An Efficient CRISPR/Cas9 Platform for Rapidly Generating Simultaneous Mutagenesis of Multiple Gene Homoeologs in Allotetraploid Oilseed Rape.
An Efficient CRISPR/Cas9 Platform for Rapidly Generating Simultaneous Mutagenesis of Multiple Gene Homoeologs in Allotetraploid Oilseed Rape.
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一种高效的 CRISPR/Cas9 平台，用于快速生成异源四倍体油菜中多基因同源物的同时诱变
DOI：
10.3389/fpls.2018.00442
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发表时间：
2018
影响因子：
5.6
通讯作者：
Hu Q
中科院分区：
文献类型：
--
作者：
Li C;Hao M;Wang W;Wang H;Chen F;Chu W;Zhang B;Mei D;Cheng H;Hu Q
随着序列特异性核酸酶（SSN）基因组靶向技术的快速发展，成簇规则间隔短回文重复序列/CRISPR相关蛋白9（CRISPR/Cas9）被认为是作物功能遗传研究和遗传改良的最有前途的方法。然而，异源四倍体油菜基因组中的基因冗余特征是在第一代中同时修饰不同同源物的主要障碍之一。此外，使用常规的基于限制性内切酶的方法来大规模筛选以鉴定突变的转基因植物是非常耗时和费力的。本研究通过合成预制的U6-26驱动的sgRNA表达盒和优化基于聚丙烯酰胺凝胶电泳（PAGE）的筛选方法，开发了一个精简的油菜CRISPR-Cas9基因组编辑平台。在我们的实验中，构建了靶向五个油菜籽SPL 3同源基因拷贝BnSPL 3-A5/BnSPL 3-A4/BnSPL 3-C3/BnSPL 3-C4/BnSPL 3-Cnn的sgRNA。高通量测序分析表明，在具有明显异源化PAGE条带的植物中，CRISPR/Cas9诱导突变的编辑频率范围为96.8%-100.0%，否则这一比例仅为0.00%-60.8%。与这些分子分析一致，Bnspl 3突变体在第一代中表现出发育延迟表型。总之，我们的数据表明，这组CRISPR/Cas9平台有资格快速产生和鉴定异源四倍体油菜籽中多个基因同源物的同时诱变。
With the rapid development of sequence specific nucleases (SSNs) for genome targeting, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is now considered the most promising method for functional genetic researches, as well as genetic improvement in crop plants. However, the gene redundancy feature within the allotetraploid rapeseed genome is one of the major obstacles for simultaneous modification of different homologs in the first generation. In addition, large scale screening to identify mutated transgenic plants is very time-and labor-consuming using the conventional restriction enzyme-based approaches. In this study, a streamlined rapeseed CRISPR-Cas9 genome editing platform was developed through synthesizing a premade U6-26 driven sgRNA expression cassette and optimizing polyacrylamide gel electrophoresis (PAGE)-based screening approach. In our experiment, a sgRNA was constructed to target five rapeseed SPL3 homologous gene copies, BnSPL3-A5/BnSPL3-A4/BnSPL3-C3/BnSPL3-C4/BnSPL3-Cnn. High-throughput sequencing analysis demonstrated that the editing frequency of CRISPR/Cas9-induced mutagenesis ranged from 96.8 to 100.0% in plants with obvious heteroduplexed PAGE bands, otherwise this proportion was only 0.00–60.8%. Consistent with those molecular analyses, Bnspl3 mutants exhibited developmental delay phenotype in the first generation. In summary, our data suggest that this set of CRISPR/Cas9 platform is qualified for rapidly generating and identifying simultaneous mutagenesis of multiple gene homologs in allotetraploid rapeseed.
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影响因子：
6.6
作者：
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通讯作者：
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