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Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice |
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| Authors: | Rongfang Xu Hao Li Ruiying Qin Lu Wang Li Li Pengcheng Wei Jianbo Yang |
| Institution: | 1. Key Laboratory of Rice Genetics Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, 230031, China 3. College of Life Sciences, Anhui University, Hefei, 230031, China 2. Institute of Agricultural Engineering, Anhui Academy of Agricultural Sciences, Hefei, 230031, China |
| Abstract: | BackgroundThe 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.FindingsHere, 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 ~2% to ~16%. Furthermore, phenotypic analysis revealed that the biallelic mutated transgenic plant was sensitive to bentazon.ConclusionsOur 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. |
| Keywords: | Gene targeting CRISPR/Cas9 Rice Agrobacterium Stable transformation Genome editing |
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