High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage
Ian M. Slaymaker,
Pablo Mesa,
Max J. Kellner,
Soumya Kannan,
Edward Brignole,
Jeremy Koob,
Patricia R. Feliciano,
Stefano Stella,
Omar O. Abudayyeh,
Jonathan S. Gootenberg,
Jonathan Strecker,
Guillermo Montoya,
Feng Zhang
Affiliations
Ian M. Slaymaker
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Corresponding author
Pablo Mesa
Protein Structure & Function Programme, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Structural Molecular Biology Group, University of Copenhagen, Copenhagen, Denmark
Max J. Kellner
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department for Biochemistry and Cell Biology, University of Vienna, Dr Bohr-Gasse 9, 1030 Vienna, Austria
Soumya Kannan
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Edward Brignole
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Jeremy Koob
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
Patricia R. Feliciano
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Stefano Stella
Protein Structure & Function Programme, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Structural Molecular Biology Group, University of Copenhagen, Copenhagen, Denmark
Omar O. Abudayyeh
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Jonathan S. Gootenberg
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Systems Biology, Harvard University, Boston, MA 02115, USA
Jonathan Strecker
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Guillermo Montoya
Protein Structure & Function Programme, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Structural Molecular Biology Group, University of Copenhagen, Copenhagen, Denmark
Feng Zhang
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Corresponding author
Summary: 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. : Slaymaker et al. present a high-resolution structure of Cas13b in combination with biochemical studies showing that Cas13b is a highly dynamic, multi-turnover enzyme distinct from other Cas13 family members. Additionally, the authors demonstrate Cas13b can be rationally engineered to change RNA cleavage specificity and create a minimal variant suitable for delivery with viral vectors. Keywords: CRISPR-Cas9 system, Cas13b, type VI CRISPR, crystal structure, RNA-targeting
