Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3
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Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3. / Carabias, Arturo; Fuglsang, Anders; Temperini, Piero; Pape, Tillmann; Sofos, Nicholas; Stella, Stefano; Erlendsson, Simon; Montoya, Guillermo.
In: Nature Communications, Vol. 12, 4476, 2021.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3
AU - Carabias, Arturo
AU - Fuglsang, Anders
AU - Temperini, Piero
AU - Pape, Tillmann
AU - Sofos, Nicholas
AU - Stella, Stefano
AU - Erlendsson, Simon
AU - Montoya, Guillermo
N1 - © 2021. The Author(s).
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
KW - Bacteriophages/enzymology
KW - CRISPR-Associated Proteins/chemistry
KW - CRISPR-Cas Systems
KW - Catalytic Domain
KW - Cryoelectron Microscopy
KW - DNA Cleavage
KW - Endodeoxyribonucleases/chemistry
KW - Escherichia coli Proteins/chemistry
KW - Gene Editing
KW - Models, Molecular
KW - Mutagenesis, Site-Directed
KW - Protein Conformation
KW - RNA, Guide/genetics
KW - RNA, Viral/genetics
U2 - 10.1038/s41467-021-24707-3
DO - 10.1038/s41467-021-24707-3
M3 - Journal article
C2 - 34294706
VL - 12
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
M1 - 4476
ER -
ID: 275949393