Journal: Nat Commun / Year: 2021 Title: Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3. Authors: Arturo Carabias / Anders Fuglsang / Piero Temperini / Tillmann Pape / Nicholas Sofos / Stefano Stella / Simon Erlendsson / Guillermo Montoya / 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.
Evidence: gel filtration, assay for oligomerization, mass photometry
Type
Name
Symmetry operation
Number
identity operation
1_555
1
Buried area
15320 Å2
ΔGint
-95 kcal/mol
Surface area
41180 Å2
-
Components
-
DNA chain , 3 types, 3 molecules EGC
#2: DNA chain
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')
Mass: 7288.730 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain
DNA (5'-D(P*GP*TP*AP*AP*TP*TP*CP*AP*G)-3')
Mass: 2754.835 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain
DNA (5'-D(P*GP*G)-3')
Mass: 613.454 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
-
Protein / RNA chain , 2 types, 2 molecules AF
#1: Protein
Cas_phi3
Mass: 87411.664 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Phage #D (virus) / Production host: Escherichia coli (E. coli)
#5: RNA chain
RNA (43-MER)
Mass: 13849.232 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
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