Establishment of an Integrated CRISPR/Cas9 Plasmid System for Simple and Efficient Genome Editing in Medaka In Vitro and In Vivo.
Establishment of an Integrated CRISPR/Cas9 Plasmid System for Simple and Efficient Genome Editing in Medaka In Vitro and In Vivo.
复制标题
建立一个综合的CRIS/CAS9质粒系统，用于在体外和体内Medaka中简单有效的基因组编辑。
作者：
目前，CRISPR/Cas9基因组编辑在鱼类中的应用有限。非常需要一种易于使用、经济有效的基因组编辑系统。在本研究中，我们建立了整合的pCas 9-U6 sgRNA质粒系统，其sgRNA由来自不同鱼类的U6启动子驱动。以青鳉为例，我们的结果表明，由内源U6启动子驱动的pCas 9-U6 sgRNA（pCas 9-mU 6sgRNA）可以非常有效地介导青鳉培养细胞中的基因敲除，而不是由外源U6启动子驱动。pCas 9-mU 6sgRNA的基因编辑效率高达93.7%，没有检测到脱靶。通过单细胞胚胎显微注射，pCas 9-mU 6sgRNA也可以有效地介导体内基因敲除。此外，通过应用pCas 9-mU 6sgRNA，在体外以及体内有效地实现了特定位点处的基因敲入。综上所述，我们成功开发了一种简单、低成本、有效的CRISPR/Cas9系统，适用于青鳉基因的体外和体内特定位点的敲除和敲入。本研究为其他鱼类基因操作提供了新的思路，对功能基因的研究具有重要的推动作用。尽管CRISPR/Cas9已被用于几种鱼类的体内基因操作，但其在鱼类培养细胞中的应用仍然受到挑战和限制。本研究建立了整合的CRISPR/Cas9质粒系统，并在体外和体内评价了该系统对青鳉（Oryzias latipes）特定位点基因敲除或敲入的效率。通过使用增强型绿色荧光蛋白报告质粒pGNtsf 1，我们证明了由内源性U6启动子驱动的pCas 9-mU 6 sgRNA（pCas 9-mU 6 sgRNA）在青鳉培养细胞中介导非常高的基因编辑效率，而不是由外源性U6启动子驱动。优化条件后，计算了8个位点对4个内源基因的基因编辑效率，最高可达94%，且未检测到脱靶。通过单细胞胚胎显微注射，pCas 9-mU 6sgRNA也介导了体内有效的基因敲除。此外，pCas 9-mU 6sgRNA在青鳉培养的细胞以及胚胎中的特定位点有效地介导基因敲入。总之，我们的研究表明，U6启动子的遗传关系是至关重要的基因编辑效率在青鳉培养细胞，并在体外和体内建立了一个简单而有效的青鳉基因组编辑系统。这项研究为其他鱼类基因组编辑提供了深入了解，并促进了基因功能分析。
The application of CRISPR/Cas9 genome editing in fish is limited to date. An easy-to-use, economical and effective genome editing system is greatly needed. In this study, we established an integrated pCas9-U6sgRNA plasmid system, the sgRNA of which was driven by U6 promoters from different fish species. Taking medaka as an example, our results suggest that pCas9-U6sgRNA driven by endogenous U6 promoter (pCas9-mU6sgRNA) can very effectively mediate gene knock-out in medaka cultured cells, but not by exogenous U6 promoter. The gene editing efficiency of pCas9-mU6sgRNA was up to 93.7% with no detectable off-target. By one-cell embryo microinjection, pCas9-mU6sgRNA can effectively mediate gene knock-out in vivo as well. Furthermore, the gene knock-in at a specific site was efficiently achieved in vitro as well as in vivo through application of pCas9-mU6sgRNA. In conclusion, we have successfully developed a simple, low-cost and effective CRISPR/Cas9 system suitable for medaka gene knock-out and knock-in at a specific site in vitro and in vivo. This study provides an insight into other fish gene manipulation and greatly promotes functional gene studies. 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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影响因子：
3
作者：
Dehler, Carola E.;Boudinot, Pierre;Martin, Samuel A. M.;Collet, Bertrand
通讯作者：
Collet, Bertrand
影响因子：
5.6
作者：
Dolskiy AA;Grishchenko IV;Yudkin DV
通讯作者：
Yudkin DV
影响因子：
46.9
作者：
通讯作者：
--
DOI：
10.1093/dnares/dss023
发表时间：
2012-10
期刊：
DNA research : an international journal for rapid publication of reports on genes and genomes
影响因子：
--
作者：
Chen J;Zhang X;Wang T;Li Z;Guan G;Hong Y
通讯作者：
Hong Y
影响因子：
14.9
作者：
Brinkman EK;Chen T;Amendola M;van Steensel B
通讯作者：
van Steensel B