title: In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus.
abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)-encoded immunity in Type I systems relies on the Cascade (CRISPR-associated complex for antiviral defence) ribonucleoprotein complex, which triggers foreign DNA degradation by an accessory Cas3 protein. To establish the mechanism for adaptive immunity provided by the Streptococcus thermophilus CRISPR4-Cas (CRISPR-associated) system (St-CRISPR4-Cas), we isolated an effector complex (St-Cascade) containing 61-nucleotide CRISPR RNA (crRNA). We show that St-Cascade, guided by crRNA, binds in vitro to a matching proto-spacer if a proto-spacer adjacent motif (PAM) is present. Surprisingly, the PAM sequence determined from binding analysis is promiscuous and limited to a single nucleotide (A or T) immediately upstream (-1 position) of the proto-spacer. In the presence of a correct PAM, St-Cascade binding to the target DNA generates an R-loop that serves as a landing site for the Cas3 ATPase/nuclease. We show that Cas3 binding to the displaced strand in the R-loop triggers DNA cleavage, and if ATP is present, Cas3 further degrades DNA in a unidirectional manner. These findings establish a molecular basis for CRISPR immunity in St-CRISPR4-Cas and other Type I systems.
authors: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius 02241, Lithuania.
authors: Tomas
authors: T
authors: Sinkunas
authors: Giedrius
authors: G
authors: Gasiunas
authors: Sakharam P
authors: SP
authors: Waghmare
authors: Mark J
authors: MJ
authors: Dickman
authors: Rodolphe
authors: R
authors: Barrangou
authors: Philippe
authors: P
authors: Horvath
authors: Virginijus
authors: V
authors: Siksnys
mesh_terms: D056704:Adaptive Immunity / Q000276:immunology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D053837:DNA Cleavage; D004265:DNA Helicases / Q000378:metabolism*; D058645:Denaturing Gradient Gel Electrophoresis; D024202:Electrophoretic Mobility Shift Assay; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics / Q000276:immunology*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics / Q000276:immunology*; D012261:Ribonucleoproteins / Q000276:immunology* / Q000378:metabolism; D012394:Rosaniline Dyes; D017422:Sequence Analysis, DNA; D048249:Streptococcus thermophilus / Q000201:enzymology / Q000276:immunology* / Q000821:virology
chemical_list: D012329:RNA, Bacterial; D012261:Ribonucleoproteins; D012394:Rosaniline Dyes; C048139:Coomassie blue; D000255:Adenosine Triphosphate; D004265:DNA Helicases