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- EMDB-51864: wtCas9 bound to off-target 1 EMX1-1 (-)SC DNA minicircle -

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Basic information

Entry
Database: EMDB / ID: EMD-51864
TitlewtCas9 bound to off-target 1 EMX1-1 (-)SC DNA minicircle
Map data
Sample
  • Complex: dCas9 EMX1-1 on-target complex on a negatively supercoiled DNA minicircle
    • RNA: EMX1-1 sgRNA
    • DNA: Target Strand Pre-Cut
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • DNA: Non-Target Strand
  • DNA: Target Strand Post-Cut
KeywordsdCas9 / Cas9 / off-target / EMX1-1 / negatively supercoiled / diamond ring / HYDROLASE
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
CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsSmith QM / Rueda D
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)UKRI MC-A658-5TY10 United Kingdom
CitationJournal: Nature / Year: 2026
Title: Structural basis of supercoiling-induced CRISPR-Cas9 off-target activity.
Authors: Quentin M Smith / Sylvia Whittle / Ricardo J Aramayo / Daniel E Rollins / Adam S B Jalal / Deborah I Egharevba / Kyle L Morris / Alice L B Pyne / David S Rueda /
Abstract: CRISPR-Cas9 is a powerful genome-editing tool, but genome-wide off-target activity can hinder therapeutic applications. Negative supercoiling ((-)SC) has been implicated in off-target activity, but a ...CRISPR-Cas9 is a powerful genome-editing tool, but genome-wide off-target activity can hinder therapeutic applications. Negative supercoiling ((-)SC) has been implicated in off-target activity, but a molecular-level understanding is lacking. Here, using (-)SC DNA minicircles, we observe supercoiling-driven structural defects in the DNA that are resolved by Cas9 binding. Cryo-electron microscopy structures of Cas9 bound in both the on-target and off-target configurations highlight that the Cas9 HNH domain is poised in a more catalytically competent conformation. New DNA-RNA mismatch geometries are accommodated across the protospacer and structural plasticity in the protospacer adjacent motif distal region of the protospacer is topology dependent. Together, our study reveals the molecular basis for (-)SC-induced Cas9 targeting and provides a framework for the design of next-generation high-fidelity CRISPR effectors with topological context.
History
DepositionOct 20, 2024-
Header (metadata) releaseApr 1, 2026-
Map releaseApr 1, 2026-
UpdateApr 8, 2026-
Current statusApr 8, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51864.png

Downloads & links

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Map

FileDownload / File: emd_51864.map.gz / Format: CCP4 / Size: 105.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 302 pix.
= 256.7 Å		0.85 Å/pix.
x 302 pix.
= 256.7 Å		0.85 Å/pix.
x 302 pix.
= 256.7 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.007
Minimum - Maximum-0.018885985 - 0.04180613
Average (Standard dev.)0.00018887887 (±0.0016426573)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions302302302
Spacing302302302
CellA=B=C: 256.7 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_51864_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_51864_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : dCas9 EMX1-1 on-target complex on a negatively supercoiled DNA mi...

EntireName: dCas9 EMX1-1 on-target complex on a negatively supercoiled DNA minicircle
Components
  • Complex: dCas9 EMX1-1 on-target complex on a negatively supercoiled DNA minicircle
    • RNA: EMX1-1 sgRNA
    • DNA: Target Strand Pre-Cut
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • DNA: Non-Target Strand
  • DNA: Target Strand Post-Cut

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Supramolecule #1: dCas9 EMX1-1 on-target complex on a negatively supercoiled DNA mi...

