title: crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus.
abstract: The Cas9-crRNA complex of the Streptococcus thermophilus DGCC7710 CRISPR3-Cas system functions as an RNA-guided endonuclease with crRNA-directed target sequence recognition and protein-mediated DNA cleavage. We show here that an additional RNA molecule, tracrRNA (trans-activating CRISPR RNA), co-purifies with the Cas9 protein isolated from the heterologous E. coli strain carrying the S. thermophilus DGCC7710 CRISPR3-Cas system. We provide experimental evidence that tracrRNA is required for Cas9-mediated DNA interference both in vitro and in vivo. We show that Cas9 specifically promotes duplex formation between the precursor crRNA (pre-crRNA) transcript and tracrRNA, in vitro. Furthermore, the housekeeping RNase III contributes to primary pre-crRNA-tracrRNA duplex cleavage for mature crRNA biogenesis. RNase III, however, is not required in the processing of a short pre-crRNA transcribed from a minimal CRISPR array containing a single spacer. Finally, we show that an in vitro-assembled ternary Cas9-crRNA-tracrRNA complex cleaves DNA. This study further specifies the molecular basis for crRNA-based re-programming of Cas9 to specifically cleave any target DNA sequence for precise genome surgery. The processes for crRNA maturation and effector complex assembly established here will contribute to the further development of the Cas9 re-programmable system for genome editing applications.
authors.affiliation: Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Tautvydas
authors.initials: T
authors.lastname: Karvelis
authors.forename: Giedrius
authors.initials: G
authors.lastname: Gasiunas
authors.forename: Algirdas
authors.initials: A
authors.lastname: Miksys
authors.forename: Rodolphe
authors.initials: R
authors.lastname: Barrangou
authors.forename: Philippe
authors.initials: P
authors.lastname: Horvath
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013297:Streptococcus pyogenes / Q000235:genetics / Q000378:metabolism; D048249:Streptococcus thermophilus / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D012329:RNA, Bacterial; D004247:DNA; D043244:Ribonuclease III
keywords: CRISPR; DNA silencing; Type II CRISPR-Cas systems
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