Abstract
The prokaryotic antiviral defense systems CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) employs short crRNAs (CRISPR RNAs) to target invading viral nucleic acids. A short spacer sequence of these crRNAs can be derived from a viral genome and recognizes a reoccurring attack of a virus via base complementarity. We analyzed the effect of spacer sequences on the maturation of crRNAs of the subtype I-B Methanococcus maripaludis C5 CRISPR cluster. The responsible endonuclease, termed Cas6b, bound non-hydrolyzable repeat RNA as a dimer and mature crRNA as a monomer. Comparative analysis of Cas6b processing of individual spacer-repeat-spacer RNA substrates and crRNA stability revealed the potential influence of spacer sequence and length on these parameters. Correlation of these observations with the variable abundance of crRNAs visualized by deep-sequencing analyses is discussed. Finally, insertion of spacer and repeat sequences with archaeal poly-T termination signals is suggested to be prevented in archaeal CRISPR/Cas systems.
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