title: An archaeal CRISPR type III-B system exhibiting distinctive RNA targeting features and mediating dual RNA and DNA interference.
abstract: 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-α and Cmr-β) 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-α or Cmr-β 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-β complexes targeted transcripts and could be recycled in RNA cleavage, Cmr-α complexes probably targeted nascent RNA transcripts and remained associated with the substrate. Moreover, Cmr-β exhibited much stronger RNA cleavage activity than Cmr-α. Since we previously showed that S. islandicus Cmr-α mediated transcription-dependent DNA interference, the Cmr-α constitutes the first CRISPR system exhibiting dual targeting of RNA and DNA.
authors.affiliation: State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University, 430070 Wuhan, China Archaea Centre, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
authors.forename: Wenfang
authors.initials: W
authors.lastname: Peng
authors.affiliation: State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University, 430070 Wuhan, China.
authors.forename: Mingxia
authors.initials: M
authors.lastname: Feng
authors.affiliation: State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University, 430070 Wuhan, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Feng
authors.affiliation: State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University, 430070 Wuhan, China.
authors.forename: Yun Xiang
authors.initials: YX
authors.lastname: Liang
authors.affiliation: State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University, 430070 Wuhan, China Archaea Centre, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark qunxin@bio.ku.dk.
authors.forename: Qunxin
authors.initials: Q
authors.lastname: She
mesh_terms: D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D019641:DNA, Archaeal / Q000737:chemistry; D059372:Nucleotide Motifs; D010957:Plasmids / Q000235:genetics; D059367:RNA Cleavage; D034622:RNA Interference; D019642:RNA, Archaeal / Q000737:chemistry / Q000378:metabolism*; D016944:Sulfolobus / Q000235:genetics
chemical_list: D019641:DNA, Archaeal; D019642:RNA, Archaeal