Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system.
Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system.
复制标题
DOI：
10.1038/srep44304
复制
发表时间：
2017-03-13
影响因子：
4.6
通讯作者：
Ye W
中科院分区：
文献类型：
--
作者：
Chen X;Lu X;Shu N;Wang S;Wang J;Wang D;Guo L;Ye W
CRISPR/Cas9系统由于其简单、高效和设计灵活，已被广泛用于各种植物的基因组编辑。然而，据我们所知，目前还没有关于CRISPR/Cas9介导的靶向诱变在棉花中应用的报道。在这里，我们报告了高地棉（Gossypium hirsutum L.，下文称为棉花）。我们设计了两个向导RNA，以靶向棉花Cloroplaesthetalterados 1（GhCLA 1）和液泡H+-焦磷酸酶（GhVP）基因的不同位点。在棉花原生质体中检测到这两个基因的突变。棉花原生质体中的突变主要为核苷酸替换，其中GhCLA 1有1个核苷酸插入和1个核苷酸替换，GhVP有1个核苷酸缺失。随后，通过农杆菌介导的转化，将这两个载体转化到棉花茎尖中，导致高效的目的基因编辑。转基因棉花的突变以核苷酸缺失为主，突变效率为47.6-81.8%。使用限制性酶-PCR测定和序列分析的评价未检测到脱靶突变。我们的结果表明，CRISPR/Cas9系统是棉花基因组靶向诱变的有效和特异性工具。
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H+-pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6–81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome.