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- EMDB-51863: dCas9 bound to off-target 2 EMX1-1 (-)SC DNA minicircle -

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Basic information

Entry
Database: EMDB / ID: EMD-51863
TitledCas9 bound to off-target 2 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
    • DNA: Non-Target Strand
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
KeywordsdCas9 / Cas9 / on-target / 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: 3.9 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_51863.png

Downloads & links

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Map

FileDownload / File: emd_51863.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 150 pix.
= 165. Å		1.1 Å/pix.
x 150 pix.
= 165. Å		1.1 Å/pix.
x 150 pix.
= 165. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0134
Minimum - Maximum-0.04641702 - 0.10627112
Average (Standard dev.)0.0011626274 (±0.005492231)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 165.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_51863_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_51863_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
    • DNA: Non-Target Strand
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1

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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: #2-#4, #1
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 160 KDa

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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 / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 158.3525 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 RLSDYDVDHI 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 LG

UniProtKB: CRISPR-associated endonuclease Cas9/Csn1

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Macromolecule #2: EMX1-1 sgRNA

MacromoleculeName: EMX1-1 sgRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 28.810205 KDa
SequenceString:
CAGAAGAAGA AGUUUUAGAG CUAGAAAUAG CAAGUUAAAA UAAGGCUAGU CCGUUAUCAA CUUGAAAAAG UGGCACCGAG UCGGUGCUU

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Macromolecule #3: Target Strand

MacromoleculeName: Target Strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.320427 KDa
SequenceString:
(DT)(DC)(DT)(DC)(DC)(DC)(DC)(DT)(DT)(DC) (DT)(DT)(DC)(DT)(DC)(DC)(DT)(DG)

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Macromolecule #4: Non-Target Strand

MacromoleculeName: Non-Target Strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.515679 KDa
SequenceString:
(DA)(DA)(DA)(DG)(DG)(DT)(DG)(DG)

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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: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 667106
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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