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Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage

Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR–Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC domain. In this study, we determined the crystal structure of apo-Cas12g, the cryo-EM structure of the C...

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Autores principales: Liu, Mengxi, Li, Zekai, Chen, Jing, Lin, Jinying, Lu, Qiuhua, Ye, Yangmiao, Zhang, Hongmin, Zhang, Bo, Ouyang, Songying
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10511118/
https://www.ncbi.nlm.nih.gov/pubmed/37729124
http://dx.doi.org/10.1371/journal.pgen.1010930
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author Liu, Mengxi
Li, Zekai
Chen, Jing
Lin, Jinying
Lu, Qiuhua
Ye, Yangmiao
Zhang, Hongmin
Zhang, Bo
Ouyang, Songying
author_facet Liu, Mengxi
Li, Zekai
Chen, Jing
Lin, Jinying
Lu, Qiuhua
Ye, Yangmiao
Zhang, Hongmin
Zhang, Bo
Ouyang, Songying
author_sort Liu, Mengxi
collection PubMed
description Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR–Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC domain. In this study, we determined the crystal structure of apo-Cas12g, the cryo-EM structure of the Cas12g-sgRNA binary complex and investigated conformational changes that occur during the transition from the apo state to the Cas12g-sgRNA binary complex. The conserved zinc finger motifs in Cas12g undergo an ordered-to-disordered transition from the apo to the sgRNA-bound state and their mutations negatively impact on target RNA cleavage. Moreover, we identified a lid motif in the RuvC domain that undergoes transformation from a helix to loop to regulate the access to the RuvC active site and subsequent cleavage of the RNA substrate. Overall, our study provides valuable insights into the mechanisms by which Cas12g recognizes sgRNA and the conformational changes it undergoes from sgRNA binding to the activation of the RNase active site, thereby laying a foundation for the potential repurposing of Cas12g as a tool for RNA-editing.
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spelling pubmed-105111182023-09-21 Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage Liu, Mengxi Li, Zekai Chen, Jing Lin, Jinying Lu, Qiuhua Ye, Yangmiao Zhang, Hongmin Zhang, Bo Ouyang, Songying PLoS Genet Research Article Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR–Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC domain. In this study, we determined the crystal structure of apo-Cas12g, the cryo-EM structure of the Cas12g-sgRNA binary complex and investigated conformational changes that occur during the transition from the apo state to the Cas12g-sgRNA binary complex. The conserved zinc finger motifs in Cas12g undergo an ordered-to-disordered transition from the apo to the sgRNA-bound state and their mutations negatively impact on target RNA cleavage. Moreover, we identified a lid motif in the RuvC domain that undergoes transformation from a helix to loop to regulate the access to the RuvC active site and subsequent cleavage of the RNA substrate. Overall, our study provides valuable insights into the mechanisms by which Cas12g recognizes sgRNA and the conformational changes it undergoes from sgRNA binding to the activation of the RNase active site, thereby laying a foundation for the potential repurposing of Cas12g as a tool for RNA-editing. Public Library of Science 2023-09-20 /pmc/articles/PMC10511118/ /pubmed/37729124 http://dx.doi.org/10.1371/journal.pgen.1010930 Text en © 2023 Liu et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Liu, Mengxi
Li, Zekai
Chen, Jing
Lin, Jinying
Lu, Qiuhua
Ye, Yangmiao
Zhang, Hongmin
Zhang, Bo
Ouyang, Songying
Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage
title Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage
title_full Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage
title_fullStr Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage
title_full_unstemmed Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage
title_short Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage
title_sort structural transitions upon guide rna binding and their importance in cas12g-mediated rna cleavage
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10511118/
https://www.ncbi.nlm.nih.gov/pubmed/37729124
http://dx.doi.org/10.1371/journal.pgen.1010930
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