Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking

Abstract Tandem paired nicking (TPN) is a method of genome editing that enables precise and relatively efficient targeted knock-in without appreciable restraint by p53-mediated DNA damage response. TPN is initiated by introducing two site-specific nicks on the same DNA strand using Cas9 nickases in...

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Autores principales:	Md. Lutfur Rahman, Toshinori Hyodo, Sivasundaram Karnan, Akinobu Ota, Muhammad Nazmul Hasan, Yuko Mihara, Md Wahiduzzaman, Shinobu Tsuzuki, Yoshitaka Hosokawa, Hiroyuki Konishi
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b018edf2c7bd485b94b5fd71b1d3fa74
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spelling	oai:doaj.org-article:b018edf2c7bd485b94b5fd71b1d3fa742021-11-21T12:23:43ZExperimental strategies to achieve efficient targeted knock-in via tandem paired nicking10.1038/s41598-021-01978-w2045-2322https://doaj.org/article/b018edf2c7bd485b94b5fd71b1d3fa742021-11-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-01978-whttps://doaj.org/toc/2045-2322Abstract Tandem paired nicking (TPN) is a method of genome editing that enables precise and relatively efficient targeted knock-in without appreciable restraint by p53-mediated DNA damage response. TPN is initiated by introducing two site-specific nicks on the same DNA strand using Cas9 nickases in such a way that the nicks encompass the knock-in site and are located within a homologous region between a donor DNA and the genome. This nicking design results in the creation of two nicks on the donor DNA and two in the genome, leading to relatively efficient homology-directed recombination between these DNA fragments. In this study, we sought to identify the optimal design of TPN experiments that would improve the efficiency of targeted knock-in, using multiple reporter systems based on exogenous and endogenous genes. We found that efficient targeted knock-in via TPN is supported by the use of 1700–2000-bp donor DNAs, exactly 20-nt-long spacers predicted to be efficient in on-target cleavage, and tandem-paired Cas9 nickases nicking at positions close to each other. These findings will help establish a methodology for efficient and precise targeted knock-in based on TPN, which could broaden the applicability of targeted knock-in to various fields of life science.Md. Lutfur RahmanToshinori HyodoSivasundaram KarnanAkinobu OtaMuhammad Nazmul HasanYuko MiharaMd WahiduzzamanShinobu TsuzukiYoshitaka HosokawaHiroyuki KonishiNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Md. Lutfur Rahman Toshinori Hyodo Sivasundaram Karnan Akinobu Ota Muhammad Nazmul Hasan Yuko Mihara Md Wahiduzzaman Shinobu Tsuzuki Yoshitaka Hosokawa Hiroyuki Konishi Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking
description	Abstract Tandem paired nicking (TPN) is a method of genome editing that enables precise and relatively efficient targeted knock-in without appreciable restraint by p53-mediated DNA damage response. TPN is initiated by introducing two site-specific nicks on the same DNA strand using Cas9 nickases in such a way that the nicks encompass the knock-in site and are located within a homologous region between a donor DNA and the genome. This nicking design results in the creation of two nicks on the donor DNA and two in the genome, leading to relatively efficient homology-directed recombination between these DNA fragments. In this study, we sought to identify the optimal design of TPN experiments that would improve the efficiency of targeted knock-in, using multiple reporter systems based on exogenous and endogenous genes. We found that efficient targeted knock-in via TPN is supported by the use of 1700–2000-bp donor DNAs, exactly 20-nt-long spacers predicted to be efficient in on-target cleavage, and tandem-paired Cas9 nickases nicking at positions close to each other. These findings will help establish a methodology for efficient and precise targeted knock-in based on TPN, which could broaden the applicability of targeted knock-in to various fields of life science.
format	article
author	Md. Lutfur Rahman Toshinori Hyodo Sivasundaram Karnan Akinobu Ota Muhammad Nazmul Hasan Yuko Mihara Md Wahiduzzaman Shinobu Tsuzuki Yoshitaka Hosokawa Hiroyuki Konishi
author_facet	Md. Lutfur Rahman Toshinori Hyodo Sivasundaram Karnan Akinobu Ota Muhammad Nazmul Hasan Yuko Mihara Md Wahiduzzaman Shinobu Tsuzuki Yoshitaka Hosokawa Hiroyuki Konishi
author_sort	Md. Lutfur Rahman
title	Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking
title_short	Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking
title_full	Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking
title_fullStr	Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking
title_full_unstemmed	Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking
title_sort	experimental strategies to achieve efficient targeted knock-in via tandem paired nicking
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/b018edf2c7bd485b94b5fd71b1d3fa74
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Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking

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