CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea

Abstract

Sequence-directed genetic interference pathways control gene expression and preserve genome integrity in all kingdoms of life. The importance of such pathways is highlighted by the extensive study of RNA interference (RNAi) and related processes in eukaryotes. In many bacteria and most archaea, clustered, regularly interspaced short palindromic repeats (CRISPRs) are involved in a more recently discovered interference pathway that protects cells from bacteriophages and conjugative plasmids. CRISPR sequences provide an adaptive, heritable record of past infections and express CRISPR RNAs - small RNAs that target invasive nucleic acids. Here, we review the mechanisms of CRISPR interference and its roles in microbial physiology and evolution. We also discuss potential applications of this novel interference pathway.

Figure 1. Features of CRISPR loci

Typically, clustered, regularly interspaced short palindromic repeats (CRISPRs, white boxes) are preceded by a leader sequence (black box) that is AT-rich but otherwise not conserved. The number of repeats can vary substantially, from a minimum of two to a few hundred. Repeat length, however, is restricted to 23 to 50 nucleotides. Repeats are separated by similarly sized, non-repetitive spacers (coloured boxes) that share sequence identity with fragments of plasmids and bacteriophage genomes and specify the targets of CRISPR interference. A set of CRISPR-associated (cas) genes immediately precedes or follows the repeats. These genes are conserved, can be classified into different families and subtypes, and encode the protein machinery responsible for CRISPR activity.

Figure 2. Acquisition of new repeat-spacer units

During the adaptation phase of clustered, regularly interspaced short palindromic repeat (CRISPR) immunity, new spacers derived from the invading DNA are incorporated into CRISPR loci. The mechanism of spacer acquisition is unknown but possibly uses the nuclease activity of CRISPR-associated 1 (Cas1) to generate short fragments of invading DNA. How the CRISPR machinery recognizes phage or plasmid DNA as invasive and therefore suitable for CRISPR locus incorporation is also unknown. The addition of new spacers is thought to involve an integration or conversion event and occurs at the leader-proximal end of the cluster. CRISPRs are shown as white boxes, the leader sequence is shown as a black box, non-repetitive spacers are shown as coloured boxes and cas genes are shown as grey arrows.

Figure 3. CRISPR interference

a | In the interference or defence phase of clustered, regularly interspaced short palindromic repeat (CRISPR) immunity, repeats and spacers are transcribed into a long precursor that is processed by a complex called CRISPR-associated complex for antiviral defence (Cascade) in Escherichia coli or CRISPR-associated 6 (Cas6) in Pyrococcus furiosus, which generates small CRISPR RNAs (crRNAs). Processing occurs near the 3′ end of the repeat sequence, leaving a short (~8 nucleotides) repeat sequence 5′ of the crRNA spacer. crRNAs have a more heterogeneous 3′ terminus that sometimes contains the palindromic sequence of the downstream repeat and has the potential to form a stem–loop structure. CRISPR loci transcription seems to be constitutive and the leader sequence may act as a promoter (arrow). CRISPRs are shown as white boxes, the leader sequence is shown as a black box, non-repetitive spacers are shown as coloured boxes and cas genes are shown as grey arrows. b | RNAs serve as guides for an effector complex, presumably composed of Cas proteins, that recognizes invading DNA and blocks infection (white cross) by an unknown mechanism.

Figure 4. self versus non-self discrimination during CRISPR immunity

The spacer sequences of clustered, regularly interspaced short palindromic repeat (CRISPR) RNAs (crRNAs) have perfect complementarity with the CRISPR locus from which they are transcribed, which confers the potential to target CRISPR DNA. In Staphylococcus epidermidis, this autoimmune response is prevented by differential base pairing of spacer/target 5′ flanking sequences. A similar role for 3′ flanking sequences in other organisms cannot be excluded. Whereas interference requires crRNA/target mismatches outside the spacer sequence, complementarity between crRNA and repeat sequences in the CRISPR DNA prevents autoimmunity. The figure is adapted, with permission, from Nature REF. © (2010) Macmillan Publishers Ltd. All rights reserved.