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- EMDB-27801: Cryo-EM structure of Acidibacillus sulfuroxidans Cas12f in comple... -

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Basic information

Entry
Database: EMDB / ID: EMD-27801
TitleCryo-EM structure of Acidibacillus sulfuroxidans Cas12f in complex with sgRNA and target DNA
Map data
Sample
  • Complex: AsCas12f-sgRNA-DNA
    • Protein or peptide: OrfB_Zn_ribbon domain-containing protein
    • DNA: target DNA strand
    • DNA: Non-target DNA strand
    • RNA: sgRNA
  • Ligand: ZINC ION
KeywordsCRISPR / Cas12 / gene editing / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyTransposase IS605, OrfB, C-terminal / Putative transposase DNA-binding domain / endonuclease activity / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding / CRISPR-associated endodeoxyribonuclease Cas12f1
Function and homology information
Biological speciesAcidibacillus sulfuroxidans (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsLiu C / Zhao M
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseJul 12, 2023-
Map releaseJul 12, 2023-
UpdateNov 8, 2023-
Current statusNov 8, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27801.png

Downloads & links

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Map

FileDownload / File: emd_27801.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.065 Å
Density
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.03330448 - 0.098283775
Average (Standard dev.)-0.000030770836 (±0.0021748214)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 272.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_27801_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

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Half map: #2

Fileemd_27801_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_27801_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : AsCas12f-sgRNA-DNA

EntireName: AsCas12f-sgRNA-DNA
Components
  • Complex: AsCas12f-sgRNA-DNA
    • Protein or peptide: OrfB_Zn_ribbon domain-containing protein
    • DNA: target DNA strand
    • DNA: Non-target DNA strand
    • RNA: sgRNA
  • Ligand: ZINC ION

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Supramolecule #1: AsCas12f-sgRNA-DNA

SupramoleculeName: AsCas12f-sgRNA-DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Acidibacillus sulfuroxidans (bacteria)

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Macromolecule #1: OrfB_Zn_ribbon domain-containing protein

MacromoleculeName: OrfB_Zn_ribbon domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Acidibacillus sulfuroxidans (bacteria)
Molecular weightTheoretical: 51.424703 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAHHHHHHSA ALEVLFQGPG YQDPMIKVYR YEIVKPLDLD WKEFGTILRQ LQQETRFALN KATQLAWEWM GFSSDYKDNH GEYPKSKDI LGYTNVHGYA YHTIKTKAYR LNSGNLSQTI KRATDRFKAY QKEILRGDMS IPSYKRDIPL DLIKENISVN R MNHGDYIA ...String:
MAHHHHHHSA ALEVLFQGPG YQDPMIKVYR YEIVKPLDLD WKEFGTILRQ LQQETRFALN KATQLAWEWM GFSSDYKDNH GEYPKSKDI LGYTNVHGYA YHTIKTKAYR LNSGNLSQTI KRATDRFKAY QKEILRGDMS IPSYKRDIPL DLIKENISVN R MNHGDYIA SLSLLSNPAK QEMNVKRKIS VIIIVRGAGK TIMDRILSGE YQVSASQIIH DDRKNKWYLN ISYDFEPQTR VL DLNKIMG IALGVAVAVY MAFQHTPARY KLEGGEIENF RRQVESRRIS MLRQGKYAGG ARGGHGRDKR IKPIEQLRDK IAN FRDTTN HRYSRYIVDM AIKEGCGTIQ MEDLTNIRDI GSRFLQNWTY YDLQQKIIYK AEEAGIKVIK IDPQYTSQRC SECG NIDSG NRIGQAIFKC RACGYEANAD YNAARNIAIP NIDKIIAESI K

UniProtKB: CRISPR-associated endodeoxyribonuclease Cas12f1

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Macromolecule #2: target DNA strand

MacromoleculeName: target DNA strand / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.983318 KDa
SequenceString:
(DT)(DT)(DC)(DC)(DG)(DG)(DC)(DC)(DT)(DG) (DG)(DA)(DT)(DT)(DG)(DT)(DG)(DG)(DG)(DT) (DC)(DT)(DT)(DG)(DA)(DG)(DA)(DG)(DC) (DA)(DA)(DA)(DA)(DA)(DC)(DC)(DT)(DG)(DT) (DT) (DT)(DT)

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Macromolecule #3: Non-target DNA strand

MacromoleculeName: Non-target DNA strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.877307 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DC)(DA)(DG)(DG)(DT)(DT) (DT)(DT)(DT)(DG)(DC)(DT)(DC)(DT)(DC)(DA) (DA)(DG)(DA)(DC)(DC)(DC)(DA)(DC)(DA) (DA)(DT)(DC)(DC)(DA)(DG)(DG)(DC)(DC)(DG) (DG) (DA)(DA)

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Macromolecule #4: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Acidibacillus sulfuroxidans (bacteria)
Molecular weightTheoretical: 62.289094 KDa
SequenceString: GGAUUCGUCG GUUCAGCGAC GAUAAGCCGA GAAGUGCCAA UAAAACUGUU AAGUGGUUUG GUAACGCUCG GUAAGGUAGC CAAAAGGCU GAAACUCCGU GCACAAAGAC CGCACGGACG CUUCACAUAU AGCUCAUAAA CAAGGGUUUG CGAGCUAGCU U GUGGAGUG ...String:
GGAUUCGUCG GUUCAGCGAC GAUAAGCCGA GAAGUGCCAA UAAAACUGUU AAGUGGUUUG GUAACGCUCG GUAAGGUAGC CAAAAGGCU GAAACUCCGU GCACAAAGAC CGCACGGACG CUUCACAUAU AGCUCAUAAA CAAGGGUUUG CGAGCUAGCU U GUGGAGUG UGAACCUCUC AAGACCCACA AUCCA

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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 / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: Ab initio reconstruction from CryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 490190
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION

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