title: An active immune defense with a minimal CRISPR (clustered regularly interspaced short palindromic repeats) RNA and without the Cas6 protein.
abstract: The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3' handle are still active in triggering an interference reaction. The complete 3' handle could be removed without loss of activity. However, manipulations of the 5' handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference.
authors.affiliation: From the Department of Biology II, Ulm University, 89069 Ulm.
authors.forename: Lisa-Katharina
authors.initials: LK
authors.lastname: Maier
authors.affiliation: From the Department of Biology II, Ulm University, 89069 Ulm.
authors.forename: Aris-Edda
authors.initials: AE
authors.lastname: Stachler
authors.affiliation: the Bioinformatics Group, Department of Computer Science, Albert-Ludwigs-University Freiburg, Georges-Köhler-Allee 106, 79110 Freiburg, and.
authors.forename: Sita J
authors.initials: SJ
authors.lastname: Saunders
authors.affiliation: the Bioinformatics Group, Department of Computer Science, Albert-Ludwigs-University Freiburg, Georges-Köhler-Allee 106, 79110 Freiburg, and the BIOSS Centre for Biological Signalling Studies, Cluster of Excellence, Albert-Ludwigs-University Freiburg, Schänzlestrasse 18, 79104 Freiburg, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Backofen
authors.affiliation: From the Department of Biology II, Ulm University, 89069 Ulm, anita.marchfelder@uni-ulm.de.
authors.forename: Anita
authors.initials: A
authors.lastname: Marchfelder
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000276:immunology*; D015152:Blotting, Northern; D064113:CRISPR-Cas Systems / Q000235:genetics / Q000276:immunology*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics / Q000276:immunology*; D019848:Gene Expression Regulation, Archaeal; D005818:Genetic Engineering; D019622:Haloferax volcanii / Q000276:immunology*; D059014:High-Throughput Nucleotide Sequencing; D010957:Plasmids / Q000235:genetics*; D034622:RNA Interference; D019642:RNA, Archaeal / Q000235:genetics / Q000276:immunology*
chemical_list: D019843:Archaeal Proteins; D019642:RNA, Archaeal
keywords: Archaea; CRISPR/Cas; Cas6; Haloferax volcanii; Type I-B; crRNA