Efficiency Optimization of CRISPR/Cas9-Mediated Targeted Mutagenesis in Grape

doi:10.3389/fpls.2019.00612

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	Efficiency Optimization of CRISPR/Cas9-Mediated Targeted Mutagenesis in Grape
	Ren, Fengrui 2; Ren, Chong 2; Zhang, Zhan 2; Duan, Wei 2; Lecourieux, David 3; Li, Shaohua 2; Liang, Zhenchang 4
刊名	FRONTIERS IN PLANT SCIENCE
	2019
卷号	10
关键词	CRISPR/Cas9 optimization GC content grape gene expression gene editing efficiency
ISSN号	1664-462X
DOI	10.3389/fpls.2019.00612
文献子类	Article
英文摘要	Clustered regularly interspersed short palindromic repeats (CRISPR)/Cas system is an efficient targeted genome editing method. Although CRISPR/Cas9-mediated mutagenesis has been applied successfully in grape, few studies have examined the technique's efficiency. To optimize CRISPR/Cas9 editing efficiency in Vitis vinifera, we surveyed three key parameters: GC content of single guide RNA (sgRNA), variety of transformant cells used, and SpCas9 expression levels in transgenic cell mass. Four sgRNAs with differing GC content were designed to target exon sites of the V. vinifera phytoene desaturase gene. Suspension cells of 'Chardonnay' and '41B' varieties were used as the transgenic cell mass. Both T7EI and PCR/RE assays showed that CRISPR/Cas9 editing efficiency increases proportionally with sgRNA GC content with 65% GC content yielding highest editing efficiency in both varieties. Additionally, gene editing was more efficient in '41B' than in 'Chardonnay.' CRISPR/Cas9 systems with different editing efficiency showed different SpCas9 expression level, but compared with GC content of sgRNA, SpCas9 expression level has less influence on editing efficiency. Taken together, these results help optimize of CRISPR/Cas9 performance in grape.
学科主题	Plant Sciences
出版地	LAUSANNE
WOS关键词	GENETIC-TRANSFORMATION ; GENOME ; PLANTS ; ARABIDOPSIS ; GENERATION ; MULTIPLEX ; BINDING
WOS研究方向	Plant Sciences
语种	英语
出版者	FRONTIERS MEDIA SA
WOS记录号	WOS:000468131600001
资助机构	National Key Research and Development Program of China ; STS project of Chinese Academy of Sciences [KFJ-STS-ZDTP-025] ; Major Science and Technology Program of Ningxia Hui Autonomous Region [2016BZ06] ; Agricultural Breeding Project of Ningxia Hui Autonomous Region [NXNYYZ20150203] ; National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [31772266]
内容类型	期刊论文
源URL	[http://ir.ibcas.ac.cn/handle/2S10CLM1/19605]
专题	中科院北方资源植物重点实验室
作者单位	1.Chinese Acad Sci, Sino Africa Joint Res Ctr, Wuhan, Hubei, Peoples R China 2.Chinese Acad Sci, Inst Bot, Lab Plant Resources, Beijing Key Lab Grape Sci & Enol, Beijing, Peoples R China 3.Univ Chinese Acad Sci, Coll Life Sci, Beijing, Peoples R China 4.Univ Bordeaux, INRA, Bordeaux Sci Agro, EGFV, Villenave Dornon, France
推荐引用方式 GB/T 7714	Ren, Fengrui,Ren, Chong,Zhang, Zhan,et al. Efficiency Optimization of CRISPR/Cas9-Mediated Targeted Mutagenesis in Grape[J]. FRONTIERS IN PLANT SCIENCE,2019,10.
APA	Ren, Fengrui.,Ren, Chong.,Zhang, Zhan.,Duan, Wei.,Lecourieux, David.,...&Liang, Zhenchang.(2019).Efficiency Optimization of CRISPR/Cas9-Mediated Targeted Mutagenesis in Grape.FRONTIERS IN PLANT SCIENCE,10.
MLA	Ren, Fengrui,et al."Efficiency Optimization of CRISPR/Cas9-Mediated Targeted Mutagenesis in Grape".FRONTIERS IN PLANT SCIENCE 10(2019).