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- PDB-8wfb: Cryo-EM structure of the dPspCas13b-ADAR2-crRNA-target RNA complex -

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Basic information

Entry
Database: PDB / ID: 8wfb
TitleCryo-EM structure of the dPspCas13b-ADAR2-crRNA-target RNA complex
Components
  • crRNA
  • dPspCas13b-ADAR2DD
  • target RNA
KeywordsRNA BINDING PROTEIN/RNA / CRISPR / RNase / RNA BINDING PROTEIN-RNA COMPLEX
Function / homologyINOSITOL HEXAKISPHOSPHATE / RNA / RNA (> 10)
Function and homology information
Biological speciesPrevotella sp. (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.71 Å
AuthorsIshikawa, J. / Kato, K. / Yamashita, K. / Nishizawa, T. / Nishimasu, H.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)23KJ0720 Japan
Japan Agency for Medical Research and Development (AMED)22am0401005h0004 Japan
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Structural insights into RNA-guided RNA editing by the Cas13b-ADAR2 complex.
Authors: Junichiro Ishikawa / Kazuki Kato / Soumya Kannan / Sae Okazaki / Soh Ishiguro / Keitaro Yamashita / Nozomu Yachie / Tomohiro Nishizawa / Feng Zhang / Hiroshi Nishimasu /
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 ...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.
History
DepositionSep 19, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 19, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: dPspCas13b-ADAR2DD
B: crRNA
C: target RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)209,6635
Polymers208,9383
Non-polymers7252
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein dPspCas13b-ADAR2DD


Mass: 176101.406 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Prevotella sp. (bacteria) / Production host: Spodoptera frugiperda (fall armyworm)
#2: RNA chain crRNA


Mass: 21092.453 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#3: RNA chain target RNA


Mass: 11744.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#4: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: The dPspCas13b-ADAR2-crRNA-target RNA complex / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Prevotella sp. (bacteria)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 47.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 70123 / Symmetry type: POINT

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