RNA-guided genetic silencing systems in bacteria and archaea

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Clustered regularly interspaced short palindromic repeat (CRISPR) are essential components of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea. Similar to RNA interference (RNAi) pathways in eukaryotes, CRISPR-mediated immune systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids, including viruses and plasmids. However, the mechanism of RNA-based bacterial immunity is distinct from RNAi. Understanding how small RNAs are used to find and destroy foreign nucleic acids will provide new insights into the diverse mechanisms of RNA-controlled genetic silencing systems.

At a glance


  1. Parallels and distinctions between CRISPR RNA-guided silencing systems and RNAi.
    Figure 1: Parallels and distinctions between CRISPR RNA-guided silencing systems and RNAi.

    CRISPR systems and RNAi recognize long RNA precursors that are processed into small RNAs, which act as sequence-specific guides for targeting complementary nucleic acids. In CRISPR systems, foreign DNA is integrated into the CRISPR locus, and long transcripts from these loci are processed by a CRISPR-associated (Cas) or RNase III family nuclease16, 17, 18, 19, 20, 21, 64. The short CRISPR-derived RNAs (crRNAs) assemble with Cas proteins into large surveillance complexes that target destruction of invading genetic material15, 22, 24, 25, 26, 27, 48. In some eukaryotes, long double-stranded RNAs are recognized as foreign, and a specialized RNase III family endoribonuclease (Dicer) cleaves these RNAs into short-interfering RNAs (siRNAs) that guide the immune system to invading RNA viruses76. PIWI-interacting RNAs (piRNAs) are transcribed from repetitive clusters in the genome that often contain many copies of retrotransposons and primarily act by restricting transposon mobility76, 77, 78. The biogenesis of piRNAs is not yet fully understood. MicroRNAs (miRNAs) are also encoded on the chromosome, and primary miRNA transcripts form stable hairpin structures that are sequentially processed (shown by red triangles) by two RNase III family endoribonucleases (Drosha and Dicer)79. miRNAs do not participate in genome defence but are major regulators of endogenous gene expression80. Like crRNAs, eukaryotic piRNAs, siRNAs and miRNAs associate with proteins that facilitate complementary interactions with invading nucleic acid targets27, 60, 69, 79. In eukaryotes, the Argonaute proteins pre-order the 5′ region of the guide RNA into a helical configuration, reducing the entropy penalty of interactions with target RNAs69. This high-affinity binding site, called the 'seed' sequence, is essential for target sequence interactions. Recent studies indicate that the CRISPR system may use a similar seed-binding mechanism for enhancing target sequence interactions26, 27, 53, 60.

  2. Diversity of CRISPR-mediated adaptive immune systems in bacteria and archaea.
    Figure 2: Diversity of CRISPR-mediated adaptive immune systems in bacteria and archaea.

    A diverse set of CRISPR-associated (cas) genes (grey arrows) encode proteins required for new spacer sequence acquisition (Stage 1), CRISPR RNA biogenesis (Stage 2) and target interference (Stage 3). Each CRISPR locus consists of a series of direct repeats separated by unique spacer sequences acquired from invading genetic elements (protospacers). Protospacers are flanked by a short motif called the protospacer adjacent motif (PAM, **) that is located on the 5′ (type I) or 3′ (type II) side in foreign DNA10, 51, 52, 59, 67. Long CRISPR transcripts are processed into short crRNAs by distinct mechanisms. In type I and III systems, a CRISPR-specific endoribonuclease (yellow ovals and green circles, respectively) cleaves 8 nucleotides upstream of each spacer sequence16, 18, 19, 20, 21, 64. In type III systems, the repeat sequence on the 3′ end of the crRNA is trimmed by an unknown mechanism (green pacman, right). In type II systems, a trans-acting antisense RNA (tracrRNA) with complementarity to the CRISPR RNA repeat sequence forms an RNA duplex that is recognized and cleaved by cellular RNase III (brown ovals)17. This cleavage intermediate is further processed at the 5′ end resulting in a mature, approximately 40-nucleotide crRNA with an approximately 20-nucleotide 3′-handle. In each system, the mature crRNA associates with one or more Cas proteins to form a surveillance complex (green rectangles). Type I systems encode a Cas3 nuclease (blue pacman), which may be recruited to the surveillance complex following target binding24, 27, 44. A short high-affinity binding site called a seed-sequence has been identified in some type I systems27, 60, and genetic experiments suggest that type II systems have a seed sequence located at the 3′end of the crRNA spacer sequence53.

