title: DNA targeting by the type I-G and type I-A CRISPR-Cas systems of Pyrococcus furiosus.
abstract: CRISPR-Cas systems silence plasmids and viruses in prokaryotes. CRISPR-Cas effector complexes contain CRISPR RNAs (crRNAs) that include sequences captured from invaders and direct CRISPR-associated (Cas) proteins to destroy corresponding invader nucleic acids. Pyrococcus furiosus (Pfu) harbors three CRISPR-Cas immune systems: a Cst (Type I-G) system with an associated Cmr (Type III-B) module at one locus, and a partial Csa (Type I-A) module (lacking known invader sequence acquisition and crRNA processing genes) at another locus. The Pfu Cmr complex cleaves complementary target RNAs, and Csa systems have been shown to target DNA, while the mechanism by which Cst complexes silence invaders is unknown. In this study, we investigated the function of the Cst as well as Csa system in Pfu strains harboring a single CRISPR-Cas system. Plasmid transformation assays revealed that the Cst and Csa systems both function by DNA silencing and utilize similar flanking sequence information (PAMs) to identify invader DNA. Silencing by each system specifically requires its associated Cas3 nuclease. crRNAs from the 7 shared CRISPR loci in Pfu are processed for use by all 3 effector complexes, and Northern analysis revealed that individual effector complexes dictate the profile of mature crRNA species that is generated.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: Elmore
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
authors.forename: Trace
authors.initials: T
authors.lastname: Deighan
authors.affiliation: Department of Genetics, University of Georgia, Athens, GA 30602, USA.
authors.forename: Jan
authors.initials: J
authors.lastname: Westpheling
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA mterns@uga.edu.
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Terns
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA Department of Genetics, University of Georgia, Athens, GA 30602, USA Department of Microbiology, University of Georgia, Athens, GA 30602, USA mterns@uga.edu.
authors.forename: Michael P
authors.identifier: http://orcid.org/0000-0001-8330-1789
authors.initials: MP
authors.lastname: Terns
mesh_terms: D064130:CRISPR-Associated Proteins / Q000378:metabolism; D064113:CRISPR-Cas Systems; D004247:DNA / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D020027:Pyrococcus furiosus / Q000235:genetics*; D019642:RNA, Archaeal / Q000378:metabolism
chemical_list: D064130:CRISPR-Associated Proteins; D019642:RNA, Archaeal; D004247:DNA; D003851:Deoxyribonucleases