High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage
Author(s)Slaymaker, Ian; Mesa, Pablo; Kellner, Max J.; Kannan, Soumya; Brignole, Edward J; Koob, Jeremy; Feliciano, Patricia Rosa; Stella, Stefano; Abudayyeh, Omar O.; Gootenberg, Jonathan S; Strecker, Jonathan; Montoya, Guillermo; Zhang, Feng; ... Show more Show less
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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.
DepartmentMcGovern Institute for Brain Research at MIT; Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences; Massachusetts Institute of Technology. Department of Biological Engineering; Massachusetts Institute of Technology. Department of Biology; Harvard University--MIT Division of Health Sciences and Technology; Broad Institute of MIT and Harvard
Slaymaker, Ian M. et al. "High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage." Cell Reports 26, 16 (March 2019): P3741-3751.e5 © 2019 The Author(s)
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