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

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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.

Original languageEnglish
Article number4476
JournalNature Communications
Number of pages12
Publication statusPublished - 2021

    Research areas

  • Bacteriophages/enzymology, CRISPR-Associated Proteins/chemistry, CRISPR-Cas Systems, Catalytic Domain, Cryoelectron Microscopy, DNA Cleavage, Endodeoxyribonucleases/chemistry, Escherichia coli Proteins/chemistry, Gene Editing, Models, Molecular, Mutagenesis, Site-Directed, Protein Conformation, RNA, Guide/genetics, RNA, Viral/genetics

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