Establishment of an Integrated CRISPR/Cas9 Plasmid System for Simple and Efficient Genome Editing in Medaka In Vitro and In Vivo

Although CRISPR/Cas9 has been used in gene manipulation of several fish species in vivo, its application in fish cultured cells is still challenged and limited. In this study, we established an integrated CRISPR/Cas9 plasmid system and evaluated its efficiency of gene knock-out or knock-in at a spec...
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Gespeichert in:

Autor*in:	Zeming Zhang [verfasserIn] Jie Wang [verfasserIn] Jianeng Li [verfasserIn] Xiang Liu [verfasserIn] Lei Liu [verfasserIn] Changle Zhao [verfasserIn] Wenjing Tao [verfasserIn] Deshou Wang [verfasserIn] Jing Wei [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2023

Schlagwörter:	CRISPR/Cas9 knock-out knock-in fish cultured cells U6 promoter medaka

Übergeordnetes Werk:	In: Biology - MDPI AG, 2012, 12(2023), 2, p 336
Übergeordnetes Werk:	volume:12 ; year:2023 ; number:2, p 336

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.3390/biology12020336

Katalog-ID:	DOAJ081005989

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authorswithroles_txt_mv	Zeming Zhang @@aut@@ Jie Wang @@aut@@ Jianeng Li @@aut@@ Xiang Liu @@aut@@ Lei Liu @@aut@@ Changle Zhao @@aut@@ Wenjing Tao @@aut@@ Deshou Wang @@aut@@ Jing Wei @@aut@@
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callnumber-first	Q - Science
author	Zeming Zhang
spellingShingle	Zeming Zhang misc QH301-705.5 misc CRISPR/Cas9 misc knock-out misc knock-in misc fish cultured cells misc U6 promoter misc medaka misc Biology (General) Establishment of an Integrated CRISPR/Cas9 Plasmid System for Simple and Efficient Genome Editing in Medaka In Vitro and In Vivo
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topic_title	QH301-705.5 Establishment of an Integrated CRISPR/Cas9 Plasmid System for Simple and Efficient Genome Editing in Medaka In Vitro and In Vivo CRISPR/Cas9 knock-out knock-in fish cultured cells U6 promoter medaka
topic	misc QH301-705.5 misc CRISPR/Cas9 misc knock-out misc knock-in misc fish cultured cells misc U6 promoter misc medaka misc Biology (General)
topic_unstemmed	misc QH301-705.5 misc CRISPR/Cas9 misc knock-out misc knock-in misc fish cultured cells misc U6 promoter misc medaka misc Biology (General)
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title	Establishment of an Integrated CRISPR/Cas9 Plasmid System for Simple and Efficient Genome Editing in Medaka In Vitro and In Vivo
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title_full	Establishment of an Integrated CRISPR/Cas9 Plasmid System for Simple and Efficient Genome Editing in Medaka In Vitro and In Vivo
author_sort	Zeming Zhang
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author_browse	Zeming Zhang Jie Wang Jianeng Li Xiang Liu Lei Liu Changle Zhao Wenjing Tao Deshou Wang Jing Wei
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title_sort	establishment of an integrated crispr/cas9 plasmid system for simple and efficient genome editing in medaka in vitro and in vivo
callnumber	QH301-705.5
title_auth	Establishment of an Integrated CRISPR/Cas9 Plasmid System for Simple and Efficient Genome Editing in Medaka In Vitro and In Vivo
abstract	Although CRISPR/Cas9 has been used in gene manipulation of several fish species in vivo, its application in fish cultured cells is still challenged and limited. In this study, we established an integrated CRISPR/Cas9 plasmid system and evaluated its efficiency of gene knock-out or knock-in at a specific site in medaka (Oryzias latipes) in vitro and in vivo. By using the enhanced green fluorescent protein reporter plasmid pGNtsf1, we demonstrate that pCas9-U6sgRNA driven by endogenous U6 promoter (pCas9-mU6sgRNA) mediated very high gene editing efficiency in medaka cultured cells, but not by exogenous U6 promoters. After optimizing the conditions, the gene editing efficiencies of eight sites targeting for four endogenous genes were calculated, and the highest was up to 94% with no detectable off-target. By one-cell embryo microinjection, pCas9-mU6sgRNA also mediated efficient gene knock-out in vivo. Furthermore, pCas9-mU6sgRNA efficiently mediated gene knock-in at a specific site in medaka cultured cells as well as embryos. Collectively, our study demonstrates that the genetic relationship of U6 promoter is critical to gene editing efficiency in medaka cultured cells, and a simple and efficient system for medaka genome editing in vitro and in vivo has been established. This study provides an insight into other fish genome editing and promotes gene functional analysis.
