The CRISPR system: small RNA-guided defense in bacteria and archaea

Abstract

All cellular systems evolve ways to combat predators and genomic parasites. In bacteria and archaea, numerous resistance mechanisms have developed against phage. Our understanding of this defensive repertoire has recently been expanded to include the CRISPR system of clustered, regularly interspaced short palindromic repeats. In this remarkable pathway, short sequence tags from invading genetic elements are actively incorporated into the host's CRISPR locus to be transcribed and processed into a set of small RNAs that guide the destruction of foreign genetic material. Here we review the inner workings of this adaptable and heritable immune system and draw comparisons to small RNA-guided defense mechanisms in eukaryotic cells.

Figure 1. The structure of a CRISPR locus

(A) Repeat sequences averaging 32bp are interleaved by variable spacers of approximately the same size. (B) The number of repeat-spacer units varies greatly. A conserved leader sequence (gray) of several hundred base pairs is located on one side of the cluster. (C) CRISPR-associated (cas) genes surround the CRISPR locus. Three examples of well-studied CRISPR loci are shown. Core cas genes are depicted in red, subtype-specific genes in blue, and the RAMP module in green. Unclassified genes are shown in dark grey. Gene names follow the nomenclature of Haft et al., except cas7, which was named by Barrangou et al.

Figure 2. Diversity and acquisition of spacer content

(A) A schematic representation of the CRISPR loci in related bacterial strains. Colored spacers represent sequences that differ among strains. All strains share the same ancestral spacers at the leader-distal end (gray spacers). Deletions after divergence have occurred in some strains. Spacer content becomes more strain-specific near the leader end. Strain ancestry can be traced by the presence of common spacers (green, red). (B) Acquisition of new spacers occurs next to the leader sequence. The spacer sequence is derived from the invading nucleic acid, selected based upon the presence of a proto-spacer adjacent motif.

Figure 3. Processing of CRISPR content into crRNAs

The locus is transcribed from the leader sequence and the RNA is cleaved within the repeat by cse3 or cas6. An additional processing step yields the mature crRNA/psiRNA consisting of an 8nt repeat tag and the spacer sequence.

Figure 4. An overall model of CRISPR/cas activity

During spacer acquisition, sequence elements from invading nucleic acids become incorporated at the leader-proximal end of the CRISPR locus. In the processing stage, the locus is transcribed and processed into mature crRNA/psiRNAs containing an 8nt repeat tag and a single spacer unit. During the effector stage, the mature crRNAs in complex with associated cas proteins leads to degradation of complementary nucleic acids. See text for details.

Figure 5. Conserved themes in small RNA-guided defense

(A) The CRISPR system actively incorporates short sequence tags from invading nucleic acids into the CRISPR locus of regularly interspaced repeats. (B) In the piRNA system, genomic piRNA clusters passively acquire new content by movement of transposons into the locus. In both cases, the locus is transcribed and processed into small RNAs that guide the destruction of exogenous or parasitic sequences.