Gong Qi, Wang Bin, Lu Xubiao, Tan Jiantao, Hou Yuke, Liu Taoli, Liu Yao-Guang, Zhu Qinlong
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou 510642, China.
Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642, China.
Plants (Basel). 2020 Aug 25;9(9):1090. doi: 10.3390/plants9091090.
Plant genetic engineering vectors, such as RNA interference (RNAi) and CRISPR/Cas9 vectors, are important tools for plant functional genomics. Efficient construction of these functional vectors can facilitate the study of gene function. Although some methods for vector construction have been reported, their operations are still complicated and costly. Here, we describe a simpler and low-cost vector construction method by nicking endonucleases-mediated DNA assembly (NEMDA), which uses nicking endonucleases to generate single-strand overhanging complementary ends for rapid assembly of DNA fragments into plasmids. Using this approach, we rapidly completed the construction of four RNAi vectors and a CRISPR/Cas9 knockout vector with five single-guide RNA (sgRNA)-expression cassettes for multiplex genome editing, and successfully achieved the goal of decreasing the expression of the target genes and knocking out the target genes at the same time in rice. These results indicate the great potential of NEMDA in assembling DNA fragments and constructing plasmids for molecular biology and functional genomics.
植物基因工程载体，如RNA干扰（RNAi）和CRISPR/Cas9载体，是植物功能基因组学的重要工具。高效构建这些功能载体有助于基因功能的研究。尽管已经报道了一些载体构建方法，但其操作仍然复杂且成本高昂。在此，我们描述了一种通过切口内切酶介导的DNA组装（NEMDA）实现的更简单、低成本的载体构建方法，该方法利用切口内切酶产生单链突出互补末端，以便将DNA片段快速组装到质粒中。利用这种方法，我们快速完成了四个RNAi载体和一个带有五个用于多重基因组编辑的单向导RNA（sgRNA）表达盒的CRISPR/Cas9敲除载体的构建，并成功实现了在水稻中同时降低靶基因表达和敲除靶基因的目标。这些结果表明NEMDA在组装DNA片段和构建用于分子生物学和功能基因组学的质粒方面具有巨大潜力。
