A type III-B CRISPR–Cas effector complex mediating massive target DNA destruction
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The CRISPR (clustered regularly interspaced short palindromic repeats) system protects archaea and bacteria by eliminating nucleic acid invaders in a crRNA-guided manner. The Sulfolobus islandicus type III-B effector Cmr-α complex targets invading nucleic acid at both RNA and DNA levels and DNA targeting relies on the directional transcription of the protospacer in vivo. To gain further insight into the involved mechanism, we purified a native effector complex of III-B Cmr-α from S. islandicus and characterized it in vitro. Cmr-α cleaved RNAs complementary to crRNA present in the complex and its ssDNA destruction activity was activated by target RNA. ssDNA cleavage required mismatches between the 5’-tag of crRNA and the 3’-flanking region of target RNA. An invader plasmid assay showed that mutation either in the HD domain (a quadruple mutation) or in the GGDD motif of theCmr-2α protein resulted in attenuation of the DNA interference in vivo. However, double mutation of the HD motif only abolished the DNase activity in vitro. Furthermore, the activated Cmr-α binary complex functioned as a highly active DNase to destroy a large excess of substrate, which could provide a powerful means to rapidly degrade replicating viral DNA.
Han , W , Li , Y , Deng , L , Feng , M , Peng , W , Hallstrøm , S , Zhang , J , Peng , N , Liang , Y X , White , M F & She , Q 2017 , ' A type III-B CRISPR–Cas effector complex mediating massive target DNA destruction ' Nucleic Acids Research , vol. 45 , no. 4 , pp. 1983-1993 . DOI: 10.1093/nar/gkw1274
Nucleic Acids Research
Copyright The Author(s) 2016. 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-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact email@example.com
DescriptionFunding: Biotechnology and Biological Sciences Research Council [BB/M000400/1 to MFW].
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