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-Structure paper
Title | Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3. |
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Journal, issue, pages | Nat Commun, Vol. 12, Issue 1, Page 4476, Year 2021 |
Publish date | Jul 22, 2021 |
Authors | Arturo Carabias / Anders Fuglsang / Piero Temperini / Tillmann Pape / Nicholas Sofos / Stefano Stella / Simon Erlendsson / Guillermo Montoya / |
PubMed Abstract | 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 ...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 links | Nat Commun / PubMed:34294706 / PubMed Central |
Methods | EM (single particle) |
Resolution | 2.66 Å |
Structure data | EMDB-12827, PDB-7odf: |
Chemicals | ChemComp-NI: ChemComp-ZN: ChemComp-HOH: |
Source |
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Keywords | RNA BINDING PROTEIN / CRISPR-Cas / RNA-guided endonuclease / R-loop |