title: Essential requirements for the detection and degradation of invaders by the Haloferax volcanii CRISPR/Cas system I-B.
abstract: To fend off foreign genetic elements, prokaryotes have developed several defense systems. The most recently discovered defense system, CRISPR/Cas, is sequence-specific, adaptive and heritable. The two central components of this system are the Cas proteins and the CRISPR RNA. The latter consists of repeat sequences that are interspersed with spacer sequences. The CRISPR locus is transcribed into a precursor RNA that is subsequently processed into short crRNAs. CRISPR/Cas systems have been identified in bacteria and archaea, and data show that many variations of this system exist. We analyzed the requirements for a successful defense reaction in the halophilic archaeon Haloferax volcanii. Haloferax encodes a CRISPR/Cas system of the I-B subtype, about which very little is known. Analysis of the mature crRNAs revealed that they contain a spacer as their central element, which is preceded by an eight-nucleotide-long 5' handle that originates from the upstream repeat. The repeat sequences have the potential to fold into a minimal stem loop. Sequencing of the crRNA population indicated that not all of the spacers that are encoded by the three CRISPR loci are present in the same abundance. By challenging Haloferax with an invader plasmid, we demonstrated that the interaction of the crRNA with the invader DNA requires a 10-nucleotide-long seed sequence. In addition, we found that not all of the crRNAs from the three CRISPR loci are effective at triggering the degradation of invader plasmids. The interference does not seem to be influenced by the copy number of the invader plasmid.
authors.affiliation: Biology II, University of Ulm, Ulm, Germany.
authors.forename: Lisa-Katharina
authors.initials: LK
authors.lastname: Maier
authors.forename: Sita J
authors.initials: SJ
authors.lastname: Lange
authors.forename: Britta
authors.initials: B
authors.lastname: Stoll
authors.forename: Karina A
authors.initials: KA
authors.lastname: Haas
authors.forename: Susan
authors.initials: S
authors.lastname: Fischer
authors.forename: Eike
authors.initials: E
authors.lastname: Fischer
authors.forename: Elke
authors.initials: E
authors.lastname: Duchardt-Ferner
authors.forename: Jens
authors.initials: J
authors.lastname: Wöhnert
authors.forename: Rolf
authors.initials: R
authors.lastname: Backofen
authors.forename: Anita
authors.initials: A
authors.lastname: Marchfelder
mesh_terms: D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics* / Q000276:immunology; D004247:DNA / Q000235:genetics; D019622:Haloferax volcanii / Q000235:genetics* / Q000276:immunology; D055029:Inverted Repeat Sequences; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010802:Phylogeny; D010957:Plasmids; D012323:RNA Processing, Post-Transcriptional; D019642:RNA, Archaeal / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D016415:Sequence Alignment; D017423:Sequence Analysis, RNA
chemical_list: D064130:CRISPR-Associated Proteins; D019642:RNA, Archaeal; D004247:DNA
keywords: CRISPR/Cas; Haloferax volcanii; PAM; archaea; crRNA; seed sequence