abstractGer	Although CRISPR/Cas9 has been used in gene manipulation of several fish species in vivo, its application in fish cultured cells is still challenged and limited. In this study, we established an integrated CRISPR/Cas9 plasmid system and evaluated its efficiency of gene knock-out or knock-in at a specific site in medaka (Oryzias latipes) in vitro and in vivo. By using the enhanced green fluorescent protein reporter plasmid pGNtsf1, we demonstrate that pCas9-U6sgRNA driven by endogenous U6 promoter (pCas9-mU6sgRNA) mediated very high gene editing efficiency in medaka cultured cells, but not by exogenous U6 promoters. After optimizing the conditions, the gene editing efficiencies of eight sites targeting for four endogenous genes were calculated, and the highest was up to 94% with no detectable off-target. By one-cell embryo microinjection, pCas9-mU6sgRNA also mediated efficient gene knock-out in vivo. Furthermore, pCas9-mU6sgRNA efficiently mediated gene knock-in at a specific site in medaka cultured cells as well as embryos. Collectively, our study demonstrates that the genetic relationship of U6 promoter is critical to gene editing efficiency in medaka cultured cells, and a simple and efficient system for medaka genome editing in vitro and in vivo has been established. This study provides an insight into other fish genome editing and promotes gene functional analysis.
abstract_unstemmed	Although CRISPR/Cas9 has been used in gene manipulation of several fish species in vivo, its application in fish cultured cells is still challenged and limited. In this study, we established an integrated CRISPR/Cas9 plasmid system and evaluated its efficiency of gene knock-out or knock-in at a specific site in medaka (Oryzias latipes) in vitro and in vivo. By using the enhanced green fluorescent protein reporter plasmid pGNtsf1, we demonstrate that pCas9-U6sgRNA driven by endogenous U6 promoter (pCas9-mU6sgRNA) mediated very high gene editing efficiency in medaka cultured cells, but not by exogenous U6 promoters. After optimizing the conditions, the gene editing efficiencies of eight sites targeting for four endogenous genes were calculated, and the highest was up to 94% with no detectable off-target. By one-cell embryo microinjection, pCas9-mU6sgRNA also mediated efficient gene knock-out in vivo. Furthermore, pCas9-mU6sgRNA efficiently mediated gene knock-in at a specific site in medaka cultured cells as well as embryos. Collectively, our study demonstrates that the genetic relationship of U6 promoter is critical to gene editing efficiency in medaka cultured cells, and a simple and efficient system for medaka genome editing in vitro and in vivo has been established. This study provides an insight into other fish genome editing and promotes gene functional analysis.
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title_short	Establishment of an Integrated CRISPR/Cas9 Plasmid System for Simple and Efficient Genome Editing in Medaka In Vitro and In Vivo
url	https://doi.org/10.3390/biology12020336 https://doaj.org/article/12658b1c643244e6a20b2bb0c0bc8ef5 https://www.mdpi.com/2079-7737/12/2/336 https://doaj.org/toc/2079-7737
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author2	Jie Wang Jianeng Li Xiang Liu Lei Liu Changle Zhao Wenjing Tao Deshou Wang Jing Wei
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Establishment of an Integrated CRISPR/Cas9 Plasmid System for Simple and Efficient Genome Editing in Medaka In Vitro and In Vivo

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