At the molecular level, CRISPR function can be divided into three phases: the incorporation of new spacers into CRISPR arrays, the expression and processing of CRISPR RNAs (crRNAs), and CRISPR interference33
. In the first phase, CRISPR loci incorporate additional spacers to programme their activity against invading plasmids and phages. This allows the cell to adapt rapidly to the invaders present in the environment and therefore we refer to it as the ‘adaptation’ phase of CRISPR function (). The information stored in spacers is then used to repel invaders during the ‘defence’ phase of CRISPR interference (described below).
Acquisition of new repeat-spacer units
Adaptation to plasmids and predatory phages by spacer acquisition has been shown to occur readily in several species. In the course of studies of phage therapy for the prevention of tooth decay, M102 phages were introduced into rats to eliminate Streptococcus mutans
, the principal aetiological agent of dental cavities. Bacteriophage-insensitive mutants (BIMs) were isolated that had added an M102-matching spacer sequence to one of the two CRISPR arrays in this species34
. Similar adaptation can be induced in laboratory cultures by phage challenge of S. thermophilus22,35,36
. These studies have determined that all new spacers are inserted at the leader end of the CRISPR array and that most integrations occur at the first position in the cluster. The loss of one or more repeat-spacer sequences has also been observed, which suggests that CRISPRs do not grow unchecked36,37
. The addition of a single repeat-spacer unit is most common, but up to four new units have been detected35
. Only two of the three CRISPR loci present in S. thermophilus
have been shown to acquire new spacers36
Active acquisition of new spacer sequences can also be detected by analysis of natural microbial populations. Metagenomic data (involving random sequencing of genomes from a whole community of microbes and phages) obtained from two sites within Richmond Mine (California, USA) over a period of months allowed the sequencing of distinct Leptospirillum
. The populations were essentially identical except in the spacer content of the single CRISPR locus found. Spacer diversity was highly polarized: the distal (relative to the leader) half of the cluster was more conserved among both populations and the proximal half was much more divergent. The appearance of unique (new) spacers was accompanied by the loss of more conserved ones, again indicating that CRISPR growth is limited. These observations suggest a common ancestor for populations that diverged in their CRISPR content as they acquired new spacers to adapt to the distinct predatory phage populations in their new environments.
The molecular mechanism of spacer incorporation is unknown. Cas1
are dispensable for the function of pre-existing spacers in E. coli38
, despite the apparent universality of these proteins in CRISPR–Cas systems, and therefore they are thought to participate in adaptation. Pseudomonas aeruginosa
Cas1 is a sequence-nonspecific DNase that generates ~80-nt DNA fragments (see above) that have been suggested to reflect the initial sources of new 32-nt spacers39
. How Cas1 might distinguish chromosomal from invasive DNA and how its presumed nucleolytic products integrate into CRISPR loci remain mysterious. Finally, a cas2
gene that is associated with a CRISPR locus of S. thermophilus
seems to be important for the acquisition of spacers in this bacterium, as its disruption prevents the generation of BIMs with novel spacers22
The alignment of protospacer flanking sequences in phage genomes reveals the presence of short (2–3 nt) conserved regions that have been named ‘CRISPR motifs’ or ‘protospacer-adjacent motifs’ on either side of the protospacer sequence17,29,34,35,40,41
. The presence of these motifs indicates that protospacers are not randomly selected and suggests that this conserved sequence may provide a recognition signal for the selection of target sequences that will become new spacers. In addition, phages can evade CRISPR immunity by mutating residues of the CRISPR motif35,41
, which suggests a role for flanking sequences during the defence phase as well. Adaptation is one of the most intriguing and under-explored aspects of CRISPR biology.