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Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3

Author

Listed:
  • Arturo Carabias

    (Faculty of Health and Medical Sciences University of Copenhagen)

  • Anders Fuglsang

    (Faculty of Health and Medical Sciences University of Copenhagen)

  • Piero Temperini

    (Faculty of Health and Medical Sciences University of Copenhagen)

  • Tillmann Pape

    (Faculty of Health and Medical Sciences University of Copenhagen
    University of Copenhagen)

  • Nicholas Sofos

    (Faculty of Health and Medical Sciences University of Copenhagen)

  • Stefano Stella

    (Faculty of Health and Medical Sciences University of Copenhagen
    Twelve Bio ApS)

  • Simon Erlendsson

    (Faculty of Health and Medical Sciences University of Copenhagen
    MRC Laboratory of Molecular Biology)

  • Guillermo Montoya

    (Faculty of Health and Medical Sciences University of Copenhagen)

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.

Suggested Citation

  • Arturo Carabias & Anders Fuglsang & Piero Temperini & Tillmann Pape & Nicholas Sofos & Stefano Stella & Simon Erlendsson & Guillermo Montoya, 2021. "Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3," Nature Communications, Nature, vol. 12(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-24707-3
    DOI: 10.1038/s41467-021-24707-3
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