期刊
NUCLEIC ACIDS RESEARCH
卷 43, 期 1, 页码 406-417出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gku1302
关键词
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资金
- Danish Council for Independent Research [DFF-0602-02196B, DFF-1323-00330]
- Carlsberg Foundation, theNatural Science Foundation of China [31128011]
- Scientific and Technological Self-Innovation Foundation of Huazhong Agricultural University [2014RC011]
CRISPR-Cas systems provide a small RNA-based mechanism to defend against invasive genetic elements in archaea and bacteria. To investigate the in vivo mechanism of RNA interference by two type III-B systems (Cmr-alpha and Cmr-beta) in Sulfolobus islandicus, a genetic assay was developed using plasmids carrying an artificial mini-CRISPR (AC) locus with a single spacer. After pAC plasmids were introduced into different strains, Northern analyses confirmed that mature crRNAs were produced from the plasmid-borne CRISPR loci, which then guided gene silencing to target gene expression. Spacer mutagenesis identified a trinucleotide sequence in the 3'-region of crRNA that was crucial for RNA interference. Studying mutants lacking Cmr-alpha or Cmr-beta system showed that each Cmr complex exhibited RNA interference. Strikingly, these analyses further revealed that the two Cmr systems displayed distinctive interference features. Whereas Cmr-beta complexes targeted transcripts and could be recycled in RNA cleavage, Cmr-alpha complexes probably targeted nascent RNA transcripts and remained associated with the substrate. Moreover, Cmr-beta exhibited much stronger RNA cleavage activity than Cmr-alpha. Since we previously showed that S. islandicus Cmr-alpha mediated transcription-dependent DNA interference, the Cmr-alpha constitutes the first CRISPR system exhibiting dual targeting of RNA and DNA.
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