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Molecular cell v.65 no.4, 2017년, pp.618 - 630.e7   SCI SCIE
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Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28

Smargon, Aaron A. (Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA ) ; Cox, David B.T. (Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA ) ; Pyzocha, Neena K. (Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA ) ; Zheng, Kaijie (Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA ) ; Slaymaker, Ian M. (Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA ) ; Gootenberg, Jonathan S. (Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA ) ; Abudayyeh, Omar A. (Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA ) ; Essletzbichler, Patrick (Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA ) ; Shmakov, Sergey (Skolkovo Institute of Science and Technology, Skolkovo 143025, Russia ) ; Makarova, Kira S. (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA ) ; Koonin, Eugene V. (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA ) ; Zhang, Feng (Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA ) ;
  • 초록  

    Summary CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 and Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts. Highlights CRISPR-Cas13b is a class 2 type VI-B CRISPR system lacking Cas1 and Cas2 Cas13b is a CRISPR-associated RNA-guided RNase with two crRNA variants Csx27 represses, whereas Csx28 enhances, Cas13b activity Cas13b RNA targeting is dependent on a double-sided PFS and RNA accessibility Graphical Abstract [DISPLAY OMISSION]


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