  3. Steps leading to new spacer integration.
    Figure 3: Steps leading to new spacer integration.

    a, The Cas1 protein forms a stable homodimer where the two molecules (green and grey) are related by a pseudo-two-fold axis of symmetry (PBD ID: 3GOD)54, 56. This organization creates a saddle-like structure in the N-terminal domain, in which β-hairpins (blue) from each symmetrically related molecule hang (like stirrups) that are separated by approximately 20 Å, and may interact with the phosphodiester backbone of double-stranded DNA. An electrostatic surface representation (bottom) reveals a cluster of basic residues (blue) that form a positively charged strip across the metal-binding surface of the C-terminal domain. This strip may serve as an electrostatic trap that positions DNA substrates proximally to catalytic metal ions (green sphere). b, CRISPR adaptation occurs by integrating fragments of foreign nucleic acid preferentially at the leader end of the CRISPR, forming new repeat-spacer units in the process. Protospacers are chosen non-randomly and may be selected from regions flanking the protospacer adjacent motif (PAM). Coordinated cleavage of the foreign DNA and integration of the protospacer into the leader-end of the CRISPR occurs through a mechanism that duplicates the repeat sequence and thus preserves the repeat-spacer-repeat architecture of the CRISPR locus. Although the protein components required for this process have not been conclusively identified, Cas1 and other general recombination or repair factors have been implicated (blue ovals)32, 54, 56.

  4. Diverse mechanisms of CRISPR RNA biogenesis.
    Figure 4: Diverse mechanisms of CRISPR RNA biogenesis.

    CRISPR RNA repeats are specifically recognized and cleaved by diverse mechanisms. In type I CRISPR systems, Cas6e (PDB ID: 2Y8W) and Cas6f (PDB ID: 2XLK) recognize the major groove of the crRNA stem-loop primarily through electrostatic interactions using a β-hairpin and α-helix, respectively18, 19, 20. Cleavage occurs at the double-stranded–single-stranded junction (black arrows), leaving an 8-nt 5′-handle on mature crRNAs. In type II CRISPR systems, tracrRNA hybridizes to the pre-crRNA repeat to form duplex RNAs that are substrates for endonucleolytic cleavage by host RNase III (PDB ID: 2EZ6), an activity that may also require Cas9 (ref. 17). Subsequent trimming (red arrows) by an unidentified nuclease removes leftover repeat sequences from the 5′ end. Cas6 (PDB ID: 3PKM) in type III-B CRISPR systems specifically recognizes single-stranded RNA, upstream of the scissile phosphate, on a face of the protein opposite that of the previously identified active site residues16, 21, 64. The remainder of the repeat substrate probably wraps around the protein (red dashed line) to allow cleavage 8 nucleotides upstream of the repeat-spacer junction. Subsequent 3′ trimming (red arrows) generates mature crRNAs of two discrete lengths. The N-terminal domain of all Cas 6 family proteins adopts a ferredoxin-like fold (light blue). The C-terminal domain of Cas6 and Cas6e also adopts a ferredoxin-like fold but the C-terminal domain of Cas6f is structurally distinct (dark blue).


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  1. Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, Maryland 20815-6789, USA.

    • Blake Wiedenheft &
    • Jennifer A. Doudna
  2. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.

    • Blake Wiedenheft &
    • Jennifer A. Doudna
  3. Department of Chemistry, University of California, Berkeley, California 94720, USA.

    • Samuel H. Sternberg &
    • Jennifer A. Doudna
  4. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.

    • Jennifer A. Doudna
  5. Present address: Department of Immunology and Infectious Diseases, Montana State University, Bozeman, Montana, USA

    • Blake Wiedenheft

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