Genome Editing in Cotton with the CRISPR/Cas9 System.
Genome Editing in Cotton with the CRISPR/Cas9 System.
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使用 CRISPR/Cas9 系统对棉花进行基因组编辑
DOI：
10.3389/fpls.2017.01364
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发表时间：
2017
影响因子：
5.6
通讯作者：
Song C
中科院分区：
文献类型：
--
作者：
Gao W;Long L;Tian X;Xu F;Liu J;Singh PK;Botella JR;Song C
基因组编辑是基因功能研究和作物改良的重要工具。最近开发的CRISPR/Cas9系统使用单向导RNA分子（sgRNA）来指导基因组中精确的双链断裂，有可能彻底改变农业。不幸的是，并非所有的sgRNA都是同样有效的，并且很难通过生物信息学预测它们的效率。在作物如棉花（Gossypium hirsutum L.）中，对于劳动密集型和冗长的转化程序，必须最大限度地减少使用无效sgRNA的风险，这可能导致产生没有所需CRISPR诱导突变的转基因植物。在这项研究中，我们开发了一种快速有效的方法来验证棉花中使用瞬时表达系统的sgRNA的功能。我们使用这种方法验证了三种不同基因GhPDS、GhCLA 1和GhEF 1的靶位点，并分析了CRISPR/Cas9诱导突变的性质。在我们的实验中，在棉花子叶中观察到的最常见的突变类型是缺失（约64%）。我们证明了CRISPR/Cas9系统可以有效地在棉花基因中产生突变，这是这种异源四倍体作物的重要条件。我们还表明，多个基因靶向可以在棉花中实现，同时表达几种sgRNA，并在两个靶位点产生了GhPDS和GhEF 1的突变。此外，我们使用CRISPR/Cas9系统在GhPDS基因座中产生靶向基因片段缺失。最后，我们获得了在GhCLA 1基因中含有CRISPR/Cas9诱导的基因编辑突变的转基因棉花植物。突变效率非常高，80.6%的转基因株系在GhCLA 1靶位点含有突变，由于干扰叶绿体生物发生而导致强烈的白化病表型。
Genome editing is an important tool for gene functional studies as well as crop improvement. The recent development of the CRISPR/Cas9 system using single guide RNA molecules (sgRNAs) to direct precise double strand breaks in the genome has the potential to revolutionize agriculture. Unfortunately, not all sgRNAs are equally efficient and it is difficult to predict their efficiency by bioinformatics. In crops such as cotton (Gossypium hirsutum L.), with labor-intensive and lengthy transformation procedures, it is essential to minimize the risk of using an ineffective sgRNA that could result in the production of transgenic plants without the desired CRISPR-induced mutations. In this study, we have developed a fast and efficient method to validate the functionality of sgRNAs in cotton using a transient expression system. We have used this method to validate target sites for three different genes GhPDS, GhCLA1, and GhEF1 and analyzed the nature of the CRISPR/Cas9-induced mutations. In our experiments, the most frequent type of mutations observed in cotton cotyledons were deletions (∼64%). We prove that the CRISPR/Cas9 system can effectively produce mutations in homeologous cotton genes, an important requisite in this allotetraploid crop. We also show that multiple gene targeting can be achieved in cotton with the simultaneous expression of several sgRNAs and have generated mutations in GhPDS and GhEF1 at two target sites. Additionally, we have used the CRISPR/Cas9 system to produce targeted gene fragment deletions in the GhPDS locus. Finally, we obtained transgenic cotton plants containing CRISPR/Cas9-induced gene editing mutations in the GhCLA1 gene. The mutation efficiency was very high, with 80.6% of the transgenic lines containing mutations in the GhCLA1 target site resulting in an intense albino phenotype due to interference with chloroplast biogenesis.
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影响因子：
4.6
作者：
Chen X;Lu X;Shu N;Wang S;Wang J;Wang D;Guo L;Ye W
通讯作者：
Ye W
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影响因子：
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作者：
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作者：
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通讯作者：
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