Here, our study provides important structure-based mechanistic insights into PAM-dependent spacer acquisition.
These observations indicate that both spacer acquisition and deletion are active in S. pyogenes CRISPR.
In S. thermophilus, csn2 (cas7) gene was shown to be involved in the spacer acquisition [11].
Despite the fact that S. pyogenes CRISPR appear to be active in spacer acquisition, the number of spacers is small.
In the spacer acquisition stage, it is known that Cas1 functions to integrate novel spacers into the CRISPR loci [45].
There is also a report that S. mutans UA159 has a truncating mutation in its cas1 gene and its CRISPR contains only one spacer [42], supporting the importance of Cas1 in spacer acquisition.
In the subset of fully sequenced isolates, we were able to search for leader end spacers from other isolates located internally in the fully sequenced loci and did not find any matches, indicating that the leader end sequences are truly unique and result from independent spacer acquisition (data not shown).
A new repeat-spacer unit is added to the CRISPR between the leader and the previous unit, which suggests this particular sequence is likely to include a binding site for the proteins (probably Cas proteins) responsible for repeat duplication and/or spacer acquisition.
This is conceptually similar to the theoretical model described by Rodriguez-Valera et al. in which diversity is maintained due to the rapid evolution of virus receptors in genomic islands [6], however it provides a mechanism for the rapid generation of variation that directly results from virus infection (CRISPR spacer acquisition) and is therefore dependent on viral density.
The G might be added during the spacer acquisition process, rather than by replacement mutation after the spacers' insertion into the CRISPR locus, in which case we would expect to observe the two variant spacers in the population, as seen in sets 11, 12, and 13 (Table 1).
