Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28

Aaron A Smargon; David B T Cox; Neena K Pyzocha; Kaijie Zheng; Ian M Slaymaker; Jonathan S Gootenberg; Omar A Abudayyeh; Patrick Essletzbichler; Sergey Shmakov; Kira S Makarova; Eugene V Koonin; Feng Zhang

doi:10.1016/j.molcel.2016.12.023

Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28

Mol Cell. 2017 Feb 16;65(4):618-630.e7. doi: 10.1016/j.molcel.2016.12.023. Epub 2017 Jan 5.

Authors

Affiliations

¹ 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.
² 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.
³ 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.
⁴ 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.
⁵ 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.
⁶ 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.
⁷ 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.
⁸ National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
⁹ 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.

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.

MeSH terms

Bacterial Proteins / genetics
Bacterial Proteins / metabolism*
CRISPR-Associated Proteins / genetics
CRISPR-Associated Proteins / metabolism*
CRISPR-Cas Systems*
Computational Biology
Data Mining
Databases, Genetic
Escherichia coli / enzymology*
Escherichia coli / genetics
Gene Editing / methods*
Gene Expression Regulation, Bacterial
RNA Interference*
RNA, Bacterial / genetics
RNA, Bacterial / metabolism*
RNA, Guide, CRISPR-Cas Systems / genetics
RNA, Guide, CRISPR-Cas Systems / metabolism*
Ribonucleases / genetics
Ribonucleases / metabolism*

Substances

Bacterial Proteins
CRISPR-Associated Proteins
RNA, Bacterial
RNA, Guide, CRISPR-Cas Systems
Ribonucleases

Abstract

MeSH terms

Substances

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