CRISPR/Cas9-Mediated Multiply Targeted Mutagenesis in Orange and Purple Carrot Plants
CRISPR/Cas9-Mediated Multiply Targeted Mutagenesis in Orange and Purple Carrot Plants
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CRISPR/Cas9 介导的橙色和紫色胡萝卜植物多重靶向诱变
DOI：
10.1007/s12033-018-00150-6
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发表时间：
2019-03-01
影响因子：
2.6
通讯作者：
Xiong, Ai-Sheng
中科院分区：
文献类型：
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作者：
Xu, Zhi-Sheng;Feng, Kai;Xiong, Ai-Sheng
最近，成簇规则间隔短回文重复序列 (CRISPR) 相关蛋白 9 (Cas9) 系统已成功用于许多植物物种（包括胡萝卜细胞）的精确基因组编辑。然而，迄今为止，还没有利用CRISPR/Cas9系统获得稳定的基因编辑胡萝卜植物。在本研究中，使用农杆菌介导的遗传转化将四个 sgRNA 表达盒（分别由四个不同的启动子驱动并组装在单个 CRISPR/Cas9 载体中）转化到胡萝卜中。选择DcPDS和DcMYB113样基因的四个位点作为靶标。敲除橙色胡萝卜‘Kurodagosun’中的DcPDS，产生白化胡萝卜植株，编辑效率约为35.3%。在紫色胡萝卜‘深紫色’中也成功编辑了DcMYB113-like，产生了紫色脱色胡萝卜植株，突变率约为36.4%。测序分析表明，靶位点发生插入、缺失和取代，产生杂合、双等位基因和嵌合突变。在 DCPDS 和 DCMYB113 样敲除 T0 植物中，在 AtU6-29 驱动的 sgRNA 靶向的位点中观察到最高的诱变效率，这些位点总是在靶位点诱导双链断裂。我们的结果证明CRISPR/Cas9系统可以用于产生稳定的基因编辑胡萝卜植物。
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system has been successfully used for precise genome editing in many plant species, including in carrot cells, very recently. However, no stable gene-editing carrot plants were obtained with CRISPR/Cas9 system to date. In the present study, four sgRNA expression cassettes, individually driven by four different promoters and assembled in a single CRISPR/Cas9 vector, were transformed into carrots usingAgrobacterium-mediated genetic transformation. Four sites ofDcPDSandDcMYB113-likegenes were chosen as targets. Knockout ofDcPDSin orange carrot ‘Kurodagosun’ resulted in the generation of albino carrot plantlets, with about 35.3% editing efficiency.DcMYB113-likewas also successfully edited in purple carrot ‘Deep purple’, resulting in purple depigmented carrot plants, with about 36.4% rate of mutation. Sequencing analyses showed that insertion, deletion, and substitution occurred in the target sites, generating heterozygous, biallelic, and chimeric mutations. The highest efficiency of mutagenesis was observed in the sites targeted by AtU6-29-driven sgRNAs in bothDcPDS- andDcMYB113-like-knockoutT0plants, which always induced double-strand breaks in the target sites. Our results proved that CRISPR/Cas9 system could be for generating stable gene-editing carrot plants.