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- PDB-9tq4: Asp2Cas12l-sgRNA bound to target DNA -

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Basic information

Entry
Database: PDB / ID: 9tq4
TitleAsp2Cas12l-sgRNA bound to target DNA
Components
  • Asp2Cas12l
  • DNA (26-MER)
  • DNA (34-MER)
  • sgRNA (149-MER)
KeywordsHYDROLASE / CRISPR-Cas / Cas12
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesArmatimonadota (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.51 Å
AuthorsSasnauskas, G. / Tamulaitiene, G. / Urbaitis, T. / Gasiunas, G.
Funding support1items
OrganizationGrant numberCountry
Not funded
Citation
Journal: CRISPR J / Year: 2026
Title: A Potent CRISPR-Cas12l Double-Strand Break Gene Editor.
Authors: Tomas Urbaitis / Laima Trinkuniene / Ieva Lenkaite / Monika Petrauskyte / Renatas Krasauskas / Migle Stitilyte / Modestas Sabaliauskas / Giedrius Sasnauskas / Giedre Tamulaitiene / Joshua K ...Authors: Tomas Urbaitis / Laima Trinkuniene / Ieva Lenkaite / Monika Petrauskyte / Renatas Krasauskas / Migle Stitilyte / Modestas Sabaliauskas / Giedrius Sasnauskas / Giedre Tamulaitiene / Joshua K Young / Virginijus Siksnys / Giedrius Gasiunas /
Abstract: Recently, a new family of CRISPR-Cas12 endonucleases from an unexplored phylum of bacteria, , was discovered. Named Cas12l, they are compact (800-900 aa), recognize a 5' C-rich protospacer adjacent ...Recently, a new family of CRISPR-Cas12 endonucleases from an unexplored phylum of bacteria, , was discovered. Named Cas12l, they are compact (800-900 aa), recognize a 5' C-rich protospacer adjacent motif, and present an N-terminal domain that stretches from the beginning to the end of the ribonucleoprotein-bound DNA target site, effectively locking it in place. Here, structure-guided rational design supplemented with AI-based large protein language model predictions was used to improve rates of DNA target cleavage of a family member, Asp2Cas12l. Compared to the wild-type, engineered variants exhibited an approximately 10-fold increase in double-strand break (DSB) editing efficiency in human cells with less target-to-target variation. Moreover, frequencies of editing were comparable to those of SpCas9 at overlapping target sites, and their DSBs efficiently corrected by homology-directed repair (39-56% of editing outcomes). Altogether, this study extends our understanding of CRISPR-Cas12 protein engineering and offers a potent new alternative for DSB-mediated genome editing in human cells.
#1: Journal: Acta Crystallogr., Sect. D: Biol. Crystallogr. / Year: 2018
Title: Real-space refinement in PHENIX for cryo-EM and crystallography
Authors: Urbaitis, T. / Sasnauskas, G. / Tamulaitiene, G. / Gasiunas, G.
History
DepositionDec 19, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 19, 2026Provider: repository / Type: Initial release
Revision 1.0Aug 19, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 19, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Aug 19, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Aug 19, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 19, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 19, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 19, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Asp2Cas12l
B: sgRNA (149-MER)
C: DNA (26-MER)
D: DNA (34-MER)


Theoretical massNumber of molelcules
Total (without water)181,1204
Polymers181,1204
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Asp2Cas12l


Mass: 99348.562 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Armatimonadota (bacteria) / Production host: Escherichia (bacteria) / Strain (production host): T7 Express
#2: RNA chain sgRNA (149-MER)


Mass: 52888.461 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Armatimonadota (bacteria)
#3: DNA chain DNA (26-MER)


Mass: 14401.233 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (34-MER)


Mass: 14481.281 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
Has protein modificationN

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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: Asp2Cas12l-crRNA bound to DNA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Armatimonadota (bacteria)
Source (recombinant)Organism: Escherichia (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal magnification: 92000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 30 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21.2_5419model refinement
13cryoSPARC4.4.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.51 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 990101 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 105.42 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004111626
ELECTRON MICROSCOPYf_angle_d0.530516675
ELECTRON MICROSCOPYf_chiral_restr0.03841947
ELECTRON MICROSCOPYf_plane_restr0.00411400
ELECTRON MICROSCOPYf_dihedral_angle_d20.39225416

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