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- PDB-8hud: Cryo-EM structure of the EvCas9-sgRNA-target DNA ternary complex -

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Basic information

Entry
Database: PDB / ID: 8hud
TitleCryo-EM structure of the EvCas9-sgRNA-target DNA ternary complex
Components
  • CRISPR-associated endonuclease Cas9
  • Non-target DNA strand
  • Target DNA strand
  • sgRNA
KeywordsRNA BINDING PROTEIN/RNA/DNA / RNA / DNA / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. ...Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / 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) / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesEubacterium ventriosum ATCC 27560 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.43 Å
AuthorsTang, N. / Wu, Z. / Gao, Y. / Chen, W. / Su, M. / Wang, Z. / Ji, Q.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)21922705 China
National Natural Science Foundation of China (NSFC)22277078 China
CitationJournal: ACS Synth Biol / Year: 2024
Title: Molecular Basis and Genome Editing Applications of a Compact CRISPR-Cas9 System.
Authors: Na Tang / Zhaowei Wu / Yan Gao / Weizhong Chen / Zixiao Wang / Mengjiao Su / Wenxin Ji / Quanjiang Ji /
Abstract: CRISPR-Cas9 systems have been widely harnessed for diverse genome editing applications because of their ease of use and high efficiency. However, the large molecular sizes and strict PAM requirements ...CRISPR-Cas9 systems have been widely harnessed for diverse genome editing applications because of their ease of use and high efficiency. However, the large molecular sizes and strict PAM requirements of commonly used CRISPR-Cas9 systems restrict their broad applications in therapeutics. Here, we report the molecular basis and genome editing applications of a novel compact type II-A CRISPR-Cas9 system (EvCas9) with 1107 residues and distinct 5'-NNGDGN-3' (where D represents A, T, or G) PAM specificity. We determine the cryo-EM structure of EvCas9 in a complex with an sgRNA and a target DNA, revealing the detailed PAM recognition and sgRNA and target DNA association mechanisms. Additionally, we demonstrate the robust genome editing capacity of EvCas9 in bacteria and human cells with superior fidelity compared to SaCas9 and SpCas9, and we engineer it to be efficient base editors by fusing a cytidine or adenosine deaminase. Collectively, our results facilitate further understanding of CRISPR-Cas9 working mechanisms and expand the compact CRISPR-Cas9 toolbox.
History
DepositionDec 23, 2022Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Dec 27, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 7, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Oct 9, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _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
B: sgRNA
C: Target DNA strand
D: Non-target DNA strand


Theoretical massNumber of molelcules
Total (without water)166,1964
Polymers166,1964
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 131110.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Eubacterium ventriosum ATCC 27560 (bacteria)
Gene: csn1, cas9, EUBVEN_00141 / Plasmid: pET28a / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: A5Z395, Hydrolases; Acting on ester bonds
#2: RNA chain sgRNA


Mass: 23976.240 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain Target DNA strand


Mass: 8641.558 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain Non-target DNA strand


Mass: 2467.629 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
Has protein modificationY

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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: Ternary complex of EvCas9-sgRNA-dsDNA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.1565 MDa / Experimental value: NO
Source (natural)Organism: Eubacterium ventriosum ATCC 27560 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pET28a
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1300 mMsodium chlorideNaCl1
25 mMmagnesium chlorideMgCl21
320 mMTRIS hydrochlorideTris-HCl1
41 mMDithiothreitolDTT1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Homemade
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281.15 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: 1800 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 16.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv3.2.0particle selection
4cryoSPARCv3.2.0CTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 300599
3D reconstructionResolution: 3.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33599 / Symmetry type: POINT

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