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

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NARCas62014.pdf (5.014Mb)
Date
02/06/2014
Author
Sokolowski, Richard David
Graham, Shirley
White, Malcolm F
Funder
BBSRC
BBSRC
BBSRC
Grant ID
BB/K000314/1
BB/M000400/1
BB/J005665/1
Keywords
CRISPR-Cas
Prokaryotic immune system
CRISPR RNA (crRNA)
Cas6
Sulfolobus solfataricus
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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.
Citation
Sokolowski , R D , Graham , S & White , M F 2014 , ' Cas6 specificity and CRISPR RNA loading in a complex CRISPR-Cas system ' , Nucleic Acids Research , vol. 42 , no. 10 , pp. 6532-6541 . https://doi.org/10.1093/nar/gku308
Publication
Nucleic Acids Research
Status
Peer reviewed
DOI
https://doi.org/10.1093/nar/gku308
ISSN
0305-1048
Type
Journal article
Rights
© 2014, The Authors. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited
Description
This research was funded in part by Biotechnology and Biological Sciences Research Council [BB/K000314/1]. The APC was paid through RCUK OA block grant funds.
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  • University of St Andrews Research
URI
http://hdl.handle.net/10023/4582

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