title: Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice.
abstract: 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.
  
  
  RESULTS
  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.
authors.affiliation: Key Laboratory of Rice Genetics Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China ; College of Life Sciences, Anhui University, Hefei 230031, China.
authors.forename: Rongfang
authors.initials: R
authors.lastname: Xu
authors.affiliation: Key Laboratory of Rice Genetics Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China ; Institute of Agricultural Engineering, Anhui Academy of Agricultural Sciences, Hefei 230031, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Li
authors.affiliation: Key Laboratory of Rice Genetics Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China.
authors.forename: Ruiying
authors.initials: R
authors.lastname: Qin
authors.affiliation: Key Laboratory of Rice Genetics Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of Rice Genetics Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China ; College of Life Sciences, Anhui University, Hefei 230031, China.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.affiliation: Key Laboratory of Rice Genetics Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China ; Institute of Agricultural Engineering, Anhui Academy of Agricultural Sciences, Hefei 230031, China.
authors.forename: Pengcheng
authors.initials: P
authors.lastname: Wei
authors.affiliation: Key Laboratory of Rice Genetics Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China.
authors.forename: Jianbo
authors.initials: J
authors.lastname: Yang
keywords: Agrobacterium; CRISPR/Cas9; Gene targeting; Genome editing; Rice; Stable transformation