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

Junichiro Ishikawa; Kazuki Kato; Soumya Kannan; Sae Okazaki; Soh Ishiguro; Keitaro Yamashita; Nozomu Yachie; Tomohiro Nishizawa; Feng Zhang; Hiroshi Nishimasu

doi:10.1038/s41594-025-01529-1

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

Nat Struct Mol Biol. 2025 Aug;32(8):1567-1578. doi: 10.1038/s41594-025-01529-1. Epub 2025 Apr 11.

Authors

Junichiro Ishikawa¹, Kazuki Kato², Soumya Kannan^{3

4

5

6}, Sae Okazaki⁷, Soh Ishiguro⁸, Keitaro Yamashita⁷, Nozomu Yachie^{8

9}, Tomohiro Nishizawa¹⁰, Feng Zhang^{3

4

5

6

11}, Hiroshi Nishimasu^{12

13

14}

Affiliations

¹ Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan.
² Department of Molecular and Mechanistic Immunology, Tokyo Medical and Dental University, Tokyo, Japan.
³ Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁴ McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁵ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁶ Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁷ Structural Biology Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan.
⁸ School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia, Canada.
⁹ Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University, Osaka, Japan.
¹⁰ Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan.
¹¹ Howard Hughes Medical Institute, Cambridge, MA, USA.
¹² Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan. nisimasu@g.ecc.u-tokyo.ac.jp.
¹³ Structural Biology Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan. nisimasu@g.ecc.u-tokyo.ac.jp.
¹⁴ Inamori Research Institute for Science, Kyoto, Japan. nisimasu@g.ecc.u-tokyo.ac.jp.

PMID: 40217120
DOI: 10.1038/s41594-025-01529-1

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.

MeSH terms

Adenosine Deaminase* / chemistry
Adenosine Deaminase* / genetics
Adenosine Deaminase* / metabolism
Bacterial Proteins* / chemistry
Bacterial Proteins* / genetics
Bacterial Proteins* / metabolism
CRISPR-Associated Proteins* / chemistry
CRISPR-Associated Proteins* / metabolism
CRISPR-Cas Systems
Cryoelectron Microscopy
Humans
Models, Molecular
RNA Editing*
RNA, Guide, CRISPR-Cas Systems* / chemistry
RNA, Guide, CRISPR-Cas Systems* / genetics
RNA, Guide, CRISPR-Cas Systems* / metabolism
RNA-Binding Proteins* / chemistry
RNA-Binding Proteins* / genetics
RNA-Binding Proteins* / metabolism

Substances

RNA, Guide, CRISPR-Cas Systems
Adenosine Deaminase
RNA-Binding Proteins
ADARB1 protein, human
Bacterial Proteins
CRISPR-Associated Proteins