title: The role of Cas8 in type I CRISPR interference.
abstract: CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use 'cascade' [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5-Cas7-crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA.
authors.affiliation: School of Life Sciences, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, U.K.
authors.forename: Simon D B
authors.initials: SD
authors.lastname: Cass
authors.affiliation: Biology II, Ulm University, 89069 Ulm, Germany.
authors.forename: Karina A
authors.initials: KA
authors.lastname: Haas
authors.affiliation: Biology II, Ulm University, 89069 Ulm, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Stoll
authors.affiliation: Bioinformatics group, Department of Computer Science, University of Freiburg, Georges-Köhler-Allee 106, 79110 Freiburg, Germany.
authors.forename: Omer S
authors.initials: OS
authors.lastname: Alkhnbashi
authors.affiliation: Max Planck Institute of Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen, Germany.
authors.forename: Kundan
authors.initials: K
authors.lastname: Sharma
authors.affiliation: Max Planck Institute of Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen, Germany Bioanalytics Research Group, Institute of Clinical Chemistry, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.affiliation: Bioinformatics group, Department of Computer Science, University of Freiburg, Georges-Köhler-Allee 106, 79110 Freiburg, Germany Centre for Biological Signalling Studies (BIOSS), Cluster of Excellence, University of Freiburg, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Backofen
authors.affiliation: Biology II, Ulm University, 89069 Ulm, Germany ed.bolt@nottingham.ac.uk anita.marchfelder@uni-ulm.de.
authors.forename: Anita
authors.initials: A
authors.lastname: Marchfelder
authors.affiliation: School of Life Sciences, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, U.K. ed.bolt@nottingham.ac.uk anita.marchfelder@uni-ulm.de.
authors.forename: Edward L
authors.initials: EL
authors.lastname: Bolt
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D019641:DNA, Archaeal / Q000737:chemistry / Q000378:metabolism; D019622:Haloferax volcanii / Q000235:genetics*; D017013:Methanobacteriaceae / Q000235:genetics*; D009154:Mutation; D060066:Protein Interaction Maps; D034622:RNA Interference
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal
keywords: CRISPR-associated (Cas)8; CRISPR-associated complex for antiviral defence (Cascade); archaea; clustered regularly interspaced short palindromic repeat (CRISPR); nuclease