SupramoleculeName: dCas9 EMX1-1 on-target complex on a negatively supercoiled DNA minicircle
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2, #4, #3
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 160 KDa

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Macromolecule #1: EMX1-1 sgRNA

MacromoleculeName: EMX1-1 sgRNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 31.765967 KDa
SequenceString:
GAGUCCGAGC AGAAGAAGAA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAG UCGGUGCUU

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Macromolecule #2: Target Strand Pre-Cut

MacromoleculeName: Target Strand Pre-Cut / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.946929 KDa
SequenceString:
(DT)(DC)(DT)(DC)(DC)(DC)(DC)(DT)(DT)(DG)

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Macromolecule #3: Non-Target Strand

MacromoleculeName: Non-Target Strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.547677 KDa
SequenceString:
(DA)(DG)(DG)(DG)(DG)(DT)(DG)(DG)

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Macromolecule #5: Target Strand Post-Cut

MacromoleculeName: Target Strand Post-Cut / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.110305 KDa
SequenceString:
(DT)(DT)(DC)(DT)(DC)(DT)(DT)(DG)(DC)(DT) (DC)(DT)(DG)(DA)(DC)(DT)(DT)

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Macromolecule #4: CRISPR-associated endonuclease Cas9/Csn1

MacromoleculeName: CRISPR-associated endonuclease Cas9/Csn1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 158.588781 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNE MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA DLRLIYLALA H MIKFRGHF ...String:
MDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNE MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA DLRLIYLALA H MIKFRGHF LIEGDLNPDN SDVDKLFIQL VQTYNQLFEE NPINASGVDA KAILSARLSK SRRLENLIAQ LPGEKKNGLF GN LIALSLG LTPNFKSNFD LAEDAKLQLS KDTYDDDLDN LLAQIGDQYA DLFLAAKNLS DAILLSDILR VNTEITKAPL SAS MIKRYD EHHQDLTLLK ALVRQQLPEK YKEIFFDQSK NGYAGYIDGG ASQEEFYKFI KPILEKMDGT EELLVKLNRE DLLR KQRTF DNGSIPHQIH LGELHAILRR QEDFYPFLKD NREKIEKILT FRIPYYVGPL ARGNSRFAWM TRKSEETITP WNFEE VVDK GASAQSFIER MTNFDKNLPN EKVLPKHSLL YEYFTVYNEL TKVKYVTEGM RKPAFLSGEQ KKAIVDLLFK TNRKVT VKQ LKEDYFKKIE CFDSVEISGV EDRFNASLGT YHDLLKIIKD KDFLDNEENE DILEDIVLTL TLFEDREMIE ERLKTYA HL FDDKVMKQLK RRRYTGWGRL SRKLINGIRD KQSGKTILDF LKSDGFANRN FMQLIHDDSL TFKEDIQKAQ VSGQGDSL H EHIANLAGSP AIKKGILQTV KVVDELVKVM GRHKPENIVI EMARENQTTQ KGQKNSRERM KRIEEGIKEL GSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDA IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNA KLITQRKFDN LTKAERGGLS ELDKAGFIKR QLVETRQITK HVAQILDSRM NTKYDENDKL IREVKVITLK S KLVSDFRK DFQFYKVREI NNYHHAHDAY LNAVVGTALI KKYPKLESEF VYGDYKVYDV RKMIAKSEQE IGKATAKYFF YS NIMNFFK TEITLANGEI RKRPLIETNG ETGEIVWDKG RDFATVRKVL SMPQVNIVKK TEVQTGGFSK ESILPKRNSD KLI ARKKDW DPKKYGGFDS PTVAYSVLVV AKVEKGKSKK LKSVKELLGI TIMERSSFEK NPIDFLEAKG YKEVKKDLII KLPK YSLFE LENGRKRMLA SAGELQKGNE LALPSKYVNF LYLASHYEKL KGSPEDNEQK QLFVEQHKHY LDEIIEQISE FSKRV ILAD ANLDKVLSAY NKHRDKPIRE QAENIIHLFT LTNLGAPAAF KYFDTTIDRK RYTSTKEVLD ATLIHQSITG LYETRI DLS QLGGD

UniProtKB: CRISPR-associated endonuclease Cas9/Csn1

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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

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 41.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 496737
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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