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Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice

文献类型： 外文期刊

作者： Xu, Rongfang 1 ; Li, Hao 1 ; Qin, Ruiying 1 ; Wang, Lu 1 ; Li, Li 1 ; Wei, Pengcheng 1 ; Yang, Jianbo 1 ;

作者机构： 1.Anhui Acad Agr Sci, Rice Res Inst, Key Lab Rice Genet Breeding Anhui Prov, Hefei 230031, Peoples R China

2.Anhui Acad Agr Sci, Inst Agr Engn, Hefei 230031, Peoples R China

3.Anhui Univ, Coll Life Sci, Hefei 230031, Peoples R China

关键词： Gene targeting;CRISPR/Cas9;Rice;Agrobacterium;Stable transformation;Genome editing

期刊名称：RICE （ 影响因子：4.783； 五年影响因子：5.23 ）

ISSN： 1939-8425

年卷期： 2014 年 7 卷

页码：

收录情况： SCI

摘要： Background: The type II clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a novel molecular tool for site-specific genome modification. The CRISPR-Cas9 system was recently introduced into plants by transient or stable transformation. Findings: Here, we report gene targeting in rice via the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system. Three 20-nt CRISPR RNAs were designed to pair with diverse sites followed by the protospacer adjacent motif (PAM) of the rice herbicide resistance gene BEL. After integrating the single-guide RNA (sgRNA) and Cas9 cassette in a single binary vector, transgenic rice plants harboring sgRNA: Cas9 were generated by A. tumefaciens-mediated stable transformation. By analyzing the targeting site on the genome of corresponding transgenic plants, the mutations were determined. The mutagenesis efficiency was varied from similar to 2% to similar to 16%. Furthermore, phenotypic analysis revealed that the biallelic mutated transgenic plant was sensitive to bentazon. Conclusions: Our results indicate that the agricultural trait could be purposely modified by sgRNA:Cas9-induced gene targeting. CRISPR-Cas9 system could be exploited as a powerful tool for trait improvements in crop breeding.

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