PT  - JOURNAL ARTICLE
AU  - Aaron Smargon
AU  - David B.T. Cox
AU  - Neena Pyzocha
AU  - Kaijie Zheng
AU  - Ian M. Slaymaker
AU  - Jonathan S. Gootenberg
AU  - Omar A. Abudayyeh
AU  - Patrick Essletzbichler
AU  - Sergey Shmakov
AU  - Kira S. Makarova
AU  - Eugene V. Koonin
AU  - Feng Zhang
TI  - Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28
AID  - 10.1101/092577
DP  - 2016 Jan 01
TA  - bioRxiv
PG  - 092577
4099  - http://biorxiv.org/content/early/2016/12/09/092577.short
4100  - http://biorxiv.org/content/early/2016/12/09/092577.full
AB  - 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 or 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.