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 1.65Å 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.
Highlights
A number of new Cas enzymes and CRISPR systems with novel properties have been discovered recently
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 structure
The direct repeat of the Cas13b CRISPR RNA (crRNA) is at the 30 end, which is the opposite orientation found in other type VI systems (Abudayyeh et al, 2016; Cox et al, 2017; Konermann et al, 2018; Smargon et al, 2017; Yan et al, 2018)
Summary
A number of new Cas enzymes and CRISPR systems with novel properties have been discovered recently Notable among these new discoveries are the class 2 type VI CRISPR-Cas systems, which use a single enzyme to target RNA using a programmable CRISPR RNA (crRNA) guide (Abudayyeh et al, 2016; Konermann et al, 2018; Shmakov et al, 2015, 2017; Smargon et al, 2017; Yan et al, 2018). This structural information together with biochemical characterization of RNA targeting provide a model for target recognition and specificity by Cas13b that is distinct from other known programmable nucleic acid targeting systems
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