Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes.
Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes.
复制标题
使用 CRISPR/Cas9 核糖核蛋白复合物对面包小麦进行高效的无 DNA 基因组编辑
DOI：
10.1038/ncomms14261
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发表时间：
2017-01-18
影响因子：
16.6
通讯作者：
Gao C
中科院分区：
文献类型：
--
作者：
Liang Z;Chen K;Li T;Zhang Y;Wang Y;Zhao Q;Liu J;Zhang H;Liu C;Ran Y;Gao C
目前正在做出大量努力来优化CRISPR/Cas9系统以进行精准作物育种。避免转基因整合和减少脱靶突变是优化的最重要目标。在这里，我们描述了一种使用CRISPR/Cas9核糖核蛋白（RNP）的面包小麦的有效基因组编辑方法。从RNP制备开始，整个方案仅需7至9周，从100个小麦幼胚中产生了4至5个独立的突变体。深度测序表明，小麦细胞中脱靶突变的机会在RNP介导的基因组编辑中比在CRISPR/Cas9 DNA编辑中低得多。与这一发现一致，在突变体植物中未检测到脱靶突变。由于在CRISPR/Cas9 RNP介导的基因组编辑中不使用外源DNA，因此获得的突变体完全不含转基因。该方法可广泛应用于生产基因组编辑的作物植物，具有良好的商业化前景。
Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.