Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression.
Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression.
复制标题
通过改进 sgRNA 表达优化棉花 CRISPR/Cas9 基因组编辑
DOI：
10.1186/s13007-018-0353-0
复制
发表时间：
2018
期刊：
影响因子：
5.1
通讯作者：
Song CP
中科院分区：
文献类型：
--
作者：
Long L;Guo DD;Gao W;Yang WW;Hou LP;Ma XN;Miao YC;Botella JR;Song CP
在开发用于作物的CRISPR/Cas9系统时，投入时间表征CRISPR/Cas9盒的基因组编辑效率至关重要，特别是如果转化系统困难或耗时。棉花是生产纤维、石油和生物燃料的重要作物。然而，棉花稳定转化通常使用根癌农杆菌进行，需要8至12个月以产生T0植物。此外，棉花是异源四倍体，并且靶向诱变被认为是困难的，因为许多基因在这个复杂的基因组中重复。CRISPR/Cas9在棉花中的应用受到漫长且技术上具有挑战性的遗传转化过程的严重阻碍，因此必须最大限度地提高其效率。在这项研究中，我们提供了一个新的系统来评估和验证CRISPR/Cas9盒在棉花中使用瞬时表达系统的效率。通过使用该系统，我们可以在稳定转化之前选择最有效的CRISPR/Cas9盒。我们还优化了现有的棉花CRISPR/Cas9系统，通过引入内源性GhU 6启动子来实现大幅提高的诱变效率，该启动子相对于拟南芥AtU 6 -29启动子增加sgRNA表达水平。克隆了300 bp的GhU6.3启动子，并利用瞬时表达系统进行了验证。当sgRNA在CRISPR/Cas9盒中的GhU6.3启动子的控制下表达时，表达水平比AtU 6 -29启动子提供的表达水平高6-7倍，CRISPR/Cas9介导的突变效率提高4-6倍。这项研究通过降低CRISPR/Cas9方法在棉花靶向诱变中应用的风险和工作量，为最大化CRISPR/Cas9介导的突变效率提供了必要的改进。本文的在线版本（10.1186/s13007-018-0353-0）包含补充材料，可供授权用户使用。
When developing CRISPR/Cas9 systems for crops, it is crucial to invest time characterizing the genome editing efficiency of the CRISPR/Cas9 cassettes, especially if the transformation system is difficult or time-consuming. Cotton is an important crop for the production of fiber, oil, and biofuel. However, the cotton stable transformation is usually performed using Agrobacterium tumefaciens taking between 8 and 12 months to generate T0 plants. Furthermore, cotton is a heterotetraploid and targeted mutagenesis is considered to be difficult as many genes are duplicated in this complex genome. The application of CRISPR/Cas9 in cotton is severely hampered by the long and technically challenging genetic transformation process, making it imperative to maximize its efficiency. In this study, we provide a new system to evaluate and validate the efficiency of CRISPR/Cas9 cassettes in cotton using a transient expression system. By using this system, we could select the most effective CRISPR/Cas9 cassettes before the stable transformation. We have also optimized the existing cotton CRISPR/Cas9 system to achieve vastly improved mutagenesis efficiency by incorporating an endogenous GhU6 promoter that increases sgRNA expression levels over the Arabidopsis AtU6-29 promoter. The 300 bp GhU6.3 promoter was cloned and validated using the transient expression system. When sgRNAs were expressed under the control of the GhU6.3 promoter in CRISPR/Cas9 cassettes, expression levels were 6–7 times higher than those provided by the AtU6-29 promoter and CRISPR/Cas9-mediated mutation efficiency was improved 4–6 times. This study provides essential improvements to maximize CRISPR/Cas9-mediated mutation efficiency by reducing risk and workload for the application of CRISPR/Cas9 approaches in the targeted mutagenesis of cotton. The online version of this article (10.1186/s13007-018-0353-0) contains supplementary material, which is available to authorized users.
登录
查看更多内容
影响因子：
5.1
作者：
Johnson, Ross A.;Gurevich, Vyacheslav;Levy, Avraham A.
通讯作者：
Levy, Avraham A.
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
影响因子：
5.1
作者：
Mikami M;Toki S;Endo M
通讯作者：
Endo M
影响因子：
17.3
作者：
Gaj, Thomas;Gersbach, Charles A.;Barbas, Carlos F., III
通讯作者：
Barbas, Carlos F., III
影响因子：
27.5
作者：
Ma, Xingliang;Zhang, Qunyu;Liu, Yao-Guang
通讯作者：
Liu, Yao-Guang