title: Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28.
abstract: 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.
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Smargon
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, Boston, MA 02115, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: David B T
authors.initials: DBT
authors.lastname: Cox
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Neena K
authors.initials: NK
authors.lastname: Pyzocha
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Kaijie
authors.initials: K
authors.lastname: Zheng
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Slaymaker
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Gootenberg
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Omar A
authors.initials: OA
authors.lastname: Abudayyeh
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Essletzbichler
authors.affiliation: Skolkovo Institute of Science and Technology, Skolkovo 143025, Russia; National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
authors.forename: Sergey
authors.initials: S
authors.lastname: Shmakov
authors.affiliation: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
authors.forename: Kira S
authors.initials: KS
authors.lastname: Makarova
authors.affiliation: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
authors.forename: Eugene V
authors.initials: EV
authors.lastname: Koonin
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: zhang@broadinstitute.org.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D019295:Computational Biology; D057225:Data Mining; D030541:Databases, Genetic; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D000072669:Gene Editing / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D034622:RNA Interference; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism*; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D012329:RNA, Bacterial; D000094704:RNA, Guide, CRISPR-Cas Systems; D012260:Ribonucleases