title: Target DNA recognition and cleavage by a reconstituted Type I-G CRISPR-Cas immune effector complex.
abstract: CRISPR-Cas immune systems defend prokaryotes against viruses and plasmids. CRISPR RNAs (crRNAs) associate with various CRISPR-associated (Cas) protein modules to form structurally and functionally diverse (Type I-VI) crRNP immune effector complexes. Previously, we identified three, co-existing effector complexes in Pyrococcus furiosus -Type I-A (Csa), Type I-G (Cst), and Type III-B (Cmr)-and demonstrated that each complex functions in vivo to eliminate invader DNA. Here, we reconstitute functional Cst crRNP complexes in vitro from recombinant Cas proteins and synthetic crRNAs and investigate mechanisms of crRNP assembly and invader DNA recognition and destruction. All four known Cst-affiliated Cas proteins (Cas5t, Cst1, Cst2, and Cas3) are required for activity, but each subunit plays a distinct role. Cas5t and Cst2 comprise a minimal set of proteins that selectively interact with crRNA. Further addition of Cst1, enables the four subunit crRNP (Cas5t, Cst1, Cst2, crRNA) to specifically bind complementary, double-stranded DNA targets and to recruit the Cas3 effector nuclease, which catalyzes cleavages at specific sites within the displaced, non-target DNA strand. Our results indicate that Type I-G crRNPs selectively bind target DNA in a crRNA and, protospacer adjacent motif dependent manner to recruit a dedicated Cas3 nuclease for invader DNA destruction.
authors: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA.
authors: Sonali
authors: S
authors: Majumdar
authors: Department of Microbiology, University of Georgia, Athens, GA, 30602, USA.
authors: Marianne
authors: M
authors: Ligon
authors: Department of Biology, University of Georgia, Athens, GA, 30602, USA.
authors: William Colby
authors: WC
authors: Skinner
authors: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA.
authors: Rebecca M
authors: RM
authors: Terns
authors: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA. mterns@uga.edu.|Department of Genetics, University of Georgia, Athens, GA, 30602, USA. mterns@uga.edu.|Department of Microbiology, University of Georgia, Athens, GA, 30602, USA. mterns@uga.edu.
authors: Michael P
authors: MP
authors: Terns
mesh_terms: D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D011485:Protein Binding; D019711:Pyrococcus / Q000235:genetics / Q000378:metabolism*
chemical_list: D064130:CRISPR-Associated Proteins
keywords: CRISPR; Cas; Cas3; Cst; Pyrococcus furiosus; Type I-G