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- PDB-8dzj: Cryo-EM structure of Acidibacillus sulfuroxidans Cas12f in comple... -

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Basic information

Entry
Database: PDB / ID: 8dzj
TitleCryo-EM structure of Acidibacillus sulfuroxidans Cas12f in complex with sgRNA and target DNA
Components
  • Non-target DNA strand
  • OrfB_Zn_ribbon domain-containing protein
  • sgRNA
  • target DNA strand
KeywordsRNA BINDING PROTEIN/RNA/DNA / CRISPR / Cas12 / gene editing / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


endonuclease activity / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
Transposase IS605, OrfB, C-terminal / Putative transposase DNA-binding domain
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endodeoxyribonuclease Cas12f1
Similarity search - Component
Biological speciesAcidibacillus sulfuroxidans (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsLiu, C. / Zhao, M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM143052 United States
CitationJournal: Nat Chem Biol / Year: 2023
Title: An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity.
Authors: Tong Wu / Chang Liu / Siyuan Zou / Ruitu Lyu / Bowei Yang / Hao Yan / Minglei Zhao / Weixin Tang /
Abstract: Compact CRISPR-Cas systems offer versatile treatment options for genetic disorders, but their application is often limited by modest gene-editing activity. Here we present enAsCas12f, an engineered ...Compact CRISPR-Cas systems offer versatile treatment options for genetic disorders, but their application is often limited by modest gene-editing activity. Here we present enAsCas12f, an engineered RNA-guided DNA endonuclease up to 11.3-fold more potent than its parent protein, AsCas12f, and one-third of the size of SpCas9. enAsCas12f shows higher DNA cleavage activity than wild-type AsCas12f in vitro and functions broadly in human cells, delivering up to 69.8% insertions and deletions at user-specified genomic loci. Minimal off-target editing is observed with enAsCas12f, suggesting that boosted on-target activity does not impair genome-wide specificity. We determine the cryo-electron microscopy (cryo-EM) structure of the AsCas12f-sgRNA-DNA complex at a resolution of 2.9 Å, which reveals dimerization-mediated substrate recognition and cleavage. Structure-guided single guide RNA (sgRNA) engineering leads to sgRNA-v2, which is 33% shorter than the full-length sgRNA, but with on par activity. Together, the engineered hypercompact AsCas12f system enables robust and faithful gene editing in mammalian cells.
History
DepositionAug 7, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 12, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 8, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: OrfB_Zn_ribbon domain-containing protein
B: OrfB_Zn_ribbon domain-containing protein
C: target DNA strand
D: Non-target DNA strand
E: sgRNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)191,0656
Polymers190,9995
Non-polymers651
Water00
1


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

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Components

#1: Protein OrfB_Zn_ribbon domain-containing protein / AsCas12f


Mass: 51424.703 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acidibacillus sulfuroxidans (bacteria) / Gene: BM613_13600 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2U3D0N8
#2: DNA chain target DNA strand


Mass: 12983.318 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: DNA chain Non-target DNA strand


Mass: 12877.307 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#4: RNA chain sgRNA


Mass: 62289.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Acidibacillus sulfuroxidans (bacteria)
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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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: AsCas12f-sgRNA-DNA / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Source (natural)Organism: Acidibacillus sulfuroxidans (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM softwareName: RELION / Category: final Euler assignment
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 490190 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039605
ELECTRON MICROSCOPYf_angle_d0.55213864
ELECTRON MICROSCOPYf_dihedral_angle_d16.8672887
ELECTRON MICROSCOPYf_chiral_restr0.0361642
ELECTRON MICROSCOPYf_plane_restr0.0041064

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