The CRISPR-Cas system represents an RNA-based adaptive immune response system in prokaryotes and archaea. CRISPRs (clustered regularly interspaced short palindromic repeats) consist of arrays of short repeat-sequences interspaced by nonrepetitive short spacers, some of which show sequence similarity to foreign phage genetic elements. Their cistronic transcripts are processed to produce the mature CRISPR RNAs (crRNAs), the elements that confer immunity by base-pairing with exogenous nucleic acids. We characterized the expression and processing patterns of Thermus thermophilus HB8 CRISPRs by using differential deep-sequencing, which differentiates between 5′ monophosphate and 5′ non-monophosphate-containing RNAs and/or between 3′ hydroxyl and 3′ non-hydroxyl-containing RNAs.

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The genome of T. Thermophilus HB8 encodes 11 CRISPRs, classified into three distinct repeat-sequence types, all of which were constitutively expressed without deliberately infecting the bacteria with phage. Analysis of the differential deep sequencing data suggested that crRNAs are generated by endonucleolytic cleavage, leaving fragments with 5′ hydroxyl and 3′ phosphate or 2′,3′-cyclic phosphate termini. The 5′ ends of all crRNAs are generated by site-specific cleavage 8 nucleotides upstream of the spacer first position; however, the 3′ ends are generated by two alternative, repeat-sequence-type–dependent mechanisms. These observations are consistent with the operation of multiple crRNA processing systems within a bacterial strain. INTRODUCTION Bacteria and archaea have developed various defense mechanisms against foreign mobile genetic elements (phages and plasmids), including a new mechanism utilizing regulatory RNAs originating from CRISPRs (clustered regularly interspaced short palindromic repeats) (; ).

Currently more than 500 bacteria are predicted to contain CRISPRs. A CRISPR locus comprises a 5′ leader sequence followed by short repeats, which are separated by spacer sequences (for reviews, see;;;;; ). The average length of the repeats and spacers is 31 and 36 nucleotides (nt), respectively (based on the CRISPRdb database, October 2010 download) (). The number of repeats in CRISPR arrays included in this database range from two to 588, with an average of 49 and median of 13 repeats per CRISPR array. The repeat-sequences are typically invariant within a given CRISPR array.

Paralogous and orthologous relationships of repeats were also observed (). Spacer sequences, on the other hand, are highly variable even within a CRISPR array but tend to exhibit similar lengths.

Clusters of genes encoding CRISPR-associated (Cas) proteins are often located in close proximity to the CRISPR locus, defining CRISPR-Cas modules. The CRISPR-Cas modules vary in their Cas protein composition and order and in the CRISPR repeat-sequence and RNA structure, features that are used to classify and characterize these modules into three major types and 10 major subtypes (;,).

The various Cas proteins were shown to be involved in each of the three major stages of CRISPR-based immunity: adaptation, CRISPR transcript biogenesis, and interference (for review, see ). Interestingly, different CRISPR-Cas modules may differ in the mechanism employed in one or more of these stages (for review, see ). CRISPR loci are transcribed to produce long precursor (pre-) CRISPR RNAs (crRNAs) that are subsequently cleaved at specific positions within the repeats, yielding mature crRNA fragments (for reviews, see;;;;;;;; ) The maturation of the crRNA 5′ end usually occurs by site-specific endonucleolytic cleavage by a Cas protein 8 nt upstream of the spacer within the preceding repeat region.

An exception was demonstrated in Streptococcus pyogenes, where the initial cleavage involves a trans-encoded small guide RNA with complementarity to the repeat, recruiting RNase III rather than a Cas endonuclease for processing (). The mechanism of crRNA 3′ end maturation varies among species and may include retention of the original 3′ end as in Escherichia coli (), or further trimming as in Pyrococcus furiosus (, ) and Pectobacterium atrosepticum (). Several functionally analogous Cas proteins are currently known to be involved in the endonucleolytic cleavage of pre-crRNAs. These include Cse3 of E. Coli () and of Thermus thermophilus HB8 (), Cas6 of P. Furiosus (; ), and Csy4 of Pseudomonas aeruginosa and P. Atrosepticum (; ).