Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3
| Autor: | Guillermo Montoya, Tillmann Pape, Simon Erlendsson, Stefano Stella, Anders Fuglsang, Piero Temperini, Nicholas Sofos, Arturo Carabias |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
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Molecular CRISPR-Cas systems Protein Conformation Science CRISPR-Associated Proteins General Physics and Astronomy CRISPR-Associated Proteins/chemistry Computational biology RNA Guide/genetics General Biochemistry Genetics and Molecular Biology Article 03 medical and health sciences chemistry.chemical_compound Endonuclease 0302 clinical medicine Protein structure Genome editing Catalytic Domain Bacteriophages/enzymology CRISPR Bacteriophages DNA Cleavage 030304 developmental biology Trans-activating crRNA Gene Editing 0303 health sciences Multidisciplinary Endodeoxyribonucleases biology Escherichia coli Proteins Mutagenesis Cryoelectron Microscopy RNA General Chemistry RNA Viral/genetics Endodeoxyribonucleases/chemistry chemistry Escherichia coli Proteins/chemistry biology.protein Mutagenesis Site-Directed RNA Viral CRISPR-Cas Systems Structural biology 030217 neurology & neurosurgery DNA RNA Guide Kinetoplastida |
| Zdroj: | Nature Communications Carabias, A, Fuglsang, A, Temperini, P, Pape, T, Sofos, N, Stella, S, Erlendsson, S & Montoya, G 2021, ' Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3 ', Nature Communications, vol. 12, 4476 . https://doi.org/10.1038/s41467-021-24707-3 Nature Communications, Vol 12, Iss 1, Pp 1-12 (2021) |
| ISSN: | 2041-1723 |
| DOI: | 10.1038/s41467-021-24707-3 |
| Popis: | 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. The Class 2 family of CRISPR nucleases named Cas12j, which shares only low sequence identity with other CRISPR nucleases was recently identified in the biggiephage clade of phages. Here, the authors present the cryo-EM structure of a functional Cas12j3−crRNA complex in the post-catalytic state and discuss Cas12j3 PAM recognition, hybrid stabilisation and the activation mechanism. |
| Databáze: | OpenAIRE |
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