Cas6 specificity and CRISPR RNA loading in a complex CRISPR-Cas system

Richard David Sokolowski, Shirley Graham, Malcolm F White

Research output: Contribution to journalArticlepeer-review

37 Citations (Scopus)
3 Downloads (Pure)

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.
Original languageEnglish
Pages (from-to)6532-6541
Number of pages10
JournalNucleic Acids Research
Volume42
Issue number10
Early online date20 Apr 2014
DOIs
Publication statusPublished - 2 Jun 2014

Keywords

  • CRISPR-Cas
  • Prokaryotic immune system
  • CRISPR RNA (crRNA)
  • Cas6
  • Sulfolobus solfataricus

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