Structural insights into RNA-guided RNA editing by the Cas13b–ADAR2 complex

Abstract

Cas13 is an RNA-guided RNA endonuclease derived from the type VI CRISPR–Cas system, which has been used in numerous RNA-targeting technologies, such as RNA knockdown, detection and editing. The catalytically inactive Prevotella sp. Cas13b (dPspCas13b) fused to the human adenosine deaminase acting on RNA 2 (ADAR2) deaminase domain can edit adenosine in target transcripts to inosine, in an RNA-editing technology called REPAIR (RNA editing for programmable A-to-I replacement), which has potential for gene therapy. Here we report the cryo-electron microscopy structures of the PspCas13b–guide RNA binary complex, the PspCas13b–guide RNA–target RNA ternary complex and the dPspCas13b–ADAR2–guide RNA–target RNA complex. These structures provide mechanistic insights into RNA cleavage and editing. We applied our structural insights to engineer a compact and efficient dPspCas13b–ADAR2 complex (REPAIR-mini). Overall, our findings advance the understanding of CRISPR–Cas13 effector nucleases and could enable the development of improved RNA-targeting technologies.