| description |
The CRISPR-Cas system represents an RNA-based adaptive immune response system in prokaryotes. CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) consist of arrays of short repeat sequences interspaced by non-repetitive 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 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. 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 eight nucleotides upstream of the spacer start 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.Overall design: 6 cDNA libraries, 3 untreated, 3 treated with phosphatase and T4 PNK in size ranges of 19-30 nt, 35-50 nt, and 50-100 nt. |