title: Cas6 specificity and CRISPR RNA loading in a complex CRISPR-Cas system.
abstract: CRISPR-Cas is an adaptive prokaryotic immune system, providing protection against viruses and other mobile genetic elements. In type I and type III CRISPR-Cas systems, CRISPR RNA (crRNA) is generated by cleavage of a primary transcript by the Cas6 endonuclease and loaded into multisubunit surveillance/effector complexes, allowing homology-directed detection and cleavage of invading elements. Highly studied CRISPR-Cas systems such as those in Escherichia coli and Pseudomonas aeruginosa have a single Cas6 enzyme that is an integral subunit of the surveillance complex. By contrast, Sulfolobus solfataricus has a complex CRISPR-Cas system with three types of surveillance complexes (Cascade/type I-A, CSM/type III-A and CMR/type III-B), five Cas6 paralogues and two different CRISPR-repeat families (AB and CD). Here, we investigate the kinetic properties of two different Cas6 paralogues from S. solfataricus. The Cas6-1 subtype is specific for CD-family CRISPR repeats, generating crRNA by multiple turnover catalysis whilst Cas6-3 has a broader specificity and also processes a non-coding RNA with a CRISPR repeat-related sequence. Deep sequencing of crRNA in surveillance complexes reveals a biased distribution of spacers derived from AB and CD loci, suggesting functional coupling between Cas6 paralogues and their downstream effector complexes.
authors.affiliation: Biomedical Sciences Research Complex, School of Biology, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Sokolowski
authors.affiliation: Biomedical Sciences Research Complex, School of Biology, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK.
authors.forename: Shirley
authors.initials: S
authors.lastname: Graham
authors.affiliation: Biomedical Sciences Research Complex, School of Biology, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK mfw2@st-andrews.ac.uk.
authors.forename: Malcolm F
authors.initials: MF
authors.lastname: White
mesh_terms: D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004722:Endoribonucleases / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D022661:RNA, Untranslated / Q000378:metabolism; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics
chemical_list: D064130:CRISPR-Associated Proteins; D022661:RNA, Untranslated; D012313:RNA; D004722:Endoribonucleases
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