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High-resolution structure of cas13b and biochemical characterization of RNA targeting and cleavage

DOI: 10.1016/j.celrep.2019.02.094 DOI Help

Authors: Ian M. Slaymaker (Broad Institute of MIT and Harvard; Massachusetts Institute of Technology;) , Pablo Mesa (University of Copenhagen) , Max J. Kellner (Broad Institute of MIT and Harvard) , Soumya Kannan (Broad Institute of MIT and Harvard; Massachusetts Institute of Technology) , Edward Brignole (Massachusetts Institute of Technology) , Jeremy Koob (Broad Institute of MIT and Harvard) , Patricia R. Feliciano (Massachusetts Institute of Technology) , Stefano Stella (University of Copenhagen) , Omar O. Abudayyeh (Broad Institute of MIT and Harvard; Massachusetts Institute of Technology) , Jonathan S. Gootenberg (Broad Institute of MIT and Harvard; Massachusetts Institute of Technology) , Jonathan Strecker (Broad Institute of MIT and Harvard; Massachusetts Institute of Technology) , Guillermo Montoya (University of Copenhagen) , Feng Zhang (Broad Institute of MIT and Harvard; Massachusetts Institute of Technology)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Cell Reports , VOL 26 , PAGES 3741 - 3751.e5

State: Published (Approved)
Published: March 2019

Open Access Open Access

Abstract: Type VI CRISPR-Cas systems contain programmable single-effector RNA-guided RNases, including Cas13b, one of the four known family members. Cas13b, which has been used for both RNA editing and nucleic acid detection, is unique among type VI CRISPR effectors in its linear domain architecture and CRISPR RNA (crRNA) structure. Here, we report the crystal structure of Prevotella buccae Cas13b (PbuCas13b) bound to crRNA at 1.65 Å resolution. This structure, combined with biochemical experiments assaying the stability, kinetics, and function of Cas13b, provides a mechanistic model for Cas13b target RNA recognition and identifies features responsible for target and cleavage specificity. Based on these observations, we generated Cas13b variants with altered cleavage preferences, which may expand the utility of nuclease-based RNA detection assays and other applications of Cas13b in mammalian cells.

Journal Keywords: CRISPR-Cas9 system; Cas13b; type VI CRISPR; crystal structure; RNA-targeting

Subject Areas: Biology and Bio-materials


Instruments: I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Documents:
mgjkgk44545.pdf