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- PDB-9eda: SpCas9 with 17-bp R-loop containing 2 terminal mismatches (State ... -

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Basic information

Entry
Database: PDB / ID: 9eda
TitleSpCas9 with 17-bp R-loop containing 2 terminal mismatches (State IV - product)
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • Non-target strand
  • PAM-distal Target Strand
  • PAM-proximal Target Strand
  • sgRNA
KeywordsDNA BINDING PROTEIN/DNA/RNA / CRISPR-associated endonuclease / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA complex
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, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix 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, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix 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) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsKiernan, K.A. / Taylor, D.W.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
Welch FoundationF-1938-20200401 United States
American Cancer SocietyRSG-21-050-01-DMC United States
CitationJournal: Nat Commun / Year: 2025
Title: Visualization of a multi-turnover Cas9 after product release.
Authors: Kaitlyn A Kiernan / David W Taylor /
Abstract: While the most widely used CRISPR-Cas enzyme is the Cas9 endonuclease from Streptococcus pyogenes (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product ...While the most widely used CRISPR-Cas enzyme is the Cas9 endonuclease from Streptococcus pyogenes (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product DNA. This blocks access to DNA repair machinery and acts as a major bottleneck during CRISPR-Cas9 gene editing. Cas9 can eventually be removed from the product by extrinsic factors, such as translocating polymerases, but the mechanisms contributing to Cas9 dissociation following cleavage remain poorly understood. Here, we employ truncated guide RNAs as a strategy to weaken PAM-distal nucleic acid interactions and promote faster enzyme turnover. Using kinetics-guided cryo-EM, we examine the conformational landscape of a multi-turnover Cas9, including the first detailed snapshots of Cas9 dissociating from product DNA. We discovered that while the PAM-distal product dissociates from Cas9 following cleavage, tight binding of the PAM-proximal product directly inhibits re-binding of new targets. Our work provides direct evidence as to why Cas9 acts as a single-turnover enzyme and will guide future Cas9 engineering efforts.
History
DepositionNov 16, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 18, 2025Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 9, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: sgRNA
C: PAM-proximal Target Strand
D: Non-target strand
c: PAM-distal Target Strand
A: CRISPR-associated endonuclease Cas9/Csn1


Theoretical massNumber of molelcules
Total (without water)223,8835
Polymers223,8835
Non-polymers00
Water181
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

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DNA chain , 3 types, 3 molecules CDc

#2: DNA chain PAM-proximal Target Strand


Mass: 6743.352 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain Non-target strand


Mass: 17026.959 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain PAM-distal Target Strand


Mass: 10077.479 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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RNA chain / Protein / Non-polymers , 3 types, 3 molecules BA

#1: RNA chain sgRNA


Mass: 31335.643 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Protein CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9


Mass: 158699.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli (E. coli)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Details

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: Ternary complex - SpCas9, mm2 sgRNA, 55-bp dsDNA / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21_5207 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 180648 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6O0Z

6o0z


Accession code: 6O0Z / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00512614
ELECTRON MICROSCOPYf_angle_d0.95417657
ELECTRON MICROSCOPYf_dihedral_angle_d18.4743043
ELECTRON MICROSCOPYf_chiral_restr0.0482058
ELECTRON MICROSCOPYf_plane_restr0.0071753

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