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- PDB-8uzb: Cryo-EM structure of iGeoCas9 in complex with sgRNA and target DNA -

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Basic information

Entry
Database: PDB / ID: 8uzb
TitleCryo-EM structure of iGeoCas9 in complex with sgRNA and target DNA
Components
  • CRISPR-associated endonuclease Cas9
  • Non-target strand DNA
  • RNA (107-MER)
  • Target strand DNA (39-MER)
KeywordsHYDROLASE/RNA/DNA / RNA-guided DNA endonuclease / ternary complex / CRISPR / HYDROLASE / HYDROLASE-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, alpha-helical lobe domain / Cas9 alpha-helical lobe 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) / RNA (> 100) / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesGeobacillus stearothermophilus (bacteria)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.63 Å
AuthorsEggers, A.R. / Soczek, K.M. / Tuck, O.T. / Doudna, J.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI171110 United States
CitationJournal: Cell / Year: 2024
Title: Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9.
Authors: Amy R Eggers / Kai Chen / Katarzyna M Soczek / Owen T Tuck / Erin E Doherty / Bryant Xu / Marena I Trinidad / Brittney W Thornton / Peter H Yoon / Jennifer A Doudna /
Abstract: Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein ...Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.
History
DepositionNov 14, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 29, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 5, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 3, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first / _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: RNA (107-MER)
C: Target strand DNA (39-MER)
D: Non-target strand DNA


Theoretical massNumber of molelcules
Total (without water)202,9044
Polymers202,9044
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 127000.977 Da / Num. of mol.: 1
Mutation: D8A, H582A,E149G, T182I, N206D, P466Q, Q817R, E843K, E884G, K908R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus stearothermophilus (bacteria)
Gene: cas9 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A150MP45
#2: RNA chain RNA (107-MER)


Mass: 44488.117 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacillus stearothermophilus (bacteria)
#3: DNA chain Target strand DNA (39-MER)


Mass: 15797.221 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mus musculus (house mouse)
#4: DNA chain Non-target strand DNA


Mass: 15618.021 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mus musculus (house mouse)

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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 deactivated iGeoCas9 with sgRNA and target DNA
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.203 MDa / Experimental value: NO
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtrisC4H11NO31
2100 mMpotassium chlorideKCl1
31 mMTCEPC9H15O6P1
40.25 %glycerolC3H8O31
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.2.1particle selection
2SerialEM4.0.19image acquisition
4cryoSPARC4.2.1CTF correctionpatch ctf
7Coot0.9.8.7model fitting
9cryoSPARC4.2.1initial Euler assignment
10cryoSPARC4.2.1final Euler assignment
11cryoSPARC4.2.1classification
12cryoSPARC4.2.13D reconstruction
19PHENIX1.19.2-4158model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.63 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 228251 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building
ID 3D fitting-IDDetailsSource nameType
11v1.4.0AlphaFoldin silico model
21ModelAngelo v1.0.1Otherin silico model

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