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Staphylococcus aureus Cas9 is a multiple-turnover enzyme

Cas9 nuclease is the key effector of type II CRISPR adaptive immune systems found in bacteria. The nuclease can be programmed by a single guide RNA (sgRNA) to cleave DNA in a sequence-specific manner. This property has led to its widespread adoption as a genome editing tool in research laboratories...

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Autores principales: Yourik, Paul, Fuchs, Ryan T., Mabuchi, Megumu, Curcuru, Jennifer L., Robb, G. Brett
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298560/
https://www.ncbi.nlm.nih.gov/pubmed/30348755
http://dx.doi.org/10.1261/rna.067355.118
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author Yourik, Paul
Fuchs, Ryan T.
Mabuchi, Megumu
Curcuru, Jennifer L.
Robb, G. Brett
author_facet Yourik, Paul
Fuchs, Ryan T.
Mabuchi, Megumu
Curcuru, Jennifer L.
Robb, G. Brett
author_sort Yourik, Paul
collection PubMed
description Cas9 nuclease is the key effector of type II CRISPR adaptive immune systems found in bacteria. The nuclease can be programmed by a single guide RNA (sgRNA) to cleave DNA in a sequence-specific manner. This property has led to its widespread adoption as a genome editing tool in research laboratories and holds great promise for biotechnological and therapeutic applications. The general mechanistic features of catalysis by Cas9 homologs are comparable; however, a high degree of diversity exists among the protein sequences, which may result in subtle mechanistic differences. S. aureus (SauCas9) and especially S. pyogenes (SpyCas9) are among the best-characterized Cas9 proteins and share ∼17% sequence identity. A notable feature of SpyCas9 is an extremely slow rate of reaction turnover, which is thought to limit the amount of substrate DNA cleavage. Using in vitro biochemistry and enzyme kinetics, we directly compare SpyCas9 and SauCas9 activities. Here, we report that in contrast to SpyCas9, SauCas9 is a multiple-turnover enzyme, which to our knowledge is the first report of such activity in a Cas9 homolog. We also show that DNA cleaved with SauCas9 does not undergo any detectable single-stranded degradation after the initial double-stranded break observed previously with SpyCas9, thus providing new insights and considerations for future design of CRISPR/Cas9-based applications.
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spelling pubmed-62985602019-01-01 Staphylococcus aureus Cas9 is a multiple-turnover enzyme Yourik, Paul Fuchs, Ryan T. Mabuchi, Megumu Curcuru, Jennifer L. Robb, G. Brett RNA Report Cas9 nuclease is the key effector of type II CRISPR adaptive immune systems found in bacteria. The nuclease can be programmed by a single guide RNA (sgRNA) to cleave DNA in a sequence-specific manner. This property has led to its widespread adoption as a genome editing tool in research laboratories and holds great promise for biotechnological and therapeutic applications. The general mechanistic features of catalysis by Cas9 homologs are comparable; however, a high degree of diversity exists among the protein sequences, which may result in subtle mechanistic differences. S. aureus (SauCas9) and especially S. pyogenes (SpyCas9) are among the best-characterized Cas9 proteins and share ∼17% sequence identity. A notable feature of SpyCas9 is an extremely slow rate of reaction turnover, which is thought to limit the amount of substrate DNA cleavage. Using in vitro biochemistry and enzyme kinetics, we directly compare SpyCas9 and SauCas9 activities. Here, we report that in contrast to SpyCas9, SauCas9 is a multiple-turnover enzyme, which to our knowledge is the first report of such activity in a Cas9 homolog. We also show that DNA cleaved with SauCas9 does not undergo any detectable single-stranded degradation after the initial double-stranded break observed previously with SpyCas9, thus providing new insights and considerations for future design of CRISPR/Cas9-based applications. Cold Spring Harbor Laboratory Press 2019-01 /pmc/articles/PMC6298560/ /pubmed/30348755 http://dx.doi.org/10.1261/rna.067355.118 Text en © 2019 Yourik et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by/4.0/ This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.
spellingShingle Report
Yourik, Paul
Fuchs, Ryan T.
Mabuchi, Megumu
Curcuru, Jennifer L.
Robb, G. Brett
Staphylococcus aureus Cas9 is a multiple-turnover enzyme
title Staphylococcus aureus Cas9 is a multiple-turnover enzyme
title_full Staphylococcus aureus Cas9 is a multiple-turnover enzyme
title_fullStr Staphylococcus aureus Cas9 is a multiple-turnover enzyme
title_full_unstemmed Staphylococcus aureus Cas9 is a multiple-turnover enzyme
title_short Staphylococcus aureus Cas9 is a multiple-turnover enzyme
title_sort staphylococcus aureus cas9 is a multiple-turnover enzyme
topic Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298560/
https://www.ncbi.nlm.nih.gov/pubmed/30348755
http://dx.doi.org/10.1261/rna.067355.118
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