Recovery of the non-functional EGFP-assisted identification of mutants generated by CRISPR/Cas9

doi:10.1007/s00299-019-02465-3

	Recovery of the non-functional EGFP-assisted identification of mutants generated by CRISPR/Cas9
	Ren, Chong 1,2; Guo, Yuchen 1,2; Gathunga, Elias Kirabi 1,2,3; Duan, Wei 1; Li, Shaohua1 ; Liang, Zhenchang 1,3
	2019
发表期刊	PLANT CELL REPORTS
ISSN	0721-7714
卷号	38 期号:12 页码:1541-1549
摘要	Key message The recovery of non-functional-enhanced green fluorescence protein can be used as indicator to facilitate the identification of mutants generated by CRISPR/Cas9. The CRISPR/Cas9 system is a powerful tool for genome editing and it has been employed to knock out genes of interest in multiple plant species. Identification of desired mutants from regenerated plants is necessary prior to functional study. Current screening methods work based on the purification of genomic DNA and it would be laborious and time consuming using these methods to screen mutants from a large population of seedlings. Here, we developed the non-functional enhanced green fluorescence protein (nEGFP) reporter gene by inserting a single guide RNA (sgRNA) and the protospacer adjacent motif in the 5 ' coding region of EGFP, and the activity of nEGFP could be recovered after successful targeted editing. Using the nEGFP as the reporter gene in Nicotiana tabacum, we found that over 94% of the plants exhibiting EGFP fluorescence were confirmed to be desired mutants. The use of this nEGFP reporter construct had limited negative effect on editing efficiency, and the expression of Cas9 and sgRNA was not affected. Moreover, this method was also applied in grape by targeting the phytoene desaturase gene (PDS), and the grape cells with EGFP signal were revealed to contain targeted mutations in VvPDS. Our results show that the nEGFP gene can be used as reporter to help screen mutants according to the recovered EGFP fluorescence during the application of CRISPR/Cas9 in plants.
关键词	Selection of mutants CRISPR Cas9 Non-functional EGFP Reporter gene
学科领域	Plant Sciences
DOI	10.1007/s00299-019-02465-3
收录类别	SCI
语种	英语
WOS关键词	TARGETED MUTAGENESIS ; CLEAVAGE ; SYSTEMS ; CAS9 ; PCR
WOS研究方向	Plant Sciences
WOS记录号	WOS:000493659000008
出版者	SPRINGER
文献子类	Article
出版地	NEW YORK
EISSN	1432-203X
资助机构	major science and technology program of NingxiaHui Autonomous region [2016BZ06] ; National Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [31772266] ; STS project of Chinese Academy of Sciences [KFJ-STS-ZDTP-025] ; Agricultural Breeding Project of Ningxia Hui Autonomous Region [NXNYYZ20150203]
作者邮箱	rcarthur@126.com ; GYC1477570428@163.com ; elias@ibcas.ac.cn ; wduan@ibcas.ac.cn ; shhli@ibcas.ac.cn ; zl249@ibcas.ac.cn
引用统计	被引频次：6[WOS] [WOS记录] [WOS相关记录]
文献类型	期刊论文
条目标识符	http://ir.ibcas.ac.cn/handle/2S10CLM1/19781
专题	中科院北方资源植物重点实验室
作者单位	1.Chinese Acad Sci, Innovat Acad Seed Design, Inst Bot, Beijing Key Lab Grape Sci & Enol, Nanxin Village 20, Beijing 100093, Peoples R China 2.Chinese Acad Sci, Innovat Acad Seed Design, Inst Bot, CAS Key Lab Plant Resources, Nanxin Village 20, Beijing 100093, Peoples R China 3.Univ Chinese Acad Sci, Beijing 100049, Peoples R China 4.Chinese Acad Sci, Sino Africa Joint Res Ctr, Wuhan 430074, Hubei, Peoples R China
推荐引用方式 GB/T 7714	Ren, Chong,Guo, Yuchen,Gathunga, Elias Kirabi,et al. Recovery of the non-functional EGFP-assisted identification of mutants generated by CRISPR/Cas9[J]. PLANT CELL REPORTS,2019,38(12):1541-1549.
APA	Ren, Chong,Guo, Yuchen,Gathunga, Elias Kirabi,Duan, Wei,Li, Shaohua,&Liang, Zhenchang.(2019).Recovery of the non-functional EGFP-assisted identification of mutants generated by CRISPR/Cas9.PLANT CELL REPORTS,38(12),1541-1549.
MLA	Ren, Chong,et al."Recovery of the non-functional EGFP-assisted identification of mutants generated by CRISPR/Cas9".PLANT CELL REPORTS 38.12(2019):1541-1549.

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