Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice
Journal Article / 2014
Xu, Rongfang; Li, Hao; Qin, Ruiying; Wang, Lu; Li, Li; Wei, Pengcheng; Yang, Jianbo
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 ~2% 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.
|Plant Species||GE Technique
Type of Alteration
|Progress in Research
Wei, Peng-Chang; Yang, Jian-Bo