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- EMDB-37860: SpCas9-pegRNA-target DNA complex (pre-initiation) -

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Basic information

Entry
Database: EMDB / ID: EMD-37860
TitleSpCas9-pegRNA-target DNA complex (pre-initiation)
Map data
Sample
  • Complex: SpCas9-pegRNA-target DNA complex
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • DNA: DNA (51-MER)
    • DNA: DNA (5'-D(*TP*GP*AP*TP*GP*GP*CP*AP*GP*AP*GP*TP*AP*CP*TP*AP*G)-3')
    • RNA: RNA (137-MER)
    • DNA: DNA (34-MER)
KeywordsCRISPR-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 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. ...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 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria) / Streptococcus pyogenes serotype M1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsShuto Y / Nakagawa R / Hoki M / Omura SN / Hirano H / Itoh Y / Nureki O
Funding support Japan, 1 items
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, 2023-
Header (metadata) releaseJun 5, 2024-
Map releaseJun 5, 2024-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37860.png

Downloads & links

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Map

FileDownload / File: emd_37860.map.gz / Format: CCP4 / Size: 10 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.2969 Å
Density
Contour LevelBy AUTHOR: 0.27
Minimum - Maximum-0.49174947 - 2.3041177
Average (Standard dev.)-0.005649345 (±0.1690001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin595959
Dimensions138138138
Spacing138138138
CellA=B=C: 178.9722 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_37860_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_37860_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_37860_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : SpCas9-pegRNA-target DNA complex

EntireName: SpCas9-pegRNA-target DNA complex
Components
  • Complex: SpCas9-pegRNA-target DNA complex
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • DNA: DNA (51-MER)
    • DNA: DNA (5'-D(*TP*GP*AP*TP*GP*GP*CP*AP*GP*AP*GP*TP*AP*CP*TP*AP*G)-3')
    • RNA: RNA (137-MER)
    • DNA: DNA (34-MER)

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Supramolecule #1: SpCas9-pegRNA-target DNA complex

SupramoleculeName: SpCas9-pegRNA-target DNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Streptococcus pyogenes (bacteria)

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Macromolecule #1: CRISPR-associated endonuclease Cas9/Csn1

MacromoleculeName: CRISPR-associated endonuclease Cas9/Csn1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus pyogenes serotype M1 (bacteria)
Molecular weightTheoretical: 158.457578 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: DKKYSIGLAI GTNSVGWAVI TDEYKVPSKK FKVLGNTDRH SIKKNLIGAL LFDSGETAEA TRLKRTARRR YTRRKNRICY LQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH M IKFRGHFL ...String:
DKKYSIGLAI GTNSVGWAVI TDEYKVPSKK FKVLGNTDRH SIKKNLIGAL LFDSGETAEA TRLKRTARRR YTRRKNRICY LQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH M IKFRGHFL IEGDLNPDNS DVDKLFIQLV QTYNQLFEEN PINASGVDAK AILSARLSKS RRLENLIAQL PGEKKNGLFG NL IALSLGL TPNFKSNFDL AEDAKLQLSK DTYDDDLDNL LAQIGDQYAD LFLAAKNLSD AILLSDILRV NTEITKAPLS ASM IKRYDE HHQDLTLLKA LVRQQLPEKY KEIFFDQSKN GYAGYIDGGA SQEEFYKFIK PILEKMDGTE ELLVKLNRED LLRK QRTFD NGSIPHQIHL GELHAILRRQ EDFYPFLKDN REKIEKILTF RIPYYVGPLA RGNSRFAWMT RKSEETITPW NFEEV VDKG ASAQSFIERM TNFDKNLPNE KVLPKHSLLY EYFTVYNELT KVKYVTEGMR KPAFLSGEQK KAIVDLLFKT NRKVTV KQL KEDYFKKIEC FDSVEISGVE DRFNASLGTY HDLLKIIKDK DFLDNEENED ILEDIVLTLT LFEDREMIEE RLKTYAH LF DDKVMKQLKR RRYTGWGRLS RKLINGIRDK QSGKTILDFL KSDGFANRNF MQLIHDDSLT FKEDIQKAQV SGQGDSLH E HIANLAGSPA IKKGILQTVK VVDELVKVMG RHKPENIVIE MARENQTTQK GQKNSRERMK RIEEGIKELG SQILKEHPV ENTQLQNEKL YLYYLQNGRD MYVDQELDIN RLSDYDVDAI VPQSFLKDDS IDNKVLTRSD KNRGKSDNVP SEEVVKKMKN YWRQLLNAK LITQRKFDNL TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS K LVSDFRKD FQFYKVREIN NYHHAHDAYL NAVVGTALIK KYPKLESEFV YGDYKVYDVR KMIAKSEQEI GKATAKYFFY SN IMNFFKT EITLANGEIR KRPLIETNGE TGEIVWDKGR DFATVRKVLS MPQVNIVKKT EVQTGGFSKE SILPKRNSDK LIA RKKDWD PKKYGGFDSP TVAYSVLVVA KVEKGKSKKL KSVKELLGIT IMERSSFEKN PIDFLEAKGY KEVKKDLIIK LPKY SLFEL ENGRKRMLAS AGELQKGNEL ALPSKYVNFL YLASHYEKLK GSPEDNEQKQ LFVEQHKHYL DEIIEQISEF SKRVI LADA NLDKVLSAYN KHRDKPIREQ AENIIHLFTL TNLGAPAAFK YFDTTIDRKR YTSTKEVLDA TLIHQSITGL YETRID LSQ LGGD

UniProtKB: CRISPR-associated endonuclease Cas9/Csn1

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Macromolecule #2: DNA (51-MER)

MacromoleculeName: DNA (51-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 15.636033 KDa
SequenceString:
(DC)(DT)(DA)(DG)(DT)(DA)(DC)(DT)(DC)(DT) (DG)(DC)(DC)(DA)(DT)(DC)(DA)(DG)(DA)(DG) (DC)(DA)(DA)(DG)(DC)(DA)(DC)(DT)(DA) (DC)(DG)(DG)(DC)(DC)(DG)(DA)(DT)(DT)(DG) (DC) (DT)(DC)(DT)(DA)(DA)(DG)(DT)(DG) (DA)(DT)(DC)

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Macromolecule #3: DNA (5'-D(*TP*GP*AP*TP*GP*GP*CP*AP*GP*AP*GP*TP*AP*CP*TP*AP*G)-3')

MacromoleculeName: DNA (5'-D(*TP*GP*AP*TP*GP*GP*CP*AP*GP*AP*GP*TP*AP*CP*TP*AP*G)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 5.291446 KDa
SequenceString:
(DT)(DG)(DA)(DT)(DG)(DG)(DC)(DA)(DG)(DA) (DG)(DT)(DA)(DC)(DT)(DA)(DG)

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Macromolecule #5: DNA (34-MER)

MacromoleculeName: DNA (34-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 10.462743 KDa
SequenceString:
(DG)(DA)(DT)(DC)(DA)(DC)(DT)(DT)(DA)(DG) (DA)(DG)(DC)(DA)(DA)(DT)(DC)(DG)(DG)(DC) (DC)(DC)(DA)(DG)(DA)(DC)(DT)(DG)(DA) (DG)(DC)(DA)(DC)(DG)

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Macromolecule #4: RNA (137-MER)

MacromoleculeName: RNA (137-MER) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 44.300379 KDa
SequenceString:
GGCCGUAGUG CUUGCUCUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAG UCGGUGCAGG AGGAAGCAGU GCAACCAAAC CACAGCGUGC UCAGUCUG

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 297 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 197777
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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