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TitleStructure of the mini-RNA-guided endonuclease CRISPR-Cas12j3.
Journal, issue, pagesNat Commun, Vol. 12, Issue 1, Page 4476, Year 2021
Publish dateJul 22, 2021
AuthorsArturo Carabias / Anders Fuglsang / Piero Temperini / Tillmann Pape / Nicholas Sofos / Stefano Stella / Simon Erlendsson / Guillermo Montoya /
PubMed AbstractCRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome ...CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.
External linksNat Commun / PubMed:34294706 / PubMed Central
MethodsEM (single particle)
Resolution2.66 Å
Structure data

EMDB-12827, PDB-7odf:
Structure of the mini-RNA-guided endonuclease CRISPR-Cas_phi3
Method: EM (single particle) / Resolution: 2.66 Å

Chemicals

ChemComp-NI:
NICKEL (II) ION / Nickel

ChemComp-ZN:
Unknown entry

ChemComp-HOH:
WATER / Water

Source
  • Phage (virus)
  • synthetic construct (others)
  • phage #d (virus)
KeywordsRNA BINDING PROTEIN / CRISPR-Cas / RNA-guided endonuclease / R-loop

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