Engineering of the genome editing protein Cas9 to slide along DNA

Abstract The genome editing protein Cas9 faces engineering challenges in improving off–target DNA cleavage and low editing efficiency. In this study, we aimed to engineer Cas9 to be able to slide along DNA, which might facilitate genome editing and reduce off-target cleavage. We used two approaches...

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Autores principales:	Trishit Banerjee, Hiroto Takahashi, Dwiky Rendra Graha Subekti, Kiyoto Kamagata
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/8870460c1a504a5ab9a7fc0004817093
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spelling	oai:doaj.org-article:8870460c1a504a5ab9a7fc00048170932021-12-02T16:14:46ZEngineering of the genome editing protein Cas9 to slide along DNA10.1038/s41598-021-93685-92045-2322https://doaj.org/article/8870460c1a504a5ab9a7fc00048170932021-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-93685-9https://doaj.org/toc/2045-2322Abstract The genome editing protein Cas9 faces engineering challenges in improving off–target DNA cleavage and low editing efficiency. In this study, we aimed to engineer Cas9 to be able to slide along DNA, which might facilitate genome editing and reduce off-target cleavage. We used two approaches to achieve this: reducing the sliding friction along DNA by removing the interactions of Cas9 residues with DNA and facilitating sliding by introducing the sliding-promoting tail of Nhp6A. Seven engineered mutants of Cas9 were prepared, and their performance was tested using single-molecule fluorescence microscopy. Comparison of the mutations enabled the identification of key residues of Cas9 to enhance the sliding along DNA in the presence and absence of single guide RNA (sgRNA). The attachment of the tail to Cas9 mutants enhanced sliding along DNA, particularly in the presence of sgRNA. Together, using the proposed approaches, the sliding ability of Cas9 was improved up to eightfold in the presence of sgRNA. A sliding model of Cas9 and its engineering action are discussed herein.Trishit BanerjeeHiroto TakahashiDwiky Rendra Graha SubektiKiyoto KamagataNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Trishit Banerjee Hiroto Takahashi Dwiky Rendra Graha Subekti Kiyoto Kamagata Engineering of the genome editing protein Cas9 to slide along DNA
description	Abstract The genome editing protein Cas9 faces engineering challenges in improving off–target DNA cleavage and low editing efficiency. In this study, we aimed to engineer Cas9 to be able to slide along DNA, which might facilitate genome editing and reduce off-target cleavage. We used two approaches to achieve this: reducing the sliding friction along DNA by removing the interactions of Cas9 residues with DNA and facilitating sliding by introducing the sliding-promoting tail of Nhp6A. Seven engineered mutants of Cas9 were prepared, and their performance was tested using single-molecule fluorescence microscopy. Comparison of the mutations enabled the identification of key residues of Cas9 to enhance the sliding along DNA in the presence and absence of single guide RNA (sgRNA). The attachment of the tail to Cas9 mutants enhanced sliding along DNA, particularly in the presence of sgRNA. Together, using the proposed approaches, the sliding ability of Cas9 was improved up to eightfold in the presence of sgRNA. A sliding model of Cas9 and its engineering action are discussed herein.
format	article
author	Trishit Banerjee Hiroto Takahashi Dwiky Rendra Graha Subekti Kiyoto Kamagata
author_facet	Trishit Banerjee Hiroto Takahashi Dwiky Rendra Graha Subekti Kiyoto Kamagata
author_sort	Trishit Banerjee
title	Engineering of the genome editing protein Cas9 to slide along DNA
title_short	Engineering of the genome editing protein Cas9 to slide along DNA
title_full	Engineering of the genome editing protein Cas9 to slide along DNA
title_fullStr	Engineering of the genome editing protein Cas9 to slide along DNA
title_full_unstemmed	Engineering of the genome editing protein Cas9 to slide along DNA
title_sort	engineering of the genome editing protein cas9 to slide along dna
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/8870460c1a504a5ab9a7fc0004817093
work_keys_str_mv	AT trishitbanerjee engineeringofthegenomeeditingproteincas9toslidealongdna AT hirototakahashi engineeringofthegenomeeditingproteincas9toslidealongdna AT dwikyrendragrahasubekti engineeringofthegenomeeditingproteincas9toslidealongdna AT kiyotokamagata engineeringofthegenomeeditingproteincas9toslidealongdna
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Engineering of the genome editing protein Cas9 to slide along DNA

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