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- PDB-9waa: SuperFi Cas9 - 22nt sgRNA - DNA ternary complex Class C -

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Basic information

Entry
Database: PDB / ID: 9waa
TitleSuperFi Cas9 - 22nt sgRNA - DNA ternary complex Class C
Components
  • (DNA (50-MER)) x 2
  • CRISPR-associated endonuclease Cas9/Csn1
  • RNA (102-MER)
KeywordsIMMUNE SYSTEM / Complex / cas / SuperFi cas9
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes serotype M1 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.17 Å
AuthorsZheng, R. / Ma, L.J.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Struct Mol Biol / Year: 2026
Title: Improving the efficiency of high-fidelity Cas9 by enhancing PAM-distal interactions.
Authors: Rong Zheng / Zhike Lu / Rongwei Wei / Young-Cheul Shin / Jiang Du / Qingfeng Zhang / Jianbo Li / Xiaoqi Wang / Yi Wei / Botao Liu / Yang Chen / Lihong Ding / Heng Zhang / Hui Chen / Jing Huang / Lijia Ma /
Abstract: Engineering CRISPR enzymes for high fidelity often impairs cleavage activity. Meanwhile, a mechanistic understanding of why high-fidelity mutations reduce Cas9's cleavage activity remains unclear, ...Engineering CRISPR enzymes for high fidelity often impairs cleavage activity. Meanwhile, a mechanistic understanding of why high-fidelity mutations reduce Cas9's cleavage activity remains unclear, presenting a challenge in balancing nuclease specificity and efficiency for clinical applications. In this study, we show that extending the spacer region to 21 or 22 nucleotides restores the impaired cleavage activity of SuperFi-Cas9, a high-fidelity Cas9 variant with 7 mutations in the RuvC domain at the protospacer adjacent motif (PAM)-distal region. Cryo-electron microscopy structures and mutational analyses reveal that the negatively charged mutations in a protruding loop of the RuvC domain create repulsive forces that destabilize the nuclease-single guide (sg)RNA-DNA complex. Spacer extension enhances interactions in the PAM-distal region, effectively restoring cleavage activity and balancing editing efficiency with specificity. In addition, we develop a deep learning model, AIdit-SuperFi, to predict optimal sgRNA length for high-fidelity genome editing. Our findings introduce a straightforward strategy to enhance CRISPR complex stability and provide mechanistic insights into the impaired cleavage activity of engineered high-fidelity Cas9, presenting a pathway toward precise and efficient genome editing and clinical translation of CRISPR technologies.
History
DepositionAug 11, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Dec 31, 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: CRISPR-associated endonuclease Cas9/Csn1
B: DNA (50-MER)
C: DNA (50-MER)
D: RNA (102-MER)


Theoretical massNumber of molelcules
Total (without water)222,1294
Polymers222,1294
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: Protein CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9


Mass: 158502.375 Da / Num. of mol.: 1
Mutation: Y1010D, Y1013D, Y1016D, V1018D, R1019D, Q1027D, K1031D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli (E. coli)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#2: DNA chain DNA (50-MER)


Mass: 15438.878 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (50-MER)


Mass: 15371.812 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: RNA chain RNA (102-MER)


Mass: 32815.465 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: SuperFi Cas9 - 22nt sgRNA - DNA ternary complex Class C
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMNaClNaCl1
220 mMHEPES pH7.4C8H18N2O4S1
310 mMMgCl2MgCl21
40.5 mMC9H15O6PC9H15O6P1
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector model
111.25FEI FALCON IV (4k x 4k)
211.02FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameCategory
1EPUparticle selection
13cryoSPARC3D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 4.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 57341 / Symmetry type: POINT
RefinementHighest resolution: 4.17 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00212904
ELECTRON MICROSCOPYf_angle_d0.4518081
ELECTRON MICROSCOPYf_dihedral_angle_d17.8423180
ELECTRON MICROSCOPYf_chiral_restr0.0342088
ELECTRON MICROSCOPYf_plane_restr0.0041762

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