Recently, arrays of clustered, regularly interspaced short palindromic repeats (CRISPRs) have been implicated in a novel genetic interference pathway that limits phage infection and plasmid conjugation. CRISPR loci keep a record of past infections to provide bacteria and archaea with a 'genetic memory' that directs the rejection of invader DNA molecules; therefore these loci constitute an adaptive immune system.
CRISPRs (which are approximately 25–50 nucleotides long) are separated by similarly short sequences called spacers that match bacteriophage or plasmid sequences and specify the targets of interference. CRISPR-associated (cas) genes, a set of conserved genes that are associated with these loci, are usually present on one or the other side of the array.
CRISPR loci are transcribed as a long precursor that is processed by Cas proteins within the repeat sequences to generate small CRISPR RNAs (crRNAs). The crRNAs serve as guides for target recognition during CRISPR interference. crRNA–Cas ribonucleoprotein complexes seem to generally target invading DNA sequences during interference but may also target RNAs in some species.
Upon bacteriophage challenge of a CRISPR-containing bacterial population, mutants resistant to the infection arise through the incorporation of additional spacer sequences derived from the challenging phage. This allows the bacteria to evolve rapidly and adapt to the viruses in the environment.
Bacteriophages and plasmids can mobilize foreign genetic material between cells, a process known as horizontal gene transfer (HGT), which is a fundamental source of genetic variability for the evolution of bacteria and archaea. CRISPR interference prevents phage infection and plasmid conjugation and therefore constitutes a natural barrier to HGT. In addition, bacteriophages constantly mutate to evade CRISPR defence. Therefore, CRISPR interference has an important role in the evolution of microbial communities.
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.
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We thank R. Terns and M. Terns for communicating results before publication. L.A.M. is a fellow of The Jane Coffin Childs Memorial Fund for Medical Research. This work was supported by a grant from the US National Institutes of Health to E.J.S.
The authors declare no competing financial interests.
Genetic alteration of a cell resulting from the acquisition of genes from free DNA molecules in the surrounding environment.
The transfer of genetic information from a donor to a recipient cell by a conjugative or mobile genetic element, often a conjugative plasmid.
(Also abbreviated to 'phage'.) A virus that infects bacteria. Virulent phages kill the host (lytic infection cycle), whereas temperate phages can integrate into the host chromosome (lysogenic cycle), becoming a prophage.
The transfer of genetic information from one bacterial or archaeal cell to another by a phage particle containing chromosomal DNA.
- DNA restriction
The destruction of foreign dsDNA by a restriction endonuclease. The protection of self DNA from restriction is achieved by DNA methylation.
- Surface exclusion
A process that bars conjugative transfer of a plasmid into recipient cells that already harbour a related plasmid.
- RNA interference
A set of related pathways in eukaryotic cells that use small (20–30 nucleotides) RNAs to regulate the expression or function of cognate sequences.
Phage or plasmid sequences that match one or more clustered, regularly interspaced short palindromic repeat (CRISPR) spacer sequences and are targeted during CRISPR interference.
- Dyad symmetry
A twofold rotational symmetry relationship (in this case, a DNA arrangement in which a 5′→3′ sequence on one strand is juxtaposed with the same 5′→3′ sequence on the opposite strand). Transcripts from such regions have the capacity to form stem–loop structures.
Referring to members of the Crenarchaeota phylum, which is composed mainly of thermophilic archaeal organisms.
- Histidine-aspartate nuclease
A divalent-metal-dependent phosphohydrolase with a conserved histidine-aspartate motif.
Enzyme that uses the free energy of NTP binding and hydrolysis to drive the separation of complementary RNA or DNA strands.
- Virulence factor
A gene responsible for the production of a molecule that contributes to the establishment of disease by bacterial pathogens.
A lysogen is a bacterium that has a prophage integrated into its chromosome. A polylysogen contains many prophage sequences in its genome.
- Virulence plasmid
A plasmid that carries virulence factor genes or pathogenicity islands.
- Pathogenicity island
Genomic islands that contain genes that are required for virulence. These islands are usually absent from non-pathogenic organisms and are acquired by horizontal gene transfer.
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Marraffini, L., Sontheimer, E. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet 11, 181–190 (2010). https://doi.org/10.1038/nrg2749
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