements encoding various nucleases, and are now being used as tools for genome editing.

  • Fig. 1 Overview of the CRISPR-Cas systems.

    (A) Architecture of class 1 (multiprotein effector complexes) and class 2 (single-protein effector complexes) CRISPR-Cas systems. (B) CRISPR-Cas adaptive immunity is mediated by CRISPR RNAs (crRNAs) and Cas proteins, which form multicomponent CRISPR ribonucleoprotein (crRNP) complexes. The first stage is adaptation, which occurs upon entry of an invading mobile genetic element (in this case, a viral genome). Cas1 (blue) and Cas2 (yellow) proteins select and process the invading DNA, and thereafter, a protospacer (orange) is integrated as a new spacer at the leader end of the CRISPR array [repeat sequences (gray) that separate similar-sized, invader-derived spacers (multiple colors)]. During the second stage, expression, the CRISPR locus is transcribed and the pre-crRNA is processed into mature crRNA guides by Cas (e.g., Cas6) or non-Cas proteins (e.g., RNase III). During the final interference stage, the Cas-crRNA complex scans invading DNA for a complementary nucleic acid target, after which the target is degraded by a Cas nuclease.

  • Fig. 2 CRISPR diversity and evolution.

    (A) Modular organization of the CRISPR-Cas systems. LS, large subunit; SS, small subunit. A putative small subunit that might be fused to the large subunit in several type I subtypes is indicated by an asterisk. Cas3 is shown as fusion of two distinct genes encoding the helicase Cas3′ and the nuclease HD Cas3′′; in some type I systems, these domains are encoded by separate genes. Functionally dispensable components are indicated by dashed outlines. Cas6 is shown with a thin solid outline for type I because it is dispensable in some systems, and by a dashed line for type III because most systems lack this gene and use the Cas6 provided in trans by other CRISPR-Cas loci. The two colors for Cas4 and C2c2 and three colors for Cas9 and Cpf1 reflect the contributions of these proteins to different stages of the CRISPR-Cas response (see text). The question marks indicate currently unknown components. [Modified with permission from (30)] (B) Evolutionary scenario for the CRISPR-Cas systems. TR, terminal repeats; TS, terminal sequences; HD, HD-family endonuclease; HNH, HNH-family endonuclease; RuvC, RuvC-family endonuclease; HEPN, putative endoribonuclease of HEPN superfamily. Genes and portions of genes shown in gray denote sequences that are thought to have been encoded in the respective mobile elements but were eliminated in the course of evolution of CRISPR-Cas systems. [Modified with permission from (31)]

  • Fig. 3 Spacer acquisition.

    (A) Crystal structure of the complex of Cas1-Cas2 bound to the dual-forked DNA (PDB accession 5DQZ). The target DNA is shown in dark blue; the Cas1 and Cas2 dimers of the complex are indicated in blue and yellow, respectively. (B) Model explaining the capture of new DNA sequences from invading nucleic acid and the subsequent DNA integration into the host CRISPR array. The numbers on the left correspond to the order of events as described in the text. The dashed lines indicate nucleotides; the nucleotides C and N on the two sides of the protospacer are shown in red and green to clarify the orientation.

  • Fig. 4 Guide expression and processing.

    (A) Generation of CRISPR RNA (crRNA) guides in type I and type III CRISPR-Cas systems. Primary processing of the pre-crRNA is catalyzed by Cas6, which typically results in a crRNA with a 5′ handle of 8 nt, a central spacer sequence, and (in some subtypes) a longer 3′ handle. Shown here is the guide processing (red triangles) for subtype I-E by Cas6e. The occasional secondary processing of the 3′ end of crRNA is catalyzed by one or more unknown RNases. (B) In type II-A 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); further trimming of the 5′ end of the spacer is carried out by unknown RNase(s) (pink). (C) CRISPR with transcriptional start site (TSS) in repeats, as observed in type II-B CRISPR-Cas systems.

  • Fig. 5 CRISPR RNP complexes.

    Crystal structures of the CRISPR ribonucleoprotein (crRNP) complexes responsible for target interference. Shown are the type I-E Cascade complex (PDB accession 4QYZ) and type III-B Cmr complex (PDB accession 3X1L) from class 1, and the type II-A Cas9 complex (PDB accession 4OO8) and type V-A Cpf1 complex (PDB accession 5B43) from class 2. Colors of nucleic acid fragments are the same as in Fig. 6.

  • Fig. 6 Target interference.

    Genomic loci architecture of the components of class 1 and class 2 CRISPR-Cas systems and schematic representation of target interference for the different subtypes. The double-stranded DNA (target) is shown in black, the target RNA in gray, the CRISPR RNA (crRNA) repeat in blue, the spacer region of the crRNA in green, and the transactivating CRISPR RNA (tracrRNA) in red.

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