Abstract
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.
Highlights
With the release of more and more various plant genome data, the study of gene functions has become increasingly important
In order to use the nicking endonucleases-mediated DNA assembly (NEMDA) strategy for fast and high-throughput assembly of intron-containing hairpin RNA (ihpRNA), we developed a plant basic RNA interference (RNAi) vector pRNAi-Nicking endonucleases (NE) (Figure 1A), which was obtained by assembling a NE-RNAi fragment (~1.2 kb) into our previous plant binary vector pYLRNAi.2.0 [17] using modified
The functional element of pRNAi-NE is composed of CaMV35s promoter, first multiple cloning site (MCS1), the GUS catalase intron, MCS2 and Nos terminator (Figure 1A), flanked by BamHI sites for digestion and identification of the ihpRNA construct
Summary
With the release of more and more various plant genome data, the study of gene functions has become increasingly important. As an important tool for plant gene function research, genetic engineering vectors have affected the progress of plant functional genomics [1]. RNA interference (RNAi) vectors and the CRISPR/Cas editing system are currently the most commonly-used reverse genetic tools for studying gene functions [2]. Construction of RNAi vectors, having an intron-containing hairpin RNA (ihpRNA) structure, and CRISPR/Cas vectors, for multiplex genome editing vectors, involves the splicing of multiple DNA fragments. A number of technologies for multi-fragment assembly have been developed, such as overlap extension-PCR (OE-PCR) [3,4], BioBrick [5,6], Gateway recombination [7], sequence and ligation-independent cloning (SLIC) [8], Golden Gate cloning [9,10] and Gibson Assembly [11].
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