| CRISPR/Cas9介导柑橘CsLOB1基因启动子的多位点编辑 |
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| 引用本文： | 邹修平,范 迪,彭爱红,何永睿,许兰珍,雷天刚,姚利晓,李 强,罗克明,陈善春.CRISPR/Cas9介导柑橘CsLOB1基因启动子的多位点编辑[J].园艺学报,2019,46(2):337-344. |
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| 作者姓名： | 邹修平 范 迪 彭爱红 何永睿 许兰珍 雷天刚 姚利晓 李 强 罗克明 陈善春 |
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| 作者单位： | 1西南大学柑桔研究所，重庆 400712；2西南大学生命科学学院，重庆 400715 |
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| 基金项目： | 南方山地园艺学教育部重点实验室开放课题项目；国家现代农业产业技术体系建设专项资金项目（CARS-26）；重庆市自然科学基金项目（cstc2017jcyjBX0020） |
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| 摘 要： | 为了获得CsLOB1启动子较大片段删除的突变体,利用CRISPR/Cas9技术对CsLOB1启动子进行多位点编辑。通过在CsLOB1启动子的EBEPthA4区域及其上、下游设计不同的靶标位点,构建了2个植物表达载体pCas9CsLOB1:2sites和pCas9CsLOB1:3sites,分别对CsLOB1启动子同时进行2个位点和3个位点的编辑。测序结果表明pCas9CsLOB1:2sites和pCas9CsLOB1:3sites的基因编辑效率分别为64.7%和80.0%,突变体植株在2个sgRNA之间发生了DNA片段的删除。进一步的分析发现,不同的sgRNA具有不同的突变效率,其差异是由于不同的sgRNA对CsLOB1-识别和结合能力的差异造成的。本结果表明对CsLOB1启动子进行多位点编辑可以获得删除较大DNA片段的突变体。
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| 关 键 词： | 柑橘 CRISPR/Cas9系统 CsLOB1 多位点 基因编辑 |
CRISPR/Cas9-mediated Editing of Multiple Sites in the Citrus CsLOB1 Promoter |
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| ZOU Xiuping,FAN Di,PENG Aihong,HE Yongrui,XU Lanzhen,LEI Tiangang,YAO Lixiao,LI Qiang,LUO Keming,CHEN Shanchun,.CRISPR/Cas9-mediated Editing of Multiple Sites in the Citrus CsLOB1 Promoter[J].Acta Horticulturae Sinica,2019,46(2):337-344. |
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| Authors: | ZOU Xiuping FAN Di PENG Aihong HE Yongrui XU Lanzhen LEI Tiangang YAO Lixiao LI Qiang LUO Keming CHEN Shanchun |
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| Institution: | 1Citrus Research Institute，Southwest University，Chongqing 400712，China；2School of Life Sciences，Southwest University，Chongqing 400715，China |
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| Abstract: | To obtain mutants with large fragment deletions in the CsLOB1 promoter，in this study，we edited multiple sites of the CsLOB1 promoter using the CRISPR/Cas9 system. Two plant expression vectors，pCas9CsLOB1:2sites and pCas9CsLOB1:3sites，which were designed for the simultaneous targeting of two and three sites in the CsLOB1 promoter，respectively，were constructed to edit the EBEPthA4 region. Sequencing data revealed that the editing efficiency in pCas9CsLOB1:2sites and pCas9CsLOB1:3sites transgenic lines was 64.7% and 80.0%，respectively，and fragment deletions between two sgRNA target sites occurred in the transgenic citrus plants. Further analyses indicated the mutation efficiency differed among the sgRNAs. The differences were likely due to variability in the binding ability of sgRNAs to the targeted CsLOB1- sequence. These results showed that mutants with the deletion of large DNA fragment can be obtained through editing multiple sites within the CsLOB1 promoter. |
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| Keywords: | citrus CRISPR/Cas9 system CsLOB1 multiple sites gene editing |
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