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

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 wa...

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Autores principales: Rahman, Md. Lutfur, Hyodo, Toshinori, Karnan, Sivasundaram, Ota, Akinobu, Hasan, Muhammad Nazmul, Mihara, Yuko, Wahiduzzaman, Md, Tsuzuki, Shinobu, Hosokawa, Yoshitaka, Konishi, Hiroyuki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8604973/
https://www.ncbi.nlm.nih.gov/pubmed/34799652
http://dx.doi.org/10.1038/s41598-021-01978-w
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author Rahman, Md. Lutfur
Hyodo, Toshinori
Karnan, Sivasundaram
Ota, Akinobu
Hasan, Muhammad Nazmul
Mihara, Yuko
Wahiduzzaman, Md
Tsuzuki, Shinobu
Hosokawa, Yoshitaka
Konishi, Hiroyuki
author_facet Rahman, Md. Lutfur
Hyodo, Toshinori
Karnan, Sivasundaram
Ota, Akinobu
Hasan, Muhammad Nazmul
Mihara, Yuko
Wahiduzzaman, Md
Tsuzuki, Shinobu
Hosokawa, Yoshitaka
Konishi, Hiroyuki
author_sort Rahman, Md. Lutfur
collection PubMed
description 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.
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spelling pubmed-86049732021-11-22 Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking Rahman, Md. Lutfur Hyodo, Toshinori Karnan, Sivasundaram Ota, Akinobu Hasan, Muhammad Nazmul Mihara, Yuko Wahiduzzaman, Md Tsuzuki, Shinobu Hosokawa, Yoshitaka Konishi, Hiroyuki Sci Rep Article 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. Nature Publishing Group UK 2021-11-19 /pmc/articles/PMC8604973/ /pubmed/34799652 http://dx.doi.org/10.1038/s41598-021-01978-w 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Rahman, Md. Lutfur
Hyodo, Toshinori
Karnan, Sivasundaram
Ota, Akinobu
Hasan, Muhammad Nazmul
Mihara, Yuko
Wahiduzzaman, Md
Tsuzuki, Shinobu
Hosokawa, Yoshitaka
Konishi, Hiroyuki
Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking
title 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_short 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
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8604973/
https://www.ncbi.nlm.nih.gov/pubmed/34799652
http://dx.doi.org/10.1038/s41598-021-01978-w
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