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- PDB-8ygj: SpCas9-MMLV RT-pegRNA-target DNA complex (elongation 28-nt) -

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Basic information

Entry
Database: PDB / ID: 8ygj
TitleSpCas9-MMLV RT-pegRNA-target DNA complex (elongation 28-nt)
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • DNA (5'-D(P*TP*GP*AP*TP*GP*GP*CP*AP*GP*AP*GP*TP*AP*CP*TP*AP*G)-3')
  • DNA (51-MER)
  • DNA (62-MER)
  • Gag-Pol polyprotein
  • RNA (137-MER)
KeywordsRNA BINDING PROTEIN/DNA/RNA / CRISPR-Cas / RNA BINDING PROTEIN-DNA-RNA COMPLEX
Function / homology
Function and homology information


host cell late endosome membrane / maintenance of CRISPR repeat elements / virion assembly / 3'-5' exonuclease activity / DNA endonuclease activity / host multivesicular body / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity ...host cell late endosome membrane / maintenance of CRISPR repeat elements / virion assembly / 3'-5' exonuclease activity / DNA endonuclease activity / host multivesicular body / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / defense response to virus / DNA recombination / structural constituent of virion / Hydrolases; Acting on ester bonds / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / host cell plasma membrane / proteolysis / DNA binding / RNA binding / zinc ion binding / metal ion binding / plasma membrane
Similarity search - Function
Gag-Pol polyprotein, Zinc-finger like domain / Murine leukemia virus integrase, C-terminal / Zinc-finger like, probable DNA-binding / Murine leukemia virus (MLV) integrase (IN) C-terminal domain / Gamma-retroviral matrix protein / Gag polyprotein, inner coat protein p12 / Core shell protein Gag P30 / : / Matrix protein (MA), p15 / Gag polyprotein, inner coat protein p12 ...Gag-Pol polyprotein, Zinc-finger like domain / Murine leukemia virus integrase, C-terminal / Zinc-finger like, probable DNA-binding / Murine leukemia virus (MLV) integrase (IN) C-terminal domain / Gamma-retroviral matrix protein / Gag polyprotein, inner coat protein p12 / Core shell protein Gag P30 / : / Matrix protein (MA), p15 / Gag polyprotein, inner coat protein p12 / Gag P30 core shell protein / Gamma-retroviral matrix domain superfamily / Reverse transcriptase/retrotransposon-derived protein, RNase H-like domain / RNase H-like domain found in reverse transcriptase / 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 / RNase H / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / RNase H type-1 domain profile. / Ribonuclease H domain / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Reverse transcriptase (RNA-dependent DNA polymerase) / Retroviral matrix protein / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / Gag-Pol polyprotein / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria)
Moloney murine leukemia virus
Streptococcus pyogenes serotype M1 (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
DepositionFeb 26, 2024Deposition 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
B: RNA (137-MER)
C: DNA (51-MER)
D: DNA (5'-D(P*TP*GP*AP*TP*GP*GP*CP*AP*GP*AP*GP*TP*AP*CP*TP*AP*G)-3')
E: Gag-Pol polyprotein
F: DNA (62-MER)
A: CRISPR-associated endonuclease Cas9/Csn1


Theoretical massNumber of molelcules
Total (without water)298,3716
Polymers298,3716
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

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RNA chain , 1 types, 1 molecules B

#1: RNA chain RNA (137-MER)


Mass: 44316.379 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Production host: Teseptimavirus

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DNA chain , 3 types, 3 molecules CDF

#2: DNA chain DNA (51-MER)


Mass: 15636.033 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#3: DNA chain DNA (5'-D(P*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)
#5: DNA chain DNA (62-MER)


Mass: 19013.145 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)

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Protein , 2 types, 2 molecules EA

#4: Protein Gag-Pol polyprotein


Mass: 55656.242 Da / Num. of mol.: 1 / Mutation: D200N, D209N, T306K, W313F, T330P, D335N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Moloney murine leukemia virus / Gene: gag-pro-pol
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q8UN00
#6: Protein CRISPR-associated endonuclease Cas9/Csn1


Mass: 158457.578 Da / Num. of mol.: 1 / Mutation: H10A, 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

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Details

Has ligand of interestN

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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-MMLV RT-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 'BL21-Gold(DE3)pLysS AG' (bacteria)
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: 104057 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00320370
ELECTRON MICROSCOPYf_angle_d0.55228619
ELECTRON MICROSCOPYf_dihedral_angle_d15.84607
ELECTRON MICROSCOPYf_chiral_restr0.0373287
ELECTRON MICROSCOPYf_plane_restr0.0032792

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