7ODF

Structure of the mini-RNA-guided endonuclease CRISPR-Cas_phi3

Summary for 7ODF

• Entry DOI: 10.2210/pdb7odf/pdb
• EMDB information: 12827
• Descriptor: Cas_phi3, DNA (5'-D(P*GP*TP*AP*TP*CP*CP*CP*AP*TP*TP*AP*CP*CP*AP*GP*CP*TP*GP*AP*AP*TP*TP*AP*C)-3'), DNA (5'-D(P*GP*TP*AP*AP*TP*TP*CP*AP*G)-3'), ... (8 entities in total)
• Functional Keywords: crispr-cas, rna-guided endonuclease, r-loop, rna binding protein
• Biological source: Phage #D; More
• Total number of polymer chains: 5
• Total formula weight: 112042.02

Authors

Carabias del Rey, A.,Fugilsang, A.,Temperini, P.,Pape, T.,Sofos, N.,Stella, S.,Erledsson, S.,Montoya, G. (deposition date: 2021-04-29, release date: 2021-07-21, Last modification date: 2024-07-10)

Primary citation

Carabias, A.,Fuglsang, A.,Temperini, P.,Pape, T.,Sofos, N.,Stella, S.,Erlendsson, S.,Montoya, G.
Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3.
Nat Commun, 12:4476-4476, 2021

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 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.

PubMed: 34294706
DOI: 10.1038/s41467-021-24707-3

Experimental method

ELECTRON MICROSCOPY (2.66 Å)