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- PDB-22fn: Cryo-EM structure of AsCas12a in complex with crDNA and RNA target -

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Basic information

Entry
Database: PDB / ID: 22fn
TitleCryo-EM structure of AsCas12a in complex with crDNA and RNA target
Components
  • CRISPR-associated endonuclease Cas12a
  • DNA (41-MER)
  • RNA (5'-R(P*GP*AP*CP*AP*GP*CP*CP*CP*AP*CP*AP*UP*GP*GP*CP*AP*UP*UP*CP*CP*AP*CP*U)-3')
KeywordsDNA BINDING PROTEIN / Cas12a / crDNA / RNA
Function / homology
Function and homology information


Bacillus subtilis ribonuclease / deoxyribonuclease I / deoxyribonuclease I activity / defense response to virus / lyase activity / DNA binding / RNA binding
Similarity search - Function
: / CRISPR-associated endonuclease Cpf1 REC2 domain / CRISPR-associated endonuclease Cas12a / Cas12a, REC1 domain / Cas12a, RuvC nuclease domain / Cas12a, nuclease domain / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease domain
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas12a
Similarity search - Component
Biological speciesAcidaminococcus sp. BV3L6 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.17 Å
AuthorsLam, W.H. / Wu, X. / Hsing, I.M. / Zhai, Y.
Funding support Hong Kong, 1items
OrganizationGrant numberCountry
The University Grants Committee, Research Grants Council (RGC) Hong Kong
CitationJournal: Nat Biotechnol / Year: 2026
Title: DNA-guided CRISPR-Cas12a effectors for programmable RNA recognition and cleavage.
Authors: Xiaolong Wu / Wai Hei Lam / Zibin Zhao / Yumeng Cao / Haosi Lin / Xianzhen Feng / Yuanliang Zhai / I-Ming Hsing /
Abstract: CRISPR-Cas effectors typically rely on RNA guides to recognize target sequences. In Cas12a, the protospacer adjacent motif on DNA engages conserved protein residues, triggering target binding and ...CRISPR-Cas effectors typically rely on RNA guides to recognize target sequences. In Cas12a, the protospacer adjacent motif on DNA engages conserved protein residues, triggering target binding and nuclease activation. Here we reprogram Cas12a into a DNA-guided, RNA-targeting effector. Exploiting protospacer-adjacent motif-dependent interaction, we engineer synthetic CRISPR DNA that engages Cas12a to form a functional deoxyribonucleoprotein complex, while repurposing solely RNA as the programmable target. Structural, biophysical and biochemical analyses reveal the molecular basis of this DNA-guided, RNA-targeting configuration and support an activation pathway distinct from that of canonical RNA-guided systems. DNA-guided Cas12a enables direct RNA detection and efficient intracellular RNA knockdown, establishing a modular activation architecture for CRISPR-Cas12a and expanding the design space for programmable RNA manipulation.
History
DepositionJan 8, 2026Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 29, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: RNA (5'-R(P*GP*AP*CP*AP*GP*CP*CP*CP*AP*CP*AP*UP*GP*GP*CP*AP*UP*UP*CP*CP*AP*CP*U)-3')
A: CRISPR-associated endonuclease Cas12a
C: DNA (41-MER)


Theoretical massNumber of molelcules
Total (without water)178,9433
Polymers178,9433
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain RNA (5'-R(P*GP*AP*CP*AP*GP*CP*CP*CP*AP*CP*AP*UP*GP*GP*CP*AP*UP*UP*CP*CP*AP*CP*U)-3')


Mass: 14179.431 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Protein CRISPR-associated endonuclease Cas12a / AsCpf1 / CRISPR-associated endonuclease Cpf1


Mass: 151410.938 Da / Num. of mol.: 1 / Mutation: M537R/F870L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acidaminococcus sp. BV3L6 (bacteria) / Gene: cas12a, cpf1, HMPREF1246_0236
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: U2UMQ6, deoxyribonuclease I, Bacillus subtilis ribonuclease
#3: DNA chain DNA (41-MER)


Mass: 13352.602 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
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: Cas12a / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.156 MDa / Experimental value: NO
Source (natural)Organism: Acidaminococcus sp. BV3L6 (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8 / Details: 10mM Tris pH 8.0, 50mM NaCl
Buffer component
IDConc.NameBuffer-ID
110 mMTris1
250 mMsodium chloride1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 47170 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2030

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv4.7.1particle selection
2EPUimage acquisition
4cryoSPARCv4.7.1CTF correction
7Coot0.9.8.96model fitting
9cryoSPARCv4.7.1initial Euler assignment
10cryoSPARCv4.7.1final Euler assignment
11cryoSPARCv4.7.1classification
12cryoSPARCv4.7.13D reconstruction
13PHENIX1.21.2-5419-000model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1305848
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 54033 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 8SFO

8sfo


Accession code: 8SFO / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.17 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311485
ELECTRON MICROSCOPYf_angle_d0.5115815
ELECTRON MICROSCOPYf_dihedral_angle_d17.3922085
ELECTRON MICROSCOPYf_chiral_restr0.0391759
ELECTRON MICROSCOPYf_plane_restr0.0041806

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