Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research

Plants

Xubiao Lu¹  Qi Gong²  Yuke Hou²  Bin Wang²  Jiantao Tan²  Taoli Liu²  Yao-Guang Liu²  Qinlong Zhu² 

[1] College of Life Sciences, South China Agricultural University, Guangzhou 510642, China;State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou 510642, China;

关键词: nicking endonuclease;    DNA fragment assembly;    NEMDA;    vector construction;    RNAi;    CRISPR/Cas9;   

DOI  :  10.3390/plants9091090

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown