【摘要】 基于Golden Gate载体构建过程,对CRISPR/Cas9双靶点敲除技术的载体构建系统进行了优化,建立了一种简单快速高效且无PCR扩增的双靶点CRISPR/Cas9载体构建方法。在本方法中,只需将设计好的2个靶点DNA小片段和1个外源片段用T4DNA连接酶构建到CRISPR/Cas9载体上即可。用本方法对10个已报道的水稻基因构建了20个双靶点CRISPR/Cas9载体,阳性率为100%,测序结果显示无突变。该载体系统非常适合大批量CRISPR/Cas9载体的构建,同时也为其他植物中构建双靶点CRISPR/Cas9载体提供借鉴。更多还原

【Abstract】 A simple,fast and efficient method of constructing dual-target CRISPR/Cas9 vector was established based on the Golden Gate ligation.The two designed small target DNA fragments and one exogenous fragment were constructed together into the CRISPR/Cas9 vector with T4 DNA ligase.20 dual-target CRISPR/Cas9 vectors against 10 reported rice genes were constructed with this method,and the positive rate was 100% with no mutation in sequences.This vector system provides an easy,rapid and efficient method for constructing dual-target CRISPR/Cas9 vector for rice.It also provides a reference for the construction of dual-target CRISPR/Cas9 vectors in other plants.更多还原