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- PDB-8wuu: SpCas9-pegRNA-target DNA complex (pre-initiation) -

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Basic information

Entry
Database: PDB / ID: 8wuu
TitleSpCas9-pegRNA-target DNA complex (pre-initiation)
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • DNA (34-MER)
  • DNA (5'-D(*TP*GP*AP*TP*GP*GP*CP*AP*GP*AP*GP*TP*AP*CP*TP*AP*G)-3')
  • DNA (51-MER)
  • RNA (137-MER)
KeywordsRNA BINDING PROTEIN/RNA/DNA / CRISPR-Cas / RNA BINDING PROTEIN-RNA-DNA 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 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain ...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 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / 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)
Streptococcus pyogenes (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsShuto, Y. / Nakagawa, R. / Hoki, M. / Omura, S.N. / Hirano, H. / Itoh, Y. / Nureki, O.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP23ama121012 Japan
CitationJournal: Nature / Year: 2024
Title: Structural basis for pegRNA-guided reverse transcription by a prime editor.
Authors: Yutaro Shuto / Ryoya Nakagawa / Shiyou Zhu / Mizuki Hoki / Satoshi N Omura / Hisato Hirano / Yuzuru Itoh / Feng Zhang / Osamu Nureki /
Abstract: The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing ...The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing guide RNA (pegRNA) to facilitate a wide variety of precise genome edits in living cells. However, owing to a lack of structural information, the molecular mechanism of pegRNA-guided reverse transcription by the prime editor remains poorly understood. Here we present cryo-electron microscopy structures of the SpCas9-M-MLV RTΔRNaseH-pegRNA-target DNA complex in multiple states. The termination structure, along with our functional analysis, reveals that M-MLV RT extends reverse transcription beyond the expected site, resulting in scaffold-derived incorporations that cause undesired edits at the target loci. Furthermore, structural comparisons among the pre-initiation, initiation and elongation states show that M-MLV RT remains in a consistent position relative to SpCas9 during reverse transcription, whereas the pegRNA-synthesized DNA heteroduplex builds up along the surface of SpCas9. On the basis of our structural insights, we rationally engineered pegRNA variants and prime-editor variants in which M-MLV RT is fused within SpCas9. Collectively, our findings provide structural insights into the stepwise mechanism of prime editing, and will pave the way for the development of a versatile prime editing toolbox.
History
DepositionOct 21, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Data collection / Database references / Structure summary
Category: audit_author / citation ...audit_author / citation / citation_author / em_author_list
Item: _audit_author.name / _citation.pdbx_database_id_PubMed ..._audit_author.name / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_author_list.author
Revision 1.2Jul 17, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Revision 1.3Sep 4, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.4Sep 11, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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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
C: DNA (51-MER)
D: DNA (5'-D(*TP*GP*AP*TP*GP*GP*CP*AP*GP*AP*GP*TP*AP*CP*TP*AP*G)-3')
B: RNA (137-MER)
F: DNA (34-MER)


Theoretical massNumber of molelcules
Total (without water)234,1485
Polymers234,1485
Non-polymers00
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 CRISPR-associated endonuclease Cas9/Csn1


Mass: 158457.578 Da / Num. of mol.: 1 / Mutation: D10A, H840A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Gene: cas9
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q99ZW2
#2: DNA chain DNA (51-MER)


Mass: 15636.033 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#3: DNA chain DNA (5'-D(*TP*GP*AP*TP*GP*GP*CP*AP*GP*AP*GP*TP*AP*CP*TP*AP*G)-3')


Mass: 5291.446 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#4: RNA chain RNA (137-MER)


Mass: 44300.379 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Production host: Teseptimavirus
#5: DNA chain DNA (34-MER)


Mass: 10462.743 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)

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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: SpCas9-pegRNA-target DNA complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 297 K

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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: 1600 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 197777 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00615208
ELECTRON MICROSCOPYf_angle_d0.57621310
ELECTRON MICROSCOPYf_dihedral_angle_d15.2443450
ELECTRON MICROSCOPYf_chiral_restr0.0422454
ELECTRON MICROSCOPYf_plane_restr0.0042062

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