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.
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.
Rice (N Y). 2014 May 2;7(1):5. doi: 10.1186/s12284-014-0005-6. eCollection 2014.
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.
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.
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.
Ⅱ型簇状、规律间隔、短回文重复（CRISPR）/CRISPR 相关蛋白 9（Cas9）系统是一种用于特定基因组修饰的新型分子工具。CRISPR-Cas9 系统最近通过瞬时或稳定转化被引入植物。
在这里，我们通过农杆菌介导的 CRISPR-Cas9 系统报告了水稻中的基因靶向。设计了三个 20 个核苷酸的 CRISPR RNA 与不同的位点配对，随后是水稻除草剂抗性基因 BEL 的前导序列相邻基序（PAM）。在将单指导 RNA（sgRNA）和 Cas9 盒整合到单个二元载体中后，通过农杆菌介导的稳定转化生成了携带 sgRNA:Cas9 的转基因水稻植物。通过分析相应转基因植物基因组上的靶向位点，确定了突变。突变效率从约 2%到约 16%不等。此外，表型分析表明，双等位基因突变的转基因植物对苯达松敏感。
我们的结果表明，农业性状可以通过 sgRNA:Cas9 诱导的基因靶向来有意修饰。CRISPR-Cas9 系统可作为作物改良中改良性状的有力工具。
