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

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 stru...

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Autores principales: Carabias, Arturo, Fuglsang, Anders, Temperini, Piero, Pape, Tillmann, Sofos, Nicholas, Stella, Stefano, Erlendsson, Simon, Montoya, Guillermo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8298400/
https://www.ncbi.nlm.nih.gov/pubmed/34294706
http://dx.doi.org/10.1038/s41467-021-24707-3
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author Carabias, Arturo
Fuglsang, Anders
Temperini, Piero
Pape, Tillmann
Sofos, Nicholas
Stella, Stefano
Erlendsson, Simon
Montoya, Guillermo
author_facet Carabias, Arturo
Fuglsang, Anders
Temperini, Piero
Pape, Tillmann
Sofos, Nicholas
Stella, Stefano
Erlendsson, Simon
Montoya, Guillermo
author_sort Carabias, Arturo
collection PubMed
description 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.
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spelling pubmed-82984002021-08-12 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 Nat Commun Article 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. Nature Publishing Group UK 2021-07-22 /pmc/articles/PMC8298400/ /pubmed/34294706 http://dx.doi.org/10.1038/s41467-021-24707-3 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Carabias, Arturo
Fuglsang, Anders
Temperini, Piero
Pape, Tillmann
Sofos, Nicholas
Stella, Stefano
Erlendsson, Simon
Montoya, Guillermo
Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3
title Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3
title_full Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3
title_fullStr Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3
title_full_unstemmed Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3
title_short Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3
title_sort structure of the mini-rna-guided endonuclease crispr-cas12j3
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8298400/
https://www.ncbi.nlm.nih.gov/pubmed/34294706
http://dx.doi.org/10.1038/s41467-021-24707-